a protein-bound o forf porcinm e renal renincircres.ahajournals.org/content/35/3/426.full.pdfa...

A Protein-Bound Form of Porcine Renal Renin

By Graham W. Boyd

ABSTRACTTwo interconvertible renins were isolated from extracts of the pig renal cortex at neutral

pH and shown to be protein-bound (renin B, molecular weight 60,000) and free (renin A,molecular weight 40,000) forms of renin. The protein-bound form (renin B) gave a muchmore prolonged pressor response than did the free form (renin A) on direct bioassay in therat; it could be converted to renin A by the action of various salts or by acidification belowpH 3. The renin-binding protein associated with renin B was isolated by DEAE-cellulosecolumn chromatography under special conditions of elution and appeared to be specificfor renin. When pig kidneys were perfused in situ with Krebs-Ringer's solution,isoproterenol stimulation (1-8 tig/m\n) resulted in release of renin in the protein-boundform. It is suggested that renin-binding protein may act as a carrier for the transport ofrenin to local tissue sites of uptake under some circumstances and thus alter the mode ofexpression of the renin-angiotensin system in vivo to one of local angiotensin generationand effect.

KEY WORDS renin-binding proteinangiotensin antibodies renin releaseangiotensin I immunoassay

renin bioassayisoproterenol

• The precise extent to which the renin-angioten-sin system is involved in the pathogenesis of renalhypertension remains uncertain despite a greatdeal of investigation (1). Possibly, its importancevaries with different types of renal hypertension(2), but in at least one type, namely, that associ-ated with unilateral renal artery clipping combinedwith contralateral nephrectomy, most of the evi-dence militates against any major role for circulat-ing angiotensin II itself (2-5). This observation sug-gests either that other unknown factors are involvedor that the mode of operation of the renin-angioten-sin system under these circumstances is moresubtle or more complex than is generally assumed.

These considerations recently led me to reinves-tigate the nature of the pressor materials present inrenal extracts from a variety of species (6). Thisstudy did not identify any new nonrenin pressormaterial, but in two species, the rat and the pig,two separate forms of renal renin were found (6).The present investigation is concerned with thefurther elucidation of the nature of these two formsof renin extracted from the pig kidney cortex. Apreliminary account of this work has been pub-lished previously (7).

MethodsMATERIALS

All chemical reagents were analytical reagent grade

From the Medical Unit, St. Mary's Hospital Medical School,Norfolk Place, London, W.2, Great Britain.

Received July 10, 1973. Accepted for publication May 31,1974.

except for NaSCN, NaC104, 8-hydroxyquinoline sulfate,and ethylenediaminetetraacetic acid (EDTA), whichwere laboratory grade. EDTA was used as a 3.0Msolution adjusted to pH 7.5 with 40% NaOH. Othercompounds used were dithioerythritol (Cleland's rea-gent, Sigma), 2-mercaptoethanol (Sigma), 2, 3-dimer-capto-1-propanol (BAL, Koch Light), 2, 2-dipyridyl(Hopkins and Williams), sodium dodecyl sulfate (BritishDrug Houses), isoproterenol sulfate, pentolinium (0.5%w/v, May and Baker Ltd.), sodium pentobarbital (Ab-bott Laboratories), and neomycin sulfate (Biorex Ltd.).Enzymes and molecular weight markers used were pep-sin (1:60,000, twice crystallized, Sigma), trypsin (twicecrystallized, Sigma), Blue Dextran 2000 (PharmaciaLtd.), bovine serum albumin (Koch Light, 0142t), oval-bumin (Koch Light, 4422h), and sperm whale myoglobin(Koch Light, 4074t).

STANDARD RENINS

The pig renin standard that I used was a semipurifiedpreparation obtained by processing renal cortical ex-tracts through stage 4 of the procedure of Peart et al. (8),including the acidification step. Standard rat and rabbitrenins were prepared by processing extracts of wholekidneys from these species on DEAE-cellulose and G100Sephadex columns exactly as described for pig renalextracts (see below). Human renin was a gift1; it had aspecific activity of 0.13 Goldblatt units/mg (9).

RENIN SUBSTRATES

The pig renin substrate used throughout the presentstudy was heparinized plasma from blood collected 24hours after bilateral nephrectomy in the male pig. Theplasma was dialyzed to pH 4.5 at 4°C, heated at 32°C for30 minutes at this pH according to the method of Skinner(10), and dialyzed again to pH 7.5 in Skinner buffer C

1 Kindly supplied by Dr. E. Haas, Beaumont Research Lab-oratories, Mt. Sinai Hospital, Cleveland, Ohio.

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PROTEIN-BOUND RENIN 427

(10). This procedure rendered the preparation virtuallyfree of angiotensinase. The preparation also contained nodetectable quantities of renin; incubation of variouspreparations with an excess of standard pig renin yielded1.5-2 ixg angiotensin I/ml substrate by immunoassay.Ox renin substrate was supplied by the Biological Stan-dards Division of the National Institute for MedicalResearch, London, and was Renin Substrate M, 67/26.This material was used in pepsin studies; each ampulwas reconstituted in 2.0 ml %of 0.05M sodium phosphatebuffer, pH 6.1. Human renin substrate was heparinized(5 units/ml) plasma from a normal female who was nottaking oral contraceptives. The amount of renin itcontained was negligible in comparison with the quantityof human renin added in the present studies.

ANTIBODY AGAINST RABBIT RENAL EXTRACT

An antibody against rabbit renal extract was raised inthe goat by injecting intramuscularly every month 4 mlof extract of whole rabbit kidney (emulsified with 2 ml ofcomplete Freund's adjuvant) prepared exactly as de-scribed- below for crude pig renal cortical extract. Theplasma used was from a blood sample drawn 10 daysafter the boosting injection given at 12 months.

ASSAYS FOR PRESSOR MATERIALS

Direct Bioassay.—The anesthetized, pentolinium-blocked rat blood pressure preparation described byPeart (11) was used. Anesthesia was maintained byintermittently administering urethane (25%) rather thanpentobarbital, since this procedure enhanced the distinc-tion between the various forms of pressor materialobtained from the renal extracts (6). The working pigrenin standard used throughout the present study wascalibrated against MRC Porcine Research Standard65/119, and all results are expressed in terms of units ofthis standard (one MRC unit is approximately equiva-lent to one Goldblatt unit). Samples were assayed bybracketing the pressor response to the unknown betweenthose to known doses of standard in a way similar to thatdescribed for angiotensin assay (12); in all cases valueswere calculated from the height of the response regard-less of its shape. Bracket assay limits were usually within±20% of the mean.

The reninlike nature of any unknown pressor materialwas investigated on direct bioassay by a comparison ofits pressor response before and after the intravenousadministration of 1 ml of a potent antiangiotensin IIantiserum into the rat bioassay preparation. This anti-serum, prepared as described previously (13), was capa-ble of neutralizing 4 jtg angiotensin Il/ml (13) and ofbinding 60% of a 5-pg '"I-angiotensin II label at adilution of 1:20,000 on radioimmunoassay (3).

Indirect Renin Assay.—In some studies, renin wasassayed indirectly by measuring the rate of generation ofangiotensin I from pig renin substrate in the presence ofinhibitors of angiotensinase and converting enzyme.Normally, 0.0075M EDTA and 0.01M BAL were sufficientto inhibit these latter enzymes, but with some fractionsof renal extracts 0.003M 8-hydroxyquinoline sulfate and0.003M 2, 2-dipyridyl were required as well. In all indirectrenin assays cited, there was less than a 20% loss of addedangiotensin I over the period of incubation (see below).

Angiotensin I was generated at 37°C and pH 7.5Circulation Research, Vol. 35, September 1974

(Skinner buffer C [10]). The volume of the incubationmixture was 2.0 ml and included 0.1-0.4 ml of thevarious renal extract fractions (either alone or in combi-nation after preincubation; see Results) and 1.4-1.6 ml ofrenin substrate. Samples (0.2 ml) were removed atvarious times, and angiotensin generation was stoppedby diluting the sample 1:4 in assay buffer and heating itat 85-90°C for 10 minutes. The samples were then frozenand thawed, and the cloudy precipitate was removed bycentrifugation at 2,700 g for 10 minutes at 4°C. Thesupernatant fluid was finally assayed against l-Asp-5-Ile-angiotensin I (Schwarz-Mann) using either bioassay(11) or radioimmunoassay of angiotensin I (14). Assaybuffer for bioassay was Skinner buffer C (10) at pH 7.5containing 2.5 mg/ml of human serum albumin (ListerInstitute), 0.02M CaCl,, and 0.02% neomycin sulfate (14).When different amounts of standard pig renin wereassayed in this system, the rate of angiotensin I genera-tion over the first 20 minutes was directly proportional tothe concentration of renin up to at least 0.025 unitsrenin/ml (this dose yielded 500 ng angiotensin I/ml in 20minutes).

ANGIOTENSINASE ASSAY

Samples (0.2-0.4 ml) for assay of angiotensinaseactivity were incubated at 37°C with angiotensin I(0.2-1.2 jzg/ml) in a final volume of 2 ml (Skinner bufferC) with the angiotensinase inhibitors BAL (0.01M),

EDTA (0.0075M), 2, 2-dipyridyl (0.003M), and 8-hydrox-yquinoline sulfate (0.003M). Samples were removed atintervals, diluted, heated, and assayed for angiotensin Ias described above.

DETERMINATION OF MICHAELIS CONSTANTS FOR RENINS

For the determination of values of the Michaelisconstant (Km), fixed amounts of various renins(0.01-0.025 units, 0.3-0.7 units/mg) were incubated withvarious concentrations of a standard preparation ofplasma renin substrate from nephrectomized pigs at37°C in the presence of BAL and EDTA exactly asdescribed above. The angiotensin I that was generatedwas measured by immunoassay, bioassay, or both, andthe initial velocity of the reaction was calculated fromthe estimates taken at 0, 5, 10, and 15 minutes. Kmvalues were obtained graphically from Lineweaver-Burkplots of the data.

PREPARATION OF CRUDE PIG RENAL CORTICAL EXTRACT

Pig kidneys were taken immediately after slaughter atabattoirs and frozen without delay. After thawing, therenal cortices were dissected off, sliced, and homogenizedfor 3 minutes in a Waring blender at 4°C. The homoge-nate was then extracted with 1-2 ml/g of either 0.15MNaCl or 0.05M sodium phosphate buffer, pH 7.4, bothcontaining 0.02% neomycin sulfate. Extracts were thenfrozen and thawed twice and centrifuged at 1,000 £ for 30minutes. The supernatant fluid was centrifuged in aMSE Superspeed 40 preparative ultracentrifuge for 60minutes at 35,000 g. The pH of the supernatant fluidfrom the saline extract was 6.5 at this stage and thatfrom the phosphate buffer extract was 7.2. Extracts weresubsequently dialyzed (24/32 Visking tubing) against thestarting buffer for the DEAE-cellulose purification step(see below) and finally clarified by ultracentrifugation at75,000 g for 60 minutes. The entire preparation was


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performed at 4°C.

DEAE-CELLULOSE COLUMN CHROMATOGRAPHY

DEAE-cellulose (DEll, Whatman Co., control no.DE/2079) was pretreated (Whatman Information LeafletIL2), equilibrated with starting buffer, and packed at4°C into columns under a hydrostatic pressure of approx-imately twice the final bed height. Renal extracts wereapplied in the same starting buffer (either 0.005M or0.025M sodium phosphate buffer with a pH of either 6.9or 7.0 as indicated in the text), and the columns weredeveloped by increasing the molarity of the sodiumphosphate buffer in steps, keeping the pH constant.Buffer was pumped onto the column at a constantflowrate (20-40 ml/hour) using a roller pump (Watson-Marlow type MHRE 22), and the eluate was collected in10-25-ml fractions. All buffers contained 0.02% neomy-cin sulfate, and the entire procedure was carried out at4°C.

O10O SEPHADEX COLUMN CHROMATOGRAPHY

Both standard and superfine grades of GlOO Sephadex(Pharmacia Ltd.) were used with columns (Pharmacia K26/100) packed to a final bed height of approximately 95cm (diameter 2.6 cm). The buffer was 0.1M sodiumphosphate, pH 6.9, containing 0.02% neomycin sulfate.Samples were applied in this buffer in a volume of 4 ml,and the column was developed at 4°C under a constanthydrostatic pressure of approximately 30 cm H,0 givinga flow rate of approximately 15 ml/hour (5 ml/hour forsuperfine Sephadex). The eluate was collected in 3- or5-ml fractions using a syphoning system (Central).Columns were subsequently calibrated according to thedata of Andrews (15) by running a mixture of BlueDextran 2000, bovine serum albumin, ovalbumin, pep-sin, trypsin, and sperm whale myoglobin as molecularweight markers.

PROTEIN ESTIMATION

Ultraviolet light absorption at 280 nm was used tomonitor the appearance of protein in the column eluate.Quantitative protein determination was performed by themethod of Lowry et al. (16), using human serum albuminas a standard. Pressure Dialysis for concentration ofprotein solutions was performed at 4°C, using an Ami-con PM 10 membrane in an ultrafiltration cell (Amiconmodel 200).

PERFU8ION OF THE ISOLATED PIG KIDNEY IN SITU

Male pigs (20-30 kg) were anesthetized with sodiumpentobarbital (30 mg/kg, iv), and their left kidneys wereexposed and isolated through an abdominal incision in away similar to that described previously for the rat (17).The left renal vein and the abdominal aorta at the originof the left renal artery were cannulated and isolated fromthe circulation, and the kidney was immediately per-fused (without interruption of flow) with improvedKrebs-Ringer's I solution (18) at 37°C at a flow rate of15-25 ml/min, using a Watson-Marlow roller pump. Theperfusion fluid was equilibrated continuously with 95%0,-5% CO,. Mean perfusion pressure (monitored by aDevices model 2 recorder) varied from 70 to 110 mm Hgbetween experiments. Before starting the experiment,the left ureter was cut to avoid back-pressure on thekidney. The kidney was first perfused until the renal vein

effluent was free of all visible blood (5-20 minutes); thisprocedure was followed by a control collection period(10-15 minutes). Isoproterenol sulfate (1-8 /ig/min) wasthen infused into the arterial supply line (1-2 ml/min),and a second sample was collected over the next 12-40minutes. All collections were taken into glass containersimmersed in an ice slurry. Samples were concentrated bypressure dialysis at 4°C and finally dialyzed against asolution of 0.05M phosphate and 0.1M NaCl, pH 7.0,containing 0.02% neomycin sulfate.

ResultsBIOASSAY OF CRUDE RENAL EXTRACTS

The renin concentration of the crude renal corti-cal extracts varied from 0.5 to 1.8 units/ml. Themean specific activity of six different preparationswas 0.035 ± 0.010 (SD) units/mg.

Initial investigation showed that these crudeneutral-pH extracts of pig renal cortex gave apattern of pressor response on rat bioassay differentfrom that obtained with a semipurified pig reninstandard in that the response was much moreprolonged with a delayed peak and a more convexdown-slope. On occasion, there was an inflection onthe upstroke prior to the peak as well, giving abiphasic appearance. These features have beendescribed previously (6). This finding was investi-gated further by DEAE-cellulose chromatographyof these crude extracts.

DEAE-CELLULOSE COLUMN CHROMATOGRAPHY

In an initial experiment, 75 ml of pig renalcortical extract (1.0 units/ml, 0.035 units/mg) wasdialyzed to 0.005M with sodium phosphate buffer atpH 7.0 and applied to a 45 x 1.7-cm column ofDEAE-cellulose at 4°C. The column was thensuccessively eluted with 0.025 (250 ml), 0.05 (400ml), and 0.1M phosphate buffer at pH 7.0. Figure 1shows that the pressor activity in this experimentwaseluted in two peaks; the first (called pig pressormaterial A for convenience) emerged with the0.05M fraction and the second (pig pressor materialB) emerged with the 0.1M fraction. These twopressor materials differed not only in their posi-tions of elution on DEAE-cellulose chromatogra-phy but also in the shapes of their pressor responseson direct bioassay (Fig. 2). The pressor response tomaterial A (which resembled pig renin standard)had a fairly sharp upstroke and a concave down-stroke. By contrast, the pressor response to mate-rial B was more reminiscent of the crude renalextract (6); it had a delayed and more roundedpeak with a very slow decay phase. This finding oftwo separate peaks of pressor activity of types Aand B has been confirmed repeatedly by similarchromatography of other batches of pig renal

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cortical extracts, including those for which pHduring the initial extraction was maintained at pH7.2.G10O SEPHADEX COLUMN CHROMATOGRAPHY

Post-DEAE-cellulose pressor material A ran witha calculated molecular weight of 40,000 on GlOOSephadex (38,000-42,000 in three separate runs),and its running position was indistinguishable fromthat of the semipurified pig renin standard in thesame system (molecular weight 42,000, N = 1). Bycontrast, renin B, run separately, emerged in amuch smaller elution volume with a correspond-ingly higher calculated molecular weight (58,000, N= 1). In the experiment shown in Figure 3, samplesof pressor materials A (3 units) and B (4 units)from DEAE-cellulose chromatography were eachconcentrated thirty times to 2 ml by pressuredialysis, mixed at 4°C, and immediately chromato-graphed on GlOO Sephadex. The two pressor mate-rials were separated in this system; material Bemerged first. Calibration of the column gave anestimated molecular weight of 60,000 for materialB and 38,000 for material A.

THE NATURE OF PRESSOR MATERIALS A AND B

Both pressor materials appeared to be protein innature. Thus, (1) they were nondialyzable (8/32Visking), (2) they had apparent molecular weightsen Sephadex chromatography in the protein range,(3) their pressor activities were lost on boiling for 5minutes, and (4) complete neutralization of pressoractivity occurred with both A and B after incuba-tion for 5 minutes at 20°C with a two-fifths volume

0-4

2 0

15

I<*> 1 0N

0-5

PHOSPHATE BUFFER pH 70

5 0 0 1000ml. ELUATE

FIGURE 1

DEAE-cellulose column chromatography of crude pig renalextract eluted with increasing molarity steps of sodium phos-phate buffer {containing 0.02% neomycin) at pH 7.0 and 4°C.O.D. - optical density.

20 min.

0 04 ml.A

0 03 ml.B

FIGURE 2

004 ml.A

Rat bioassay following chromatography of a mixture of pig renal pressor materials A and B on GlOOsuperfine Sephadex; 0.04 ml was administered per injection. Molecular weights (M.W.) were derivedafter calibration of the column with suitable markers (see text). B.P. - blood pressure.



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40

S 20

0

Void vol.= 160ml.

B

M.W.= 60,000M«t»ri»l A

M.W. = 38,000

180 193 200ml.ELUATE

FIGURE 3

220 223

Rat bioassay following chromatography of a mixture of pig renal pressor materials A and B on G100superfine Sephadex; 0.04 ml was administered per injection. Molecular weigts (M. W.) were derivedafter calibration of the column with suitable markrs (see text). B.P. - blood pressure.

of an antibody raised against rabbit kidney extract.Since this antibody is known to neutralize thepressor activity of pig renin standard under theseconditions, the latter observation also suggestedthat both materials A and B might be renins.

Indeed, two lines of evidence indicate that bothmaterials A and B are renins. (1) Incubation ofboth materials separately with pig renin substrateat 37 °C in the presence of BAL and EDTA resultedin the generation of a material which was biologi-cally and immunologically (radioimmunoassay) in-distinguishable from angiotensin I. (2) The pressorresponse to both materials was virtually abolishedafter the intravenous injection of antiangiotensin IIplasma into the rat bioassay preparation (Fig. 4).

The difference in the shape of the pressor re-sponses to materials A and B did not appear to berelated to any contamination with other compo-nents of the renin-angiotensin system. The possi-

bility that the relatively shorter duration of actionof material A was due to contamination with largeamounts of angiotensinase was excluded by theobservation that a substantial reduction in angi-otensinase activity in material A after acidificationto pH 2.5 at 4°C for 30 minutes was not associatedwith any change in the shape or the duration of itspressor response. The sharper upstroke of the bloodpressure response to material A also did not resultfrom contamination with angiotensin I, since itsactivity was completely destroyed by boiling for 5minutes, whereas angiotensin I added to material Awas recovered in at least 70% yield after thisprocedure. Material B, the material with the highermolecular weight, did not appear to be a complexof renin with its substrate, since incubation ofmaterial B alone at 37°C in the presence of theangiotensinase inhibitors BAL and EDTA did notresult in the generation of any detectable angioten-

20 min. 20min.

40

20

0 L

t i l l I40ngNor. I0 1ml

A

002 ml.B

40ng. I 40 ng.Nor. | Nor.

1ml.anti-AHplasma

I.V.

FIGURE 4

01ml.B

002ml.A

40ngNor.

Loss of the rat blood pressure (B.P.) responses to pig renal pressor materials A (0.17 units/ml) and B(0.75 units/ml) resulting from the intravenous (I.V.) administration of antiangiotensin II into thebioassay preparation. The pressor response to norepinephrine (Nor.) was unaffected by thisimmunization procedure.



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sin I either on bioassay or immunoassay.

THE INTERRELATIONSHIP BETWEEN RENINS A AND B

Whereas acidification to pH 2.5 at 4°C for 30minutes did not detectably alter the height or theshape of the pressor response to post-DEAE-cel-lulose renin A, similar treatment of renin B alwaysresulted in a very striking change: the height of thepressor response was greatly increased and theduration was shortened so that the response be-came indistinguishable from that to renin A ondirect bioassay (Fig. 5). This alteration in responsewas not due to the effect of reduction in pH on thebioassay preparation per se, since the solutionswere neutralized to pH 7.0 immediately beforeinjection into the rat. Also similar acidification andneutralization of renin A or of a nonpressor fractionfrom the DEAE-cellulose column (e.g., 0.025Mprotein peak) did not result in any change inresponse.

Various preparations of post-DEAE-celluloserenin B were assayed directly against standard pigrenin before and after this acidification using abracket assay based on the heights of the pressorresponses (see Methods). Acidification increasedthe activity by a mean factor of 2.45 ± 0.35 (SD) (N= 14). (Reinvestigation of crude renal extracts atthis stage also showed a two- to threefold increasein activity after acidification with a similar changein shape of the blood pressure response curve.)

Further evidence that the change in response topost-DEAE-cellulose renin B following this acidifi-cation was due to transformation to renin A wasobtained by G100 superfine Sephadex chromatog-

raphy of acidified renin B (after neutralization);this procedure resulted in the emergence of a peakof pressor activity that was indistinguishable fromrenin A both in its position of elution (calculatedmolecular weight 41,000) and in the shape of itspressor response.

Other maneuvers were also capable of effectingthe apparent transformation of renin B to renin Aas judged by direct bioassay (Table 1). The mosteffective of these were a change in pH to below 4.5or above 10.0 at 4°C for 15-30 minutes (most of thechange occurred in the first minute) and theaddition of 3-4M NaCl, 1M Nal, 0.5M NaC104, 1MNaSCN, or 0.005% sodium dodecyl sulfate. How-ever, with most of these materials, the change ofthe pressor response of renin B to that of renin Awas not reversible by dialysis at neutral pH.Materials which did not cause any change over 1-2hours at 4°C included 3M urea (8M urea destroyedthe pressor activity of renin A), 0.4M sodiumsulfate, 1% mercaptoethanol, Cleland's reagent(0.02M dithioerythritol), and 0.015M EDTA.

Crude preparations of renin B appeared to bequite stable, but following purification on G100Sephadex this material showed spontaneous par-tial transformation to renin A under some circum-stances. Thus post-GlOO Sephadex renin B ap-peared to be most stable at pH 7.5 in 0.175MSkinner buffer C but underwent some transforma-tion toward a renin A response (in 0.05M sodiumphosphate buffer, pH 7.0, with 0.02% neomycinsulfate) under the following conditions: (1) over aperiod of several weeks at 4°C, (2) over a period ofseveral hours at 37°C, and (3) over a period of

mmHq

AB.R

3O-

15-

O-/ ~ ^

\t• Iml

B

•

f\\

t•Iml

pH2-5- pH7

r•ImlB

FIGURE 5

Change in the shape and the height of the pressor response to pig renal pressor material B (0.15units/ml) on rat bioassay after acidification with IN HCl to pH 2.5 for 30 minutes at 4°C. Theacidified material was neutralized to pH 7 before injection. B.P. - blood pressure (Reproduced withpermission from Hypertension '72, edited by J. Genest and E. Koiw, New York, Springer-Verlag,1972.)



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TABLE 1

Effect of Various Agents on Renin A, Renin B, and Renin-Binding Protein (RBP)

Agent

pH

NaCl

Nal

NaQiD.H.O

Sodium dodecyl sulfate

Concent-tration

2.53.03.54.34.96.09.0

10.010.511.0lM3M

4M

0.5M

1M

0.5M

0.5M

0.005%

Conditions

Time(min)

151515151515404030303030

3606090253030

Temper-ature(°C)

4444444444

37444

374

3737

Methodof

analysis

11

112

1 & 21&2

12122

Extent ofchange ofrenin B

to renin A(%)

> 8 0> 80

70-10060-8030-50< 1 0< 1 010-20> 8 0

> 8 5> 8 5

20-40> 8 5

50-70> 8 5> 8 5

Loss ofrenin-

bindingcapacityof RBP

(%)

> 80> 80

40-60

< 1 0< 1 5

> 8 0>80< 1 5< 1 5< 1 5

> 8 5

> 8 5> 8 5

Loss ofbiologicalactivity of

renin A(%)

< 15< 1 5< 1 0

< 10< 1 0< 2 0> 8 0< 1 5< 1 5< 1 5

< 2 0

< 1 5< 1 5

See text for explanation of methods 1 and 2.

several hours after 1:10 dilution even at 4°C. Suchpartial transformation is compatible with the exist-ence of an equilibrium reaction between the twoforms of renin (see below).

ENZYME STUDIES

Michaells-Menten Constants for Renins A andB.—Lineweaver-Burk plots of the enzyme kineticdata on post-DEAE-cellulose renins A and Bshowed their Km values to be indistinguishable,the calculated value for both being 2.0 x 10"*moles/liter.

Angiotensin I Generation Rates with Renin Bbefore and after Acidification.—A fixed amount ofpost-DEAE-cellulose renin B (approximately 0.015units, specific activity 0.07 units/mg) was incu-bated at 37°C with pig renin substrate at pH 7.5 inthe presence of the angiotensinase inhibitors BAL,EDTA, 2, 2-dipyridyl, and 8-hydroxyquinoline.The rate of angiotensin I generation was measured,and the result was compared with a similar experi-ment in which the renin B was acidified to pH 2.5at 4°C for 30 minutes (and then neutralized to pH7.5) immediately before incubation with substrateand inhibitors. Figure 6 shows the result of thisstudy: the average angiotensin I generation ratewith acidified renin B was 1.7 times that observed

with native renin B. This difference could not beexplained by the small amount of angiotensinasepresent in the unacidified renin B preparation (Fig.6, top).

THE NATURE OF THE RELATIONSHIP BETWEEN RENINS A AND B

The foregoing data on molecular weights, pH andionic effects, and enzyme studies of the two formsof renin suggested that renin B was a protein-bound form of renin A. However, other explana-tions were tenable, including the possibility thatrenin B was a dimer of renin A even though itsapparent molecular weight on Sephadex chroma-tography was somewhat less than twice that ofrenin A.

In the further elucidation of this relationship, itwas noted that, when crude renal extracts werechromatographed on DEAE-cellulose, prolongedelution with increased volumes of the low molaritybuffers resulted in the continued appearance ofrenin A in the eluate rather than single sharp peaksof this material. Since it seemed possible that thisphenomenon was due to the continued dissociationof renin A from a renin-binding substance whichitself remained adsorbed on the DEAE-cellulosecolumn, such columns were eluted with highermolarity steps and the eluates were examined very

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carefully for the presence of any renin-bindingsubstance.

In a typical experiment, 160 ml of renal corticalextract (1.1 units/ml, 0.04 units/mg) in 0.025Mphosphate, pH 6.9, was applied to a 95 x 2.6-cmcolumn of DEAE-cellulose (65 g DEll), and thecolumn was developed with low molarity buffersuntil renin A ceased to appear in the eluate. Thus,800 ml of 0.025M phosphate buffer was followed by900 ml of 0.05M buffer, 1,000 ml of 0.075M buffer,900 ml of 0.0875M buffer, and 750 ml of 0.1Mphosphate buffer all at pH 6.9. Maximum amountsof renin A appeared in the 0.075M fraction, withearly emergence of peak activity (0.25 units/ml)being followed by a long tail down to 0.04 units/mlby the end of this collection. Small amounts ofrenin A (maximum 0.06 units/ml) continued toappear in the 0.0875M fraction, but under theseelution conditions no pressor activity (either reninA or B) was detectable in the 0.1M eluate.

No pressor activity emerged from the columnwhen the eluting buffer was changed to 0.15Mphosphate, pH 6.9, under these conditions. How-ever, incubation of the protein peak from this 0.15Mstep with renin A (0.075M fraction above) for 30minutes at 37°C resulted in a striking decrease inthe height and a prolongation of the duration of therenin pressor response so that it came to appearidentical to that of renin B. This phenomenon isshown in Figure 7 in which the incubated materialwas compared with separate injections of renin Aand the 0.15M fraction given in rapid succession soas to eliminate the possibility that some artifact(e.g., angiotensinase) caused the observed changein response. (Also, no change of pressor activity wasobserved when renin A was incubated with the0.15M fraction for 30 minutes at 4°C.) This diminu-tion in the height of the pressor response wasassociated with a decrease in the rate of angioten-sin I generation from substrate in vitro exactly likethat observed for renin B in Figure 6.

Identity of the incubated material with nativerenin B was further suggested by the observationthat its position of elution on G100 Sephadexchromatography was identical to that of renin B.Also, acidification of the incubated material to pH2.5 at 4°C for 30 minutes resulted in an apparentreversion to renin A as judged by the shape of thepressor response (Fig. 7) and its position of elutionon G100 Sephadex chromatography. These andother data provide strong evidence that renin B is abound form of renin A.

Thus,' renin B appears to be dissociated onDEAE-cellulose chromatography under these con-Circulation Research, Vol. 35, September 1974

ditions to renin A and a renin-binding substance.Furthermore, this dissociation appears to be re-versible. Thus,

Renin A + Renin-Binding Substance^ Renin B. (1)

Progressive increases in the proportion of therenin-binding substance with fixed amounts ofrenin A resulted in progressive decreases in theheight of the pressor response and progressiveincreases in its duration; this finding together withthe tendency for partially purified (post-GlOO-Sephadex) renin B to dissociate spontaneouslyunder some circumstances (see above) furtheremphasizes the equilibrium nature of the relation-ship in Eq. 1.

Apart from the prolonged elution at low molari-

6 0 0

4 0 0

5 ?

2 0 0

1OOO

600 -

s i

200 •

10 20 30Incubation time (min.)

FIGURE 6

Comparison of angiotensin I generation rates resulting from theaction of native renin B (solid circles) and preuiously acidifiedrenin B (open circles) on pig renin substrate. Angiotensinbioassay, with limits showing assay brackets. The top sectionshows the rate of angiotensin I loss during incubation of each ofthe renins with 500 ng/ml of angiotensin I in the presence ofangiotensinase inhibitors.


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434 BOYD

20min

' 4 0 r

,. 20

<*)

0 005RBP

005Renin A

0005 RBP+ 0-05 Renin A

3O',37°CI

0005RBP

005Renin A

FIGURE 7

Transformation of the renin A (0.25 units/ml) response to that of renin B by incubation of renin Awith renin-binding protein (RBP) prior to injection. The incubated sample is compared with separateinjections of renin A and renin-binding protein given in rapid succession. Note the reversal of shape ofthe pressor response to the incubated material by acidification (neutralized to pH 7 immediatelybefore injection). Rat bioassay. Amounts shown are in milliliters. B.P. » blood pressure.

ties, other factors of importance in the preparationof the renin-binding material from renal extractson DEAE-cellulose were the degree of columnloading (greater loading resulted in the elution ofincreased amounts of renin B) and a tendency forvariability in the chromatographic pattern withdifferent batches of DEAE-cellulose (DE11).THE NATURE OF THE RENIN-BINDING SUBSTANCE

The renin-binding substance appeared to be aprotein, since it was nondialyzable (PM10 mem-brane, Amicon), its capacity to bind renin A wascompletely destroyed by boiling for 5 minutes, andits molecular weight was in the protein range (seebelow). Also, its activity was abolished by incuba-tion at 37°C for 10 minutes with a one-twentiethvolume of antiserum raised against it in the rabbit.

Reversibility of Combination with Renin A.—Table 1 shows the effect of pH and various reagentson renin B, renin A, and renin-binding protein. Thedata were obtained by direct bioassay either in thepresence of the reagent (method 1) or after it hadbeen removed by dialysis for 24-48 hours at 4°Cagainst 0.1M phosphate buffer, pH 6.9 (method 2).Since renin B could reform during such dialysis (byreassociation), data obtained on its transformationto renin A using method 2 were included in thetable only where a positive (or irreversible) effectwas obtained.

Table 1 shows that the apparent irreversibility ofthe transformation of renin B to renin A notedabove with certain reagents (e.g., pH below 3.0,

Nal, NaC104) NaSCN) is explained by a loss ofbinding capacity of the renin-binding protein itselfduring these maneuvers. However, almost com-plete dissociation of renin B to renin A occurred inthe presence of 3M or 4M NaCl at 4°C withoutdetectable loss of renin-binding protein (or reninA); under these circumstances, renin B was re-formed after removal of the NaCl by prolongeddialysis. G100 Sephadex chromatography in 4MNaCl and 0.025M phosphate, pH 6.9, has since beenused to prepare renin-binding protein free from anybound renin.

Molecular Weight of Renin-BindingProtein.—G100 Sephadex column chromatographyof the post-DEAE-cellulose renin-binding proteinindicated that it had a molecular weight of approx-imately 55,000, a value somewhat greater than thatwhich would be expected from the difference in themolecular weights of renins A and B on Sephadex(see Discussion).

Specificity of Renin-Binding Protein.—Whenporcine renin-binding protein (post-DEAE-cel-lulose) was incubated with standard rabbit renin(37°C, 30 minutes), there was a reduction in theheight of the pressor response of approximately thesame degree as that seen with pig renin A. Somedecrease in the height of the response also occurredwith rat renin but to a lesser extent. Human reninwas also investigated by indirect assay usinghuman renin substrate and a final renin concentra-tion of 0.04 Goldblatt units/ml. No binding of hu-



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TABLE 2

Effect of Renin-Binding Protein {RBP) on the Angiotensin I-Generating Activities of Pig Renin A andPepsin on Ox Renin Substrate

Time(min)

01020

Renin A

10136259

Angiotensin I generation

Renin A + RBP

7110169

rate (ng/ral)

Pepsin

11148223

Pepsin + RBP

13147240

man renin by porcine renin-binding protein was de-tectable.

The 0.025M phosphate eluate from DEAE-cel-lulose chromatography of pig renal cortical extract(see above) was chosen as a source of renal angi-otensinase, since (in contradistinction to the 0.15Mrenin-binding protein fraction) it contained largeamounts of angiotensinase activity which could notbe inhibited by the combination of BAL, EDTA,2,2-dipyridyl, and 8-hydroxyquinoline. The rate ofloss of angiotensin I with a 1:12 dilution of thismaterial (53%/hour) assessed in the presence ofthese inhibitors was not reduced by its preincuba-tion with renin-binding protein (60% angiotensin Iloss/hour).

The enzyme pepsin has been shown to cleaverenin substrate at precisely the same peptidelinkage (Leu-Leu) as does renin itself (19), andthus it generates angiotensin I. It was therefore ofinterest to observe whether renin-binding proteinmight inhibit this action of pepsin in the same waythat it inhibits renin. For this investigation, sam-ples of standard pig renin or 20 ng of pepsin wereincubated for 30 minutes at 37°C with and without

excess renin-binding protein in a volume of 0.4 ml(0.05M phosphate buffer, pH 6.1). Ox renin sub-strate (1.2 ml) was then added, together with BAL,EDTA, 2,2-dipyridyl, and 8-hydroxyquinoline(final volume 2 ml, pH 6.1), and the rate ofangiotensin I generation over the next 20 minuteswas assessed by immunoassay. The results areshown in Table 2; the presence of renin-bindingprotein clearly reduced the angiotensin I-generat-ing activity of renin A but had no such effect onthis action of pepsin.RENIN RELEASE DURING PERFUSION OF THE ISOLATED PIGKIDNEY IN SITU

The isolated pig kidney was perfused in situ withisoproterenol as detailed in Methods. Results ofdirect bioassay of the perfusate after concentrationby pressure dialysis are shown in Table 3. Theamounts of renin in the perfusate before iso-proterenol infusions were small, and there was noevident increase with sample acidification (pH 2.5,4°C for 30 minutes), suggesting that the reninpresent was renin A. However, the quantity ofrenin at this level was too near the assay detectionlimit to define the type of renin on this basis with

TABLE 3

Study of Renin Release in the Isolated Perfused Pig Kidney during Isoproterenol Infusion

Pif?no.

12345

Perfusateconcentration

(pressuredialysis)

x 10x 15x 40x 3 2x 8 0

Control

Renin in concentrate*(munils/ml)

Beforeacidification

< 12121230

< 2 0

Afteracidification

< 1 2121230

< 2 0

Isoproterenolinfusion rate

Oig/min)

1.02.04.08.08.0

Isoproterenol

Perfusateconcentration

x 10x 15x 4 0x 4 5x 130

infusion

Renin in concentrate*(munits/ml)

Beforeacidification

1242

12550

125

Afteracidification

1285

300160300

* Direct rat blood pressure bioassay against standard pig renin.



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20min.

40

20

0 L

I t I0-2 ml.

P

pH 2-5

0-2 ml. P

30'

0-2 ml.P

FIGURE 8

Rat blood pressure response to pig renal perfusate (P) (0.05 units/ml) collected during isoproterenolinfusion (8 ng/min) in pig no. 4. The perfusate had been concentrated forty-five times prior toinjection. The renin B nature of the sample is evident from its prolonged pressor response and theincrease in height and the change to a renin A-like response after acidification to pH 2.5. B.P. - blood

pressure.

any certainty. With 1 j*g/min of isoproterenolsulfate given intra-arterially, there was no appar-ent increase in renin, but with 2 jtg/min or more aconsistent rise was observed. Furthermore, thepressor response to this renin was rounded andprolonged on direct bioassay (Fig. 8), and a two- tothreefold increase in height with a shortening ofduration followed acidification to pH 2.5 at 4°C for30 minutes (Table 3, Fig. 8), suggesting that thisrenin was renin B. This possibility was confirmedby showing that its position of elution on G100Sephadex chromatography was identical to that ofpost-DEAE-cellulose renin B.

DiscussionThe present study was initiated by the observa-

tion of a striking difference between the shape ofthe pressor response to crude pig renal corticalextracts on rat bioassay and that to semipurifiedpig renin and has led to the isolation of twointerconvertible forms of porcine renal renin,namely, a protein-bound (renin B, molecularweight 60,000) and a free (renin A, molecularweight 40,000) form.

Careful scrutiny of the earlier literature revealsbrief mention by two groups of workers of anunusual response to crude preparations of hog (8,20) and other (20) renins, but both groups thought

that this finding was most likely the result ofimpurities. It is interesting, however, that Gold-blatt and Haas (20) did comment on a prolongedpressor response to crude renin and a change in itsshape on acidification. In addition, Haas et al. (21)found evidence for the existence of several inter-convertible forms of hog renin in separate studies ofits ultraviolet spectroscopic behavior; again, how-ever, the precise nature of this relationship was notclear.

More recently, Skeggs and his colleagues (22)isolated four different forms of renin from pigkidney extracts; two of these were interconvertible.Renin I was converted to renin II on acidification,and examination of the data suggests that renin I ofSkeggs et al. (22) is the same as renin B of thepresent study and that renin II is equivalent torenin A. The nature of the relationship betweenrenins I and II was not investigated by Skeggs et al.(21), but it seemed clear to them that the previousfailure to find more than one form of renin duringpurification in some studies (23) was due to theinclusion of an acidification step, which rapidlyconverted renin I (renin B) to renin II (renin A).

In the present study, the strongest evidence thatrenin B is a protein-bound form of renin A wasobtained on DEAE-cellulose chromatography ofcrude renal extracts under especially designed



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conditions of elution (see Results). This procedureled to the isolation of a protein which, althoughbiologically inactive in itself, was capable of trans-forming renin A to a material identical to renin Bnot only in the shape of its pressor response but alsoin its position of elution on GlOO Sephadex and inits convertibility to renin A on acidification.

The prolonged pressor response to crude pigrenal extract (6) and the increase in its heightfollowing acidification suggest that much of theextractable renin in the pig kidney is in theprotein-bound form. The preparation of largeamounts of renin-binding protein during DEAE-cellulose chromatography of crude renal extractsunder special conditions is in keeping with thisidea, and the high yield of renin A under thesecircumstances is almost certainly due to its dis-sociation from the protein-bound form of reninduring the chromatographic procedure itself (seeabove). Although these distortions during prepara-tion could give a false impression of the reninpattern in the original extract, they could also beused to great advantage in the controlled prepara-tion of either renin A, renin B, or renin-bindingprotein.

Under Results it was suggested that the protein-bound form of renin is probably in equilibrium withfree renin (renin A). If this equilibrium exists, itwould not be necessary to postulate that theprotein-renin complex has intrinsic pressor activ-ity, since such activity could be explained by virtueof its equilibrium with free renin A. The similarityof Km values for the actions of renins A and B onrenin substrate is in keeping with this hypothesis.Under these circumstances, the prolonged pressorresponse to renin B on direct bioassay would seemto be due to slow dissociation of the renin-proteincomplex in vivo.

The data on the dissociation of renin B by pHand ionic effects suggest that the binding of theprotein-renin complex is predominantly related tointeraction between polar groups. There certainlywas no indication that disulfide linkages are in-volved, although other more nonspecific forcescould be.

The renin-binding protein itself was found tohave an apparent molecular weight on Sephadex of55,000, a value somewhat greater than that ex-pected from the observed difference in the molecu-lar weights of renins A and B. Several explanationsare possible. Free renin-binding protein could be arelatively nonspherical molecule or it could aggre-gate in concentrated solution. Either possibilitywould result in a falsely high value for its molecularCirculation Research, Vol. 35, September 1974

weight in the present studies. Alternatively, it ispossible that renin B itself is retarded duringchromatography on Sephadex through a process ofcontinual dissociation and recombination in equi-librium with renin A and renin-binding protein;this effect would result in a falsely low value for themolecular weight renin B. Further studies areneeded to elucidate this point.

There is evidence to suggest that bound forms ofrenin also exist in other species. Thus, recentstudies on rabbit renal extracts in this laboratoryhave led to the isolation of a renin-binding proteinin this species similar to that observed in the pig(personal observations); this finding is in keepingwith the finding by Leckie (24) of acid activation ofrenal renin in the rabbit. Also Skinner et al. (25)have recently described discrepancies in theirhuman plasma renin assay which suggest an in-crease in the activity of renin after acidification,particularly in early pregnancy, and Morris andLumbers (26) have since observed similar acid ac-tivation of renin in human amniotic fluid. They in-terpreted their results as an enzymatic activationof renin at low pH. The exact relationship with thepresent observations in the pig remains to be deter-mined.

The physiological significance of the renin-bind-ing protein described in the present study isunknown. As mentioned previously, most of therenin extractable from the pig kidney appears to beprotein bound, and the binding capacity of renin-binding protein seems to be specific for renin astested so far with the exception that it is also ableto bind renal renins from certain other species.Incubation of free renin A with renin-bindingprotein certainly leads to a profound change in itspressor response, so that there can be little doubt ofits potential importance. Of course the finding ofmost.-interest in this context is the release of reninin the protein-bound form during isoproterenolstimulation of the isolated perfused pig kidney.The situation in more physiological circumstancesis unknown, but from the present data it is evidentthat the possibility of renin binding will have to beconsidered in interpreting any future study of reninrelease or angiotensin-renin correlations.

Whether the present findings explain some of thedifficulties in relating circulating angiotensin II torenal hypertension (1) is also unknown. However, itis conceivable that renin-binding protein could actas a transport protein to carry renin from thekidney to distant local tissue sites of action on reninsubstrate. The activity of the renin-angiotensinsystem might then be expressed outside the circu-


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lation at sites relatively sequestered from it. Thisphenomenon might explain some of the discrepan-cies referred to earlier in this paper, including thefailure of angiotensin antibodies to reduce bloodpressure in renal hypertension in some circum-stances (3-5, 27), particularly those (27) in whichlow molecular weight (and presumably more diffusi-ble) antagonists have been found to be effective (2).Local angiotensin production on such a basis iscertainly possible, since reninlike activity has beenfound recently in a wide variety of tisses.

Further elucidation of the role of renin-bindingprotein will depend, among other things, on moredetailed studies of its specificity and affinity forrenin binding, and these investigations in turn willrequire the preparation of pure renin for labelingand saturation analysis studies.

AcknowledgmentI wish to thank Miss Mary Gurney for technical assistance,

Dr. G. J. Macdonald for the antibody to rabbit renin, andProfessor W. S. Peart for discussion and criticism of themanuscript. The assistance of Mr. M. E. Snell in preparing theisolated pig kidney for perfusion studies is also gratefullyacknowledged.

References1. MACDONALD GJ, BOYD GW, PEART WS: Renal hypertension

and angiotensin antibodies. Am Heart J 83:137-139,19722. BRITNNER HR, KIRSHMAN JD, SEALEY JE, LARAGH JH: Hyper-

tension of renal origin: Evidence for two different mech-anisms. Science 174:1344-1346, 1971

3. MACDONALD GJ, Louis WJ, RENZINI V, BOYD GW, PEART WS:Renal clip hypertension in rabbits immunized againstangiotensin II. Circ Res 27:197-211, 1970

4. EIDE I, AARS H: Renal hypertension in rabbits immunizedwith angiotensin II. Scand J Clin Lab Invest 25:119-127,1970

5. JOHNSTON CL, HUTCHINSON JS, MENDELSOHN FA: Biologicalsignificance of renin angiotensin immunization. Circ Res27(suppl H):n-215-222, 1970

6. BOYD GW: Nature of renal renin. In Hypertension '72,edited by J Genest and E Koiw. New York, Springer-Verlag, 1972, p 161

7. BOYD GW: Isolation of binding protein for pig renin (abstr).J Physiol (Lond) 232:22-23P, 1973

8. PEART WS, LLOYD AM, THATCHER GN, LEVER AF, PAYNE N,STONE N: Purification of pig renin. Biochem J 99:708-716,1966

9. HAAS E, GOLDBLATT H, GIPSON EC, LEWXS L: Extraction,purification and assay of human renin free of angiotensi-nase. Circ Res 19:739-749, 1966

10. SKINNER SL: Improved assay methods for renin concentra-tion and activity in human plasma. Circ Res 20:391-402,1967

11. PEART WS: New method of large-scale preparation ofHypertensin, with a note on its assay. Biochem J59:300-302, 1955

12. LEVER AF, ROBERTSON JIS, TREE M: Estimation of renin inplasma by an enzyme kinetic technique. Biochem J91:346-352, 1964

13. BOYD GW, PEART WS: Production of high-titre antibodyagainst free angiotensin II. Lancet 2:129-133, 1968

14. BOYD GW, ADAMSON AR, Frrz AE, PEART WS: Radioimmunoassay determination of plasma-renin activity.Lancet 1:213-218, 1969

15. ANDREWS P: Estimation of molecular size and molecularweights of biological compounds by gel filtration. InMethods of Biochemical Analysis, vol 18, edited by DGlick. New York, Interscience, 1970, p 1

16. LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ: Proteinmeasurement with the phenol reagent. J Biol Chem193:265-275, 1951

17. VANDONGEN R, PEART WS, BOYD GW: Adrenergic stimula-tion of renin secretion in the isolated perfused rat kidney.Circ Res 32:290-296, 1973

18. DAWSON RMC: Physiological media. In Data for Biochemi-cal Research, edited by RMC Dawson, DC Elliott, WHElliott, and KM Jones. London, Oxford University Press,1969, p 507

19. FRANZE DE FERNANDEZ MT, PALADINI AC, DEUUS AE: Isola-tion and identification of a pepsitensin. Biochem J97:540-546, 1965

20. GOLDBLATT H, HAAS E: Renal mechanism of hypertension.In Proceedings of the 6th Annual Conference on NephroticSyndrome, edited by J Metcoff. New York, NationalNephrosis Foundation, Inc., 1955, pp 117-138

21. HAAS E, LAMFROM H, GOLDBLATT H: Ultraviolet spectroscopyof renin. Arch Biochem Biophys 44:79-94, 1953

22. SKEGGS LT, LENTZ KE, KAHN JR, HOCHSTRASSER H: Studieson the preparation and properties of renin. Circ Res21(suppl n):n-91-100, 1967

23. PEART WS: Discussion. Circ Res 21(suppl II):II-100, 196724. LECKIE B: Activation of a possible zymogen of renin in

rabbit kidney. Clin Sci 44:301-304, 197325. SKINNER SL, LUMBERS ER, SYMONDS EM: Analysis of

changes in the renin-angiotensin system during preg-nancy. Clin Sci 42:479-488, 1972

26. MORRIS BJ, LUMBERS ER: Activation of renin in humanamniotic fluid by proteolytic enzymes. Biochim BiophysActa 289:385-391, 1972

27. EIDE I: Renovascular hypertension in rats immunized withangiotensin II. Circ Res 30:149-157, 1972



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GRAHAM W. BOYDA Protein-Bound Form of Porcine Renal Renin

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