ANALYTICAL BIOCHEMISTRY 133, @%dl6 (1983)

Affinity Precipitation of Dehydrogenases

SUSANNEFLYGARE,TADHG GRIFFIN,* PER-OLOF LARSSON, ANDKLAUS MOSBACH

Pure and Applied Biochemistry, Chemical Center, University of Lund, P.O. Box 740, S-220 07 Lund, Sweden, and *Department of Biochemistry, Trinity College, Dublin University, Dublin, Ireland

Received March 7, 1983

Affinity precipitation, a novel technique closely related to immunoprecipitation and affinity chromatography, has been evaluated in systems comprised of dehydrogenases and a bifunctional NAD derivative, Bis-NAD. Lactate dehydrogenase and glutamate dehydrogenase were easily precipitated whereas yeast alcohol dehydrogenase required the presence of salt to enhance the affinity precipitation. Liver alcohol dehydrogenase did not precipitate, probably because most of the affinity complexes formed were composed of only two enzyme molecules. Affinity pre- cipitation was carried out on a preparative scale for the isolation of ox heart lactate dehydrogenase from a crude extract. The yield and purity of the enzyme and the general properties of the procedure are considered very satisfactory.

Recently we introduced a new enzyme pu-

rification method which we call affinity pre- cipitation (1). In many aspects this method resembles immunoprecipitation. The first step in affinity precipitation involves the mix- ing of a bifunctional ligand with an oligomeric enzyme. If the spacer linking the two ligand entities together is long enough to bridge the distance between the two enzyme molecules and if the binding between the ligand and en- zyme is strong enough, a precipitation may occur. This phenomenon occurs because a bis- ligand interacts simultaneously with two en- zyme molecules. Since the enzyme is oligo- meric, it is easily conceivable that an elaborate network of enzymes and bifunctional mole- cules will form. When such a network has grown to a sufficient size, it no longer remains in solution but precipitates out. To improve further the selectivity of the procedure and to increase the effective binding between ligand and enzyme, ternary complex formation may be employed (1). For example, a Bis-NAD analog together with pyruvate formed a strong complex with the active site of soluble lactate dehydrogenase enabling the enzyme to pre- cipitate. Other related examples are the affinity

complex formation obtained with bisbiotinyl compounds and avidin, as demonstrated by electron microscopy (2) and agglutination of red blood cells using bifunctional boronic acid derivatives (3). At this point it should be men- tioned that the term affinity precipitation has also been used for a technique whereby affinity complexes between an enzyme (trypsin) and a water-soluble polymer-bound ligand (ami- nobenzamidine) were precipitated by lowering the pH (4). We feel, however, that the term should be reserved for systems where precip- itation is a direct consequence of the formation of affinity complexes. In the present work af- finity precipitation of several dehydrogenases has been achieved with NAD derivatives. A procedure for preparative use of the technique is also presented.

MATERIALS AND METHODS

Lactate dehydrogenase (beef heart, type III), glutamate dehydrogenase (bovine liver, type I), and yeast alcohol dehydrogenase were ob- tained from Sigma, St. Louis, Missouri; horse liver alcohol dehydrogenase from Boehringer- Mannheim, Mannheim, West Germany. N2,

409 0003-2697183 $3.00 Copyright 0 1983 by Academtc Press, Inc.

All rights of reprcductmn m any form reserved.

410 FLYGARE ET AL.

Nz-Adipodihydrazido-his-(N6-carbonylmeth- yl-NAD) (Bis-NAD) was synthesized as de- scribed (1). The compound is now commer- cially available from Sigma.

Determination of Enzyme Concentrations and Enzyme Activities

The concentration of bovine liver glutamate dehydrogenase was determined spectropho- tometfidlY (A 1%. 280 nm = 8.9) (5). The enzyme activity was determined by following the re- duction of a-ketoglutarate by NADH (6). The concentration of beef heart lactate dehydro- genase in crude mixtures was determined from activity data, assuming that 1 mg of pure en- zyme oxidizes 175 pmol of NADH/min under the following conditions: The assay was carried out at 22C. The medium (total volume 3 ml) contained 0.05 M sodium phosphate buffer, pH 7.5, 1 mM sodium pyruvate, and 0.30 mM NADH. The reaction was initiated by the ad- dition of enzyme solution (20-100 ~1) to give a MS40 < O.l/min.

The concentration of yeast alcohol dehy- drogenase was determined spectrophotomet- ficdlY (&4o, 280 nm = 12.6) (7).

The concentration of liver alcohol dehy- drogenase was determined spectrophotomet- fiCdY (Al%, 280 nm = 4.55) (7) or by active-site titrations (see Fig. 1).

Protein was determined by the Lowry method (8).

Afinity Precipitation of Glutamate Dehydrogenase (Typical Procedure)

Glutamate dehydrogenase, 1.5 ml ( 1.8 mg/ ml), was dialyzed against 0.05 M sodium phosphate buffer, pH 7.5. To this solution 0.25 ml of 0.8 M glutarate, followed by 0.25 ml of 0.12 mM Bis-NAD was added. Upon gentle mixing, the solution became rapidly opaque. After 10 min a precipitate had formed. Leav- ing the mixture standing overnight at 4C re- sulted in a heavy precipitate accumulation in

Abbreviation used: Bis-NAD, N,,Ni-adipodihydra- zido-bis-(N6-carbonylmethyl-NAD).

the bottom of the test tube. This precipitate was isolated by centrifugation and could be redissolved by adding 0.5 ml of 10 mM NADH.

A@nity Precipitation of Yeast Alcohol Dehydrogenase (Typical Procedure)

The experimental procedure was essentially the same as that for glutamate dehydrogenase. To 1 .O ml of alcohol dehydrogenase ( 1.7 mg/ ml) in 0.05 M sodium phosphate, pH 7.5,0.25 ml of 0.8 M pyrazole was added followed by 0.5 ml of phosphate buffer. Bis-NAD (0.25 ml of 0.12 mM solution) was the final addition, with the exception of a few experiments where NaCl was added to a final concentration of 0.2 M in order to enhance the precipitation.

Preparative Afinity Precipitation of Lactate Dehydrogenase

(a) Preparation of crude extract from ox heart. Frozen ox heart was thawed and cut into small pieces, and visible fat was removed. Sodium phosphate buffer (120 ml of 0.05 M), pH 7.5, containing 1 mM mercaptoethanol was then added per 100 g of ox heart. The mixture was homogenized in a Waring blender for 5 min at 4C and the homogenate was centrifuged for 15 min at 10,OOOg. The pre- cipitate was discarded and the supematant was centrifuged for 30 min at 20,OOOg. The su- pernatant was dialyzed overnight at 4C against 0.05 M sodium phosphate buffer, pH 7.5, and centrifuged for 15 min at 2O,OOOg, and was then ready for use in the affinity pre- cipitation experiments outlined below.

(b) Determination of optimum Bis-NAD concentration (pilot precipitation). In order to obtain a high yield in the affinity precipitation step, optimum Bis-NAD concentration was first determined in a small-scale experiment. To a sample of the crude extract (2 ml), so- dium oxalate (0.25 ml, 0.1 M) and 0.25 ml Bis-NAD were added. The concentrations of the Bis-NAD were chosen such that the nom- inal ratios of NAD equivalents to lactate de- hydrogenase subunits were 1:4, 1:2, 1: 1, 2: 1,

AFFINITY PRECIPITATION 411

3:1, 4:1, and 8: 1, respectively (the nominal enzyme concentration was estimated from ac- tivity measurements). Precipitation occurred almost immediately and after 30 min the pre- cipitate was spun down at 10,OOOg for 10 min. Lactate dehydrogenase activity was deter- mined in the supernatant solutions and it was assumed that the test tube having the lowest activity had the best conditions for affinity precipitation. These conditions were then ap plied in the following large-scale preparation.

(c) Large-scale precipitation. To 200 ml of crude extract (33 mg protein/ml, 0.53 mg/ml lactate dehydrogenase according to activity measurements) 25 ml 0.1 M sodium oxalate and Bis-NAD solution in an amount deter- mined by the pilot precipitation described above, i.e., 20 ml 2.6 X lop4 M Bis-NAD, were added. After 20 h at 4C the suspension was centrifuged (lO,OOOg, 15 min), and the pre- cipitate was dissolved in 10 ml 0.60 mM NADH (in phosphate buffer) and dialyzed against phosphate buffer overnight. The so- lution was then centrifuged at 10,OOOg for 10 min in order to remove insoluble material. Lactate dehydrogenase was finally precipitated with (NH4)$04 (80% saturation), collected by centrifugation, and stored in 2.1 M (NH4)$04 at 4C.

RESULTS AND DISCUSSION

Glutamate Dehydrogenase

Earlier model studies (1) had shown that affinity precipitation of lactate dehydrogenase with bifunctional NAD derivatives is an ef- ficient procedure provided that the correct ra- tio (around unity) between enzyme subunits and NAD residues is observed. To obtain a reasonable yield, as well as to improve the specificity, adding pyruvate was beneficial since it participates in ternary complex for- mation resulting in tighter binding between enzyme and ligand.

In the present work further evidence has been found for the importance of ternary complex formations. Glutamate dehydroge- nase did not precipitate in the presence of

a bifunctional NAD derivative alone, but it did so very readily when glutarate, a competi- tive inhibitor (9), was added. The bifunc- tional NAD derivative used here, N2,N2-adi- podihydrazidobis(N6 - carbonylmethyl-NAD) or Bis-NAD, has a 17-A spacer joining the two amino groups of the adenines (-CH2- CONHNHCO(CH2)4CONHNHCOCH~-).

The efficiency of the affinity precipitation as a function of the enzyme/B&NAD ratio was investigated. In contrast to affinity pre- cipitation of lactate dehydrogenase where the ratio must be fairly close to unity ( 1) the pre- cipitation yield for glutamate dehydrogenase is very high over a wide range of ratios. This can be explained by the fact that the hexameric glutamate dehydrogenase alone spontaneously aggregates to larger structures (lo), whereas lactate dehydrogenase is a tetramer and does not polymerize on its own.

However, to allow buildup of large enzyme complexes in the presence of Bis-NAD there must be at least two connecting links to every enzyme molecule, whether or not the enzyme in question is a tetramer such as lactate de- hydrogenase, or an oligomer/polymer, as is the case with glutamate dehydrogenase. It was shown that even at a NAD/subunit ratio as low as 0.16, the precipitation of glutamate dehydrogenase was as high as 70%, a fact that requires the enzyme to be present as an oligo- mer containing at least 12 subunits.

At NAD/subunit ratios higher than unity the precipitation yield would also be expected to diminish since intermolecular crosslinking would have less chance to occur (1). An en- zyme with many subunits should however be less sensitive to deviations from a ratio of unity. This was shown to be the case. For example, at a ratio of 10 the precipitation yield was still as high as 95% for glutamate dehy- drogenase.

The precipitated glutamate dehydrogenase was difficult to dissolve, unless a high NADH concentration was used (10 mM), especially when the NAD/subunit ratio was higher than 0.5. The recovery of enzyme activity in re- dissolved material was quantitative.

412 FLYGARE ET AL.

.-.-.-. 80 -

_ f -_.,._-_I ~,--------?--------,

s /

, I?

% 60 -

,T 0

8 LJ l =NAD 5 p 40- $

o = Bis-NAD

3 ,

LA. ,

20- /-

/

0 , I I I I

0 05 IO 15 20 2.5 30

Equwalents/subunit

FIG. I. Active-site titration of liver alcohol dehydrogenase with NAD and with Bis-NAD. The assay mixture (3 ml) contained 0. I M ethanol, 0.05 M isobutyramide, 0.05 M sodium phosphate buffer, pH 7.5, and 1 pmole dehydrogenase subunits/liter. Excitation was at 330 nm and the emission was determined at 410 nm (arbitrary units). 0, fluorescence with NAD, 0, fluorescence with Bis-NAD.

Liver Alcohol Dehydrogenase

When affinity precipitation of alcohol de- hydrogenase from horse liver was attempted, no precipitation occurred even when the sys- tem contained 1 mM pyrazole, which should participate in ternary complex formation (11). The liver enzyme is a dimer and linear poly- mer chains were therefore expected to form in the presence of a bis-ligand. Efforts were made to clarify why no precipitation occurred. To this end the enzyme active sites were ti- trated with Bis-NAD and with NAD and NADH as references (Fig. 1). The titration system used ethanol and isobutyramide to re- duce the NAD entities and subsequently lock them in the active site as fluorescent enzyme- NADH-isobutyramide complexes. Figure 1 shows that NAD behaved very similarly to Bis-NAD as did NADH (results not shown): it was thus concluded that both NAD residues on a Bis-NAD molecule can simultaneously interact with alcohol dehydrogenase sites.

The distance between the two cofactor binding sites on the dimeric alcohol dehydro- genase molecule is long enough, approxi- mately 50 A (1 l), to rule out the possibility that one Bis-NAD molecule could simulta-

neously interact with both sites on one enzyme molecule (Fig. 2A). There then remain two possibilities: Bis-NAD and liver alcohol de- hydrogenase either form linear polymers that for some reason do not precipitate (Fig. 2C), or complexes containing two enzymes and two Bis-NAD molecules (Fig. 2B) that obviously are less prone to precipitate.

The question was solved by gel filtration on a TSK 3000 SW HPLC column. When a mixture of alcohol dehydrogenase and NAD was applied to the column only one peak was obtained (Fig. 3A). But when Bis-NAD was used instead of NAD another peak also ap- peared (Fig. 3B), corresponding to the double molecular weight, supporting the dimeric complex hypothesis (Fig. 2B). A somewhat disturbing observation was that the peaks both in experiments A and B corresponded to a much too low molecular weight when com- pared to a set of calibrating proteins. The rea- son is not known, but nonspecific adsorption of alcohol dehydrogenase is possible. Figure 3 also shows that the injection of enzyme + Bis-NAD also gives a minor portion of high- molecular-weight material.

The molecular weight was also estimated by analytical ultracentrifugation. The sedi-

AFFINITY PRECIPITATION 413

A

C

FIG. 2. Possible complexes between Bis-NAD and liver alcohol dehydrogenase.

mentation coefficient (sZO, ,) was 8.39 S for a stoichiometric mixture of alcohol dehydro- genase (4 mg/ml) and Bis-NAD (in 50 mM sodium phosphate, pH 7.5, containing 0.1 M pyrazole). The corresponding value for a ref- erence containing NAD instead of Bis-NAD, was calculated to be 5.30 S. These values are in good agreement with a molecular weight of 160,000 for the complex between alcohol dehydrogenase and Bis-NAD, and of 80,000 for alcohol dehydrogenase and NAD.

We therefore suggest that liver alcohol de- hydrogenase is not affinity precipitated be- cause the predominant species formed are di- merit complexes as shown in Fig. 2B.

Yeast Alcohol Dehydrogenase

When experiments were carried out with yeast alcohol dehydrogenase, which is a tet- rameric enzyme, no precipitation occurred in the presence of Bis-NAD and pyrazole. How- ever, when buffer containing 0.2 M NaCl was used, precipitation did take place. A control experiment was conducted which showed that no enzyme was precipitated when Bis-NAD was absent, ruling out a simple salting-out effect. The enzyme precipitation process was very slow compared with glutamate dehydro- genase and lactate dehydrogenase affinity pre- cipitation. After 3.5 h the sample was slightly

cloudy, alter 5.5 h this cloudiness was more pronounced, and after 20 h a clearly defined precipitate could be seen.

Preparative Applications

Preparative affinity precipitation from crude systems is a considerably more complex task than model studies would suggest due to the presence of related enzymes, other proteins, nucleic acids, and fragments thereof in the crude extract. Initial studies with affinity pre- cipitation of lactate dehydrogenase from crude extracts showed that pyruvate was a useful, although not an ideal, third component for precipitate formation. The main drawback was that the aggregates which formed also dissolved comparatively slowly, which is in line with the known kinetics of the ternary complex involved. The fact that pyruvate is a substrate of the enzyme may also cause problems.

An alternative third component, sodium oxalate, was tested and gave good results. When using oxalate, ternary complex for- mation with lactate dehydrogenase and Bis- NAD occurred rapidly, as did the growth of the initially formed aggregates, judged from the fact that precipitation of the enzyme oc- curred only minutes after mixing. The dis- solution of complexes in NADH solution after collection of the precipitate also went smoothly.

In order to establish a suitable oxalate con- centration, affinity precipitation was carried out in the presence of a range of oxalate con- centrations. From a practical point of view, the best oxalate concentration was found to be around 10 mM. At lower concentrations the precipitation yield was unsatisfactory; at higher concentrations the enzyme activity was influenced.

As can be seen from Fig. 4, affinity precip- itation gives a high yield of enzyme provided that the stoichiometry of reactants is correct. The best ratio according to Fig. 4 is higher than the theoretically calculated maximum value of one NAD entity per dehydrogenase subunit. This discrepancy may at least partially be explained by the fact that the lactate de-

414 FLYGARE ET AL.

Elution Volume (ml)

FIG. 3. Size exclusion chromatography of alcohol dehydrogenase complexed with NAD or Bis-NAD. The column used was an LKB Ultropac TSK 3000 SW column (600 X 8 mm). The applied sample (200 ~1) contained 5.4 nmol alcohol dehydrogenase and 7.6 nmol NAD (A) or 3.8 nmol Bis-NAD (B), i.e., a NAD/subunit ratio of 0.70. The flow rate was 0.5 ml - min- . The mobile phase consisted of 0.05 M sodium phosphate buffer, pH 7.5, and 0. I M isobutyramide.

hydrogenase concentrations were determined by activity measurements. Presence of inhib- itory substances would then result in a lower estimate of the enzyme concentration, the final result being that the best precipitating ratio would seem to be higher than one. To clarig this point an analysis of the actual composition of precipitated complexes was undertaken. Crude extract was mixed with different Bis- NAD concentrations and after a few hours the precipitates which had formed were iso- lated by centrifugation, dissolved in 8 M urea, and analyzed spectrophotometrically (6). The composition of the complexes was then cal- culated from the simultaneous equations:

optimum, the precipitation yield rapidly di- minished. It is thus particularly important that the ratio does not become too low. In the context of large-scale preparations the de- scribed deviations from the theoretical opti- mum ratio would cause difficulties. Fortu- nately, however, a remedy was easily devised. Before attempting the affinity precipitation of lactate dehydrogenase from a large volume of crude extract, a pilot precipitation was car- ried out as described under Materials and Methods. To small samples of crude extracts varying amounts of Bis-NAD were added in

A 290 nrn = 41,500~ + 1800~

A 266 a,,, = 37,300x + 22,500~

where x and y denote the concentrations (mole/liter) of subunits and NAD residues, respectively. The molar absorption coefficients were determined from spectra of pure lactate dehydrogenase and of Bis-NAD.

In all cases a ratio of 1.25 NAD equivalents/

8 60 - : I

\\\

.-

E , .e 60- 0 1

\ \\ l , 1 \

i 40-Y \

\ t

1 \ 20 - : \

\.

0 5 IO

NAD equivalentS/sutmit

subunit was found, regardless of the ratio in FIG. 4. Affinity precipitation as a function ofthe nominal the mixture before precipitation. This agrees NAD equivalents/lactate dehydrogenase subunit ratio. The

well with the predicted value of 1. enzyme concentration in the extract was calculated from activity measurements. The specific activity in the crude

Figure 4 shows that at ratios lower than extrac; was 3.7 U/mg protein.

AFFINITY PRECIPITATION 415

I I I

100 - - l ,-------II- I 80 - s z 60- x $ 40 -.

20 zf=p - 0, ,g I ,I L

_______ _____

0 100 200 1200

T !me (mm)

FIG. 5. Affinity precipitation as a function of precipi- tation time. Enzyme extract (2 ml), 0.25 ml 0. I M oxalate, and 0.25 ml Bis-NAD solution were gently mixed, held at 4C, and then centrifuged at 10,OOOg for 10 min and the activity of the supematant determined, 0, NAD entity/ subunit ratio = 0.30; 0, NAD entity/subunit ratio = 1.5; 0, NAD entity/subunit ratio = 6.

such a way that the nominal NAD equiva- 1ents:lactate dehydrogenase subunit ratios varied between 1:4 and 8:l (nominal lactate dehydrogenase concentrations are estimated from activity measurements). The sample where the best precipitation occurred was then taken as a model for the large-scale precipi- tation. This pilot experiment can be carried out very quickly since the precipitation is close to completion after a couple of minutes as is shown in Fig. 5. It also follows that the pre-

parative precipitation step could be carried out in just a few hours. The rapid precipitation is also demonstrated by the series of photo- graphs in Fig. 6.

Table 1 gives a protocol for a preparative affinity precipitation experiment. It is note- worthy that the affinity precipitation step gave high recovery (9 1%) as well as a good puri- fication factor (40X). Additional steps (dialysis and ammonium sulfate precipitation) in- creased the specific activity further. Analysis by sodium dodecyl sulfate electrophoresis proved the final preparation to be almost ho- mogeneous. One minor extra band (~5%) could be observed.

CONCLUSIONS

As described in this paper, affinity precip- itation is applicable to oligomeric enzymes. It may also work with monomeric enzymes hav- ing several binding sites, e.g., effector sites, provided that the bifunctional ligand is het- erofunctional. Affinity precipitation is useful as a purification method and may possibly compete with the established technique, af- finity chromatography, in cases where the proper interaction between ligand and the macromolecule is difficult to realize, e.g., steric

A B C 0 E FIG. 6. Affinity precipitation of lactate dehydrogenase. Enzyme (1 mg./ml), oxalate (10 mM), and Bis-

NAD (1.4 X lo- M) were mixed and held at room temperature. Lactate dehydrogenase, oxalate, and Bis- NAD 30 s after mixing (C), 3 min after mixing (D), and 15 min after mixing (E). (A) and (B) are blanks containing no Bis-NAD and no oxalate, respectively.

416 FLYGARE ET AL.

TABLE 1

PURIFICATION OF LACTATE DEHYDROGENASE FROM Ox HEART

Stage Volume

(ml) Protein

(mid

Total specific activity activity

(U) W/w) PllIificatiOII

(fold) Recovery

6)

Crude extract Affinity precipitate Dissolved precipitate

after dialysis Enzyme crystals in

UWMO,

200 6500 18700 3 1 100 - 141 17ooo 121 40 91

10 117 16800 144 48 90

10 loo 14950 150 50 80

hindrance by the affinity chromatographic support material. A drawback in preparative application of the technique is the cost of the his-l&and. However, since only stoichiometric amounts of ligand are used, this problem is not too great. If necessary, it should also be possible to recover the l&and. Another ap- proach is to prepare inexpensive bis-ligands. One example might be BisCibachron Blue, useful for precipitation of dehydrogenases, among other proteins.2

A prerequisite for affinity precipitation is a bis-ligand with a suitable spacer length which allows a correct spatial positioning of the en- zyme molecules. A successful precipitation thus gives some information about the ar- rangement of enzyme subunits and about the general molecular architecture. By employing spacers of different length and observing the precipitation characteristics, even more in- formation should be gained.

The dimeric enzyme, liver alcohol dehy- drogenase, was not precipitated. Elucidation of the reasons for this behavior revealed an interesting molecular arrangement. Related molecular arrangements involving two en- zyme species have recently been prepared and their kinetics studied (12). There also, Bis- NAD derivatives were used, together with ter- nary complex forming molecules, for juxta- posing the active sites of the enzymes (alcohol and lactate dehydrogenases).

* K. Mosbach et al., to be published.

ACKNOWLEDGMENTS

This work was supported by grants from the Swedish Natural Science Research Council, The Biotechnology Research Foundation, and the European Molecular Bi- ology Organization.

1.

2.

3.

4.

5.

REFERENCES

Larsson, P.-O., and Mosbach, K. (1979) FEBS Lett. 98, 333-338.

Green, N. M., Konieczny, L., Toms, E. J., and Val- entine, R. C. (1971) Biochem. J. 125, 781-791.

Burnett, T. J., Peebles, H. C., and Hageman, J. H. (1980) B&hem. Biophys. Res. Commun. %,157- 162.

Schneider, M., Guillot, C., and Lamy, B. (198 1) Ann. N. Y. Acad. Sci. 349, 257-263.

Fahien, L. A., and Kmiotek, E. (1979) J. Biol. Chem. 254,5983-5990.

6. Brodelius, P., and Kaplan, N. 0. (1979) Arch. Biochem. Biophys. 194,449-456.

7. Worthington Enzyme Manual (1972) pp. l-4, Wor- thington Biochemical Corporation, Freehold, New Jersey.

8. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and RandalI, R. J. (1951) J. Biof. Chem. 193, 265- 275.

9. Frieden, C. (1963) in The Enzymes (Boyer, P. D., Lardy, H., and Myrbiick, K., eds.), 2nd ed., Vol. 7, pp. 3-24, Academic Press, New York.

10. Smith, E. L., Austen, B. M., Blumenthal, K. M., and Nyc, J. F. (1975) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 11, pp. 293-367, Academic Press, New York.

11. Briinden, C-I., Jomvall, H., Eklund, H., and Furugren, B. (1975) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 11, pp. 103-190, Academic Press, New York.

12. M&son, M.-O., Siegbahn, N., and Mosbach, K. (1983) Proc. Nat. Acad. Sci. USA 80, 1487-1491.