monoacyl-sn-glycerol 3-phosphate acyltransferase specificity in bovine mammary microsomes

Monoacyl-sn-glycerol 3-Phosphate Acyltransferase Specificity in Bovine Mammary Microsomes JOHN E. KINSELLA, College of Agriculture and Life Sciences, Department of Food Science, Cornell University, Ithaca, New York 14853

ABSTRACT

The acyl-CoA:acyl-sn-glycerol 3-phos- p h a t e acyltransferases located in the microsomai fraction of lactating bovine mammary tissue show a preference for palmityl-CoA particularly above the apparent Km values of the acyl acceptors. Using saturating levels of monopalmityl- sn-glycerol 3-phosphate, the order of a c y l a t i o n was palmityl- > myristyl- > oleyl- > stearyl- >linoleyl-CoA. Apparent Km values for monopalmityl- and monooleyl-sn-glycerol 3-phosphate with palmityl-CoA as donor were 16 and 13/aM, respectively, while the Km values for palmityl-CoA with these two acyl acceptors were 5 and 5.2/aM, respectively. The a p p a r e n t Vmax values for the palmityl acceptor and donor were 25 and 30 nmol/min/mg protein. Phosphatidic acid was the principal product. The inclu- sion of magnesium in the assay depressed activity while the addit ion of ethylenediaminetetraacetate doubled the rate of acylation.

I NTRODUCTI ON

Bovine milk triglyeerides are very hetero- genous and differ from other animal glycerides in composit ion and location of c o m p o n e n t fat ty acids. Unlike most natural triglycerides where the fat ty acids on posit ion sn-2 tend to be unsaturated, the glycerides from ruminant mammary tissue are predominant ly saturated. This nonrandom distribution of fat ty acids (1,2) is most probably controlled by the specificity of the acyltransferases involved in glycerolipid synthesis and to some degree by the relative availability of the precursor acyl-CoA molecules.

Some selectivity in patterns of acylation of fat ty acids was observed by Pyndath and Kumar (3) and Askew et al. (4) using homogen- ares of lactating caprine and bovine mammary glands, respectively. The specificity of acyl-CoA util ization observed with rat mammary microsomes was consistent with the distribution of fat ty acids in rat milk triglyeerides (S).

A marked preference for palmityl-CoA, exhibited by the acyltransferase of bovine

m a m m a r y microsome in the acylat ion of sn-glycerol 3-phosphate (GP), was observed by Gross and Kinsella (6), However, because in the above experiments both positions sn-I and sn-2 of the GP were acylated to produce 1,2-diacyl- sn-glycerol 3-phosphate (DAGP), it was not possible to estimate the relative preference of the acyltransferase (AT) enzyme acylating posit ion sn-2 of the putative intermediate, 1-ac~,l-sn-glycerol 3-phosphate (AGP). Thus, the apparent specificity observed was a composite of the selectivity of the transferases acylating both positions. To determine if a relationship exists between the location of fat ty acids in position sn-2 of milk glycerollpids and the specificity of the AGP acyltransferases, we studied the relative activity of this enzyme from lactating bovine mammary tissue toward several acyl-CoA species.

MATERIALS AND METHODS

Lipid standards were purchased from Ap- plied Science Laboratories (State College, PA). The pure acyl-CoA substrates were purchased from P and L Biochemical (Milwaukee, WI), a n d t h e monoacyl-sn-glycerol 3-phosphates (AGP) were obtained from Serdary Research ( L o n d o n , Ontario, Canada). Radiochemicals were purchased from New England Nuclear (Boston, MA). Lipid "free" bovine serum a l b u m i n (BSA) was obtained from Miles Laboratories (Kankakee, IL). The 5,5'-dithiobis (2-nl t robenzoate) (DTNB) and ethylenediaminetetraacetate (EDTA) were purchased from Sigma Co. (St. Louis, MO) and Fisher Scientific ( P i t t s b u r g h , PA) , respectively. All other chemicals were reagent grade, and double distilled deionized water was used.

Enzyme Preparation and Assay

Mammary tissue was obtained from a cow in t h e third trimester of her first lactation. Secretory tissue devoid of adipose and connec- tive tissue was carefully excised, homogenized, and fractionated as outlined previously (7). The final microsomal pellet was washed, freeze- dried, and stored in sealed vials at -20 C. Under these conditions, the acyltransferases are stable for at feast 3 months (6). For enzyme assays, the microsomes were dispersed in Tris-HC1 buffer (65 mM, pH 7.4). Protein was quantified by

680

ACYLTRANSFERASE SPECIFICITY 681

TABLE I

Rates of Acylation of Increasing Concentration of Monopalmityl- and Monooleyl-sn-glycerol 3-Phosphate with Different Acyi-CoA Species by Microsomal

Acyltransferase from Lactating Bovine Mammary Tissue (nmol/min/mg protein)

Substrate a 12/~M 18 ~M 24 #M 48/IM 350 ~tM b (Donor) P O P O P O P O P O

Myrist y i -CoA 7.1 2.7 10.7 3.0 13.0 3.6 15.0 4.2 45 23 Palmit y l -CoA 7.0 3.0 10.1 3.2 15.0 4.2 20 .3 5.1 55 27 S tea ry i -CoA 5.7 2.2 7.0 2.6 8.7 3.1 10.0 3.8 36 15 OleyI -CoA 6.5 3.6 9 .8 4.0 11.4 4.5 9.3 5.3 36 16 L ino ley I -CoA 0.2 0.3 0 .6

aRates are initial velocities measured in the presence of substrate acyl-CoA species (10/~M) and 0.1 mg microsomal protein at 31 C. P and O refer to monopalmityl and monooleyl species of acyl-sn-glycerol 3-phosphate, respectively.

bRates measured under similar conditions with acyI-CoA concentrations at 25 #M.

the procedure of Lowry et al. (8) using bovine serum albumin as standard. Prior to enzyme assay, the microsomal dispersion was sonicated in an ultrasonic cleaner (Model 8845, Cole- Palmer, Chicago, IL) for 1 rain at 4 C.

The spectrophotometric assay method of Lands and Hart (9), in which the release of CoASH from acyl-CoA upon esterification is continuously monitored at 412 nm by the formation of a colored product with DTNB, was used for measuring enzyme activity. Prior to assay, the enzyme solution was held for 3 rain to attain 31 C. The reaction was initiated by adding the AGP, and the change in absorb- ance was recorded continuously for 5-7 min using a Perkin-Elmer Model 356 spectropho- tometer attached to a 10mV Hitachi Perkin- Elmer Model 165 recorder. The activity of acyl- CoA (palmityl-CoA) thiolase in this system was less than 0.5 nmol/min/mg microsomal protein. The standard assay contained Tris-HC1 buffer, 65 mM, pH 7.4; acyl-CoA, 2-25/aM; AGP, 5-50/aM; DTNB, 1.0 raM; and microsomal protein, 0.1 mg/ml.

Products of the reaction, formed in identical assay systems containing [1-14C] acyl-CoA, were extracted, fractionated by thin layer chromatography (TLC), and the radioactivity in various lipid classes was quantified as previously described (6).

RESULTS

Linear reaction rates were obtained at protein concentrations between 0.05 and 1.0 mg/ml. Using a microsomal protein concentration of 0.1 mg/ml, product formation was linear for at least 5 min. This enzyme concentration was used in all assays to minimize substrate binding and complications arising from monomer/micelle phase transitions (10,11). By

using radioactively labeled acyl-CoA species 'in assays and subsequent separation of products by TLC, it was confirmed that phosphatidic acid was the predominant product (~90%), with diglycerides containing the remainder of the radioactivity.

Based on the composition of milk glycerolipids (1,12) and previous enzymatic studies of acyltransferases (13), it was decided that monopalmityl-sn-glycerol 3-phosphate (PGP) and monooleyl-sn-glycerol 3-phosphate (OGP) were appropriate substrates for the scyltransferases of bovine mammary microsomes, hence these were used as acyl acceptors. At low accep to r concentrations, acyl-CoA specificity was not very marked (Table I). Above Km concentrations of the acceptors, palmityl-CoA became the preferred acyl donor for PGP, and this preference was accentuated at saturating levels of PGP. Myristyl-CoA, oleyl-CoA, stearyl-CoA, and linoleyl-CoA were incorporated into PGP at progressively lower rates (Table I). At high concentrations of acceptors (350/aM) and with donor acyl-CoA at 25/aM, i.e., substrates above the critical micellar concentration of the amphilphilic mixture, the rates of acylation were markedly enhanced, three- to fivefold, for the various acyl-CoA species studies.

The rates of acylation of OGP were markedly lower than those observed with PGP, and the acyl specificity was less apparent. Oleyl-CoA was the preferred acyl donor at all concentrations of OGP up to 48 /aM. At high concentrations, palmityl-CoA was the preferred d o n o r for OGP, with myristyl-CoA also showing a high rate of acylation.

The observed preference of the acyltransferase for PGP was consistent with the marked specificity of the sn-glycerol 3-phosphate acyltransferase for palmityl-CoA (6). The data may also indicate that in lactating bovine

LIPIDS, VOL. 11, NO. 9

682 JOHN E. KINSELLA

.2 "7

.1

, 0 5

-.06 -.02

PS~P (IGP PGP

KM = 16UM 13pM

, , i , i , i i i i i i i

.02 .Of) .I0 .lq .18 .22 ,26

MONOACYL-SN'GLYCEROL 3-PMDSPHATE (IJM -~ }

FIG. 1. Lineweaver-Burk plots showing rates of acylafion at various concentrations of monopalmityl- sn-glycerol 3-phosphate (PGP) or monooleyl-sn- glycerol 3-phosphate (OGP) with palrnityl-CoA by acyltransferase of microsomes from lactating bovine mammary tissue. Assays at 31 C contained palmityl- CoA (10 #M), 5,5'-dithiobis(2-nitrobenzoate) (1 mM), monoacyl-sn-glycerol 3-phosphate (varying levels), and 0.1 mg microsomal protein in 1 ml of Tris-HC1 buffer (66 mM, pH 7.4). Vmax has units of nmoles/min/mg protein.

"~, "~ .16 K,~ : 5.0uM 5.2uM

VMAX :31,0 20.4 ~ O

~ ~ .08

~ i i i i -0,I 0,i 0,2 0.5 0.4

PALMITYL-CoA (uM -I)

FIG. 2. Lineweaver-Butk plots showing the rates of acylation of fixed levels of monopalmityl-sn-glycerol 3-phosphate (A) and monoleyl-sn-glyeerol 3-phosphate (B) by palmityl-CoA using acyltransferase of microsomes from lactating bovine mammary cells. Assays at 31 C contained palmityl-CoA (varying con- centrat ions) , monoaeyl-sn-glycerol 3-phosphate ~40 uM). tl mM), and 0.1 mg microsomal protein in 1 ml of Tris-HC1 buffer. Vmax has units of nmoles/min/ mg protein.

mammary PGP is the preferred intermediate acyl acceptor in glycerolipid synthesis.

Using palmityl-CoA as donor, Km and Vmax values of 16/aM and 25 and 13 /aM and 19.2 nmol/min/mg protein were determined for PGP and OGP, respectively (Fig. 1). When BSA (3 mg/ml) was included in similar assays, much higher levels of substrates were required and higher apparent Km values of 40 and 41,~M for

PGP a nd OGP were obtained. This was probably caused by binding of the PGP and OGP to the BSA, thereby reducing the concentration of free acceptors (14).

At low substrate levels, in the absence of BSA, hyperbolic rate patterns were obtained with increasing levels of acyl-CoA from 2.5 to 10/~M. Lineweaver-Burk plots (Fig. 2) revealed apparent Km and Vmax values of 5/aM and 31 nmol/min/mg protein, and 5.2/aM and 20.4 nmol/min/mg protein for palmityl CoA with PGP and OGP, respectively. Comparable Km values were obtained with myristyl and oleyl- CoA, but Vmax data were lower than those obtained with palmityl-CoA.

In comparing data obtained with microsomes from lactating mammary tissue of different animals, marked variation in acylation rates was obtained (e.g., from 10 to 30 nmol palmityl-CoA acylated into PGP per min per mg protein). This may be attributed to differences in enzyme levels between animals, to the activity of acyl-CoA hydrolases, and to presence of varying amounts of inhibitory agents.

Because of the report of Jamdar and Fallon (15) that magnesium enhanced acylation and glycerolipid synthesis via stimulation of phos- phatidate phosphohydrolase, the effect of this cation on acylation of PGP was examined. However, magnesium actually depressed acyltransferase activity in the present studies (Table II). This was consistent with the findings of Kuhn (16), using guinea pig mammary tissue, but contrary to observations with bovine tissue (6). Recently it was shown that the depression of PGP acyltransferase by magnesium in microsomes from lactating rabbit mammary was not caused by cation complexation of either of the substrates (11). It thus appeared possible that endogenous cations might be depressing acyltransferase activity, and conceivably variation in cation levels between different microsomal preparations could influence the acyltransferase rates observed experimentally. Since microsomes were obtained from lactating mammary tissue, it seemed likely that calcium was present in these preparations. The presence of inhibitory cations associated with the microsomes was indicated by the marked increase in acyltransferase activity when EDTA was included in the buffer used for suspending the microsomes (Table II) .

DISCUSSION

The observed preference of the acyltransferase for palmityl-CoA was consistent with earlier observations obtained using caprine, bovine, rat, and guinea pig mammary tissue (3,5,6,16). This contrasts with many other


ACYLTRANSFERASE SPECIFICITY 683

TABLEII

Effects of Magnesium and Ethylenediaminetetraacetate (EDTA) on the PalmityI-CoA:Monopalmityl-sn-glycerol 3-Phosphate Palmityltransferase

Activity in Bovine Mammary Microsomes

Rate of acylation Enzyme preparation (nmol/min/mg protein)

Microsomes alone Microsomes + MgCI 2 (0.SmM) Microsomes + MgCl 2 (1.0mM) Microsomes + EDTA (lmM) Microsomes + EDTA (2raM) Microsomes + EDTA (3mM)

17.o 14.0 10.0 30.3 33.0 30.0

mammalian AGP acyltransferases which show a marked preference for unsaturated fatty acids (15,17-19). However, the validity of enzymatic data obtained with substrates that are anionic amphilphiles must be considered with reserva- tion. Such substrates may exist as free mono- mers in solution, as micelles, or they may tightly bind to proteins in these systems. Hence, it is difficult to know the actual substrate concentration available to the enzyme. Some of the problems associated with such systems and the manner in which they affect kinetics and specificities have been studied and discussed (7,10,11,14,17,20,21).

In the present study, relatively low concentrations of donor substrates were employed to comply with the admonition of Tipton (22) that, in order to obtain reliable evidence of enzyme specificity, it is necessary to make initial velocity measurements at substrate levels in the vicinity of apparent Km values.

A marked difference in capacity of the mammary enzymes to acylate PGP compared to the OGP was apparent at all concentrations of substrates. Though Barden and Cleland (1969) observed some differences, they concluded that the nature of the fatty acids present in the 1-acyl-sn-glycerol 3-phosphate had a negligible effect on the rate of acylation and that the specificity resided in the selectivity of the acyltransferases for the donor fatty acids. In contrast to the present data, Okuyama and Lands (1972) reported that selective acylation of AGP occurred only when concentrations of this acyl a c c e p t o r was very low, i.e., below apparent Km values.

The affinity of the bovine mammary microsomal enzymes for PGP and OGP were quite similar (Fig. 1). These Km values are within t h e range reported by Barden and Cleland (14) and were below the critical rnicellar concentration (cmc) of these amphiphiles (14). In contrast to t h e observations of Okuyama and Lands (17) and Numa and Yamashita (19), little specificity in acylation of either AGP or OGP at low con-

centrations was obtained in the present study, and selectivity for acyl donor group only became apparent at comparatively high concentrations of acceptors. The specificity for acyl- CoA's (palmityl > oleyl > myristyl-CoA) was similar to that observed by Barden and Cleland (14) for acyl-CoA concentrations ranging from 6 to 9/.tM.

The apparent Km values for the acyl-CoA species were around the cmc of these com- pounds, which ranges from 3 to 7 #M (14). These observed Km values may be somewhat high, being inflated by nonspecific binding (11). Acyltransferases apparently prefer substrates in true solution (14,17). However, recently the presence of isoenzymes of AGP acyltransferases in rabbit mammary has been reported (20). One isoenzyme utilizes substrate in monomeric form, whereas the other isoenzyme species function at much higher rates with substrate in micellar form (20,23). T h e marked increase in rate of acylation at high concentrations of PGP and OGP observed in the present study may also indicate isoenzymes in b o v i n e mammary microsomes. Conceivably these facilitate the continued synthesis of glycerolipids in the presence of relatively large concentrations of amphilphilic lipids as may occur in lactating mammary tissue.

T h e observed selectivity in acylation of t h e different acyl-CoA species by the enzyme from bovine mammary microsomes was consistent with the relative concentrations of these fatty acids in position sn-2 of milk triglycerides ( 1 ).

ACKNOWLEDGMENT

We are greateful for the technical assistance of M. Gross and the financial support of NSF Grant Nos. 37174 and PCM75-19123.

REFERENCES

1. Kuksis, A., JAOCS 50:193 (1973). 2. Jensen, R., and J. Sampugna, J. Dairy Sci. 49:460

(1966). 3. Pyndath, T., and S. Kumar, Biochim. Biophys.


684 JOHN E. KINSELLA

Acta 84:251 (1964). 4. Askew, E.W., R.S. Emery, and J.W. Thomas,

Lipids 6:777 (1971). 5. Tanioka, H., C.Y. Lin, S. Smith, and S. Abraham,

Ibid. 9:229 (1974). 6. Gross, M., and J.E. Kinseila, Ibid. 9:905 (1974). 7. McDonald, T., and J.E. Kinsella, Arch. Biochem.

Biophys. 156:223 (1973). 8. Lowry, O.M., N.J. Rosebrough, A.L. Farr, and

R.J. Randall, J. Biol. Chem. 193:265 (1951). 9. Lands, W.E.M., and P. Hart, Ibid. 240:1905

(1964). 10. Gatt, S., Y. Barenholz, ICI. Borkovski, and B.E.

Leibovitz, Adv. Exp. Med. Bio.. 19:237 (1972). 11. Caffrey, M.D., "Properties of the PalmityI-CoA:

Monopalmityl-sn-glycerol 3-Phosphate Palmityl- t r ans fe ra ses f rom Mammary Tissue," Thesis, Corneil University, Ithaca, NY, 1976, pp. 120-208.

12. Kuksis, A., Prog. Chem. Fats Other Lipids 12:1 (1972).

13. Kinsella, J.E., and M. Gross, Biochim. Biophys. Acta 316:109 (1973).

14. Barden, R.W., and W.W. Cleland, J. Biol. Chem.

244:3677 (1969). 15. Jamdar, S.C., and H.J. Fallon, J. Lipid Res.

14:509 (1973). 16. Kuhn, N., Biochem. J. 105:213 (1967). 17. Okuyama, H., and W.E.M. Lands, Ibid. 247:1414

(19"/2). 18. Hill, E.M., and W.E.M. Lands, in "Lipid Metabo-

l ism," Vol. 1, Edited by S. Wakil, Academic Press, New York, NY, 1970), p. 185.

19. Numa, S., and S. Yamashita, in "Current Topics in Cell Regulat ion," Vol. 8, Edited by B. Horecker and E. Stadtman, Academic Press, New York, NY, 1974, p. 197.

20. Caffrey, M., J.P. Infante, and J.E. Kinsella, FEBS Lett. 52:116 (19"/5).

21. Caffrey, M.C., and J.E. Kinsella, Biochim, Bio- phys. Acta (In press).

22. T i p t o n , ICF., Biochem. Pharmacol. 22:2933 (1973).

23. Caffrey, M.C., and J.E. Kinsella, Biochem. Bio- phys. Res. Commun . (in press).

[ Received March 8, 1976]


monoacyl-sn-glycerol 3-phosphate acyltransferase specificity in bovine mammary microsomes

Documents