Lipids of Cultured HepatomaCelts: IV. Effect of Serum and Lipid upon Cellular and Media Neutral Lipids RANDALL WOOD and JOHN FALCH, Division of Gastroenterology, Departments of Medicine and Biochemistry, University of Missouri School of Medicine, Columbia, Missouri 65201
lism of this neoplasm at the class level.
ABSTRACT
Minimal deviation hepatoma 7288C cells were cultured in a modified Swim's medium supplemented with decreasing levels of serum, lipid-free serum, lipid-free serum plus fatty acids, and other additives. Cellular and media neutral lipid classes were quantitated, the fatty acids of triglycerides and sterol esters analyzed, and the carbon number distribution of triglycerides determined. Cellular triglyceride biosynthesis virtually was inhibited when the medium was supplemented with bovine serum alone. This inhibition was not observed when the medium was supplemented with fetal calf serum alone or mixtures of fetal calf serum and bovine serum. Cells cultivated on medium supplemented with lipid-free serum plus palmitic or linoleic acids had much lower levels of free and esterified cholesterol. The fatty acid composition of cellular triglycerides and cholesterol esters differed d r a m a t i c a l l y from the corresponding media lipid classes. Except when linoleic acid was added to the medium, changes in the media serum and lipid levels had only marginal effects upon the fatty acid composition of cellular triglycerides and cholesterol esters. These data, in conjunction with earlier data that showed the media neutral lipid levels did not decrease during cell growth, indicate that these hepatoma cells utilize little or no serum triglycerides and cholesterol esters. Linoleic acid added to the medium dramatically reduced the level of 18:1 acids in cellular triglycerides and cholesterol esters. Palmitic acid added to the medium did not change the fatty acid compositions significantly. Comparison of experimentally determined and calculated triglyceride carbon number percentages indicated a random distribution of fatty acids in this glyceride. The fatty acid composition of cellular triglycerides was similar to the composition of the cholesterol esters. The lack of characteristic and distinguishable compositions of these two classes that occur in most normal tissues suggests a loss of specificity in the lipid metabo-
I NTRODUCTION
The importance of lipids and the advantages of using cultured minimal deviation hepatoma cells to study the changes that occur in the lipid metabolism of neoplasms has been discussed previously (1). Earlier studies of this series described the qualitative changes that occurred in cellular and media lipids and the quantitative changes observed in individual phospholipid class fatty acids from hepatoma cells cultured on media containing decreasing amounts of serum, lipid-free serum, and lipid-free serum containing added fatty acids (1,2). This report describes the quantitative analysis of the media and cellular neutral lipid classes and the detailed examination of the triglycerides and sterol esters from those experiments. EXPERIMENTAL
PROCEDURES
Minimal deviation hepatoma 7288C cells (HTC) were grown in roller cultures as monolayers on a modified Swim's 77 medium supplemented with various levels of serums and lipids (Table I), as previously described (1). Lipids were extracted from cells and media and separated into neutral lipid and polar lipid fractions (1). The quantitation of the neutral lipid classes was determined by high temperature gas liquid chromatography (GLC) of the intact neutral lipid fraction, as described by Kuksis and colleagues (3,4). Modifications of the method and equipment that have proved advantageous in our hands are described. Neutral lipid aliquots (50-200 pg) were hydrogenated (5), diluted in 50 p l i t e r N, O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) in a chromatography tube (Kontes), heated on the injector port of the chromatograph until the BSTFA refluxed, and an aliquot of the BSTFA solution analyzed. The rapid build-up of silicon on the detector of the instrument can be reduced greatly by evaporating the BSTFA and using chloroform as a solvent. A standard consisting of known quantities of free fatty acid, cholesterol, diglyceride, cholesterol ester, and triglyceride was analyzed daily to obtain a detector response factor for each lipid class, necessary for the quantitative 979
R. WOOD AND J. FALCH
980
TABLE I Media Containing Various Levels of Serum and Lipid Used To Culture Hepatoma Cells
Medium A B C D E F G H
Percent bovine serum 20 10 5
Swim's 77 medium modifications Percent fetal calf Percent lipid-free serum serum a
Other additions or deletions
5 5 5 5
5 2.5
J
+ Palmitic acid (37.5 #g/ml) {+ Linoleic acid (37.5 ug/ml) - Dextrose + Maltose + Sterculia foetida oil (50/.tg/ml) + Insulin (1 unit/ml)
5
K
5
5
L
5
5
a Fetal calf serum. calculations. Analyses were made on an Aerograph m o d e l 21 00 gas c h r o m a t o g r a p h m o d i f i e d to accept a 3 / 1 6 i n . column. A 75 c m x 4 mm (2.5 mm, inside diameter) glass c o l u m n packed with 1% OV-17 coated on 100-120 mesh Gas Chrom-Q was fitted to the i n s t r u m e n t with Pyrex to Kovar seals (6,7). Operating parameters were similar to those used previously (8). Peak areas were q u a n t i t a t e d with a digital integrator. Triglycerides and sterol esters were resolved by thin layer c h r o m a t o g r a p h y (TLC) on adsorbent layers of Silica Gel G developed in a solvent system of hexane-diethyl ether-acetic acid 80:20:1 (v/v/v). Resolved lipid bands were located by viewing c h r o m a t o p l a t e s sprayed with R h o d a m i n e 6G under UV light. A d s o r b e n t layers containing triglycerides or sterol esters were scraped directly into teflon lined screw cap culture tubes (16 x 100 ram) and c o n v e r t e d to m e t h y l esters by heating in a boiling water bath with 3 ml 2% sulfuric acid in a n h y d r o u s methanol. After 2-6 hr, an equal v o l u m e of water was added, the sulfuric acid neutralized with excess a m m o n i u m h y d r o x i d e and the methyl esters e x t r a c t e d thrice with hexane. Methyl esters were analyzed and q u a n t i t a t e d on the same i n s t r u m e n t using 180 c m x 2 m m (inside diameter) p y r e x c o l u m n s packed with 10% EGSS-X coated on 100-120 mesh Gas Chrom-P. Column oven t e m p e r a t u r e was p r o g r a m e d 140-200 C at 2 C/min. Identities of fatty acid m e t h y l esters were based u p o n analysis before and after h y d r o g e n a t i o n and c o c h r o m a t o g r a p h y with standards. LIPIDS, VOL. 9, NO. 12
The source and quality of standards, solvents, reagents, etc,, were the same as given previously (1). RESULTS Cell Growth
The growth rate and q u a n t i t y of lipids obtained f r o m HTC cells cultured on media A t h r o u g h I (Table l) have been described previously (1). The substitution o f maltose for glucose ( m e d i u m J) increased the doubling time to 4.2 days. The g r o w t h that did occur probably resulted f r o m n o n e n z y m a t i c hydrolysis o f maltose and the glucose (0.56 mole %) contaminating the maltose. The poorly growing cells contained 47 and 32 p g / 1 0 6 cells of p h o s p h o lipid and neutral lipid, respectively, higher lipid levels than those cells growing m o r e rapidly, as n o t e d previously (1). The a d d i t i o n of insulin and sterculia foetida oil to m e d i u m containing 10% serum (media K & L, Table 2) increased the cell doubling time m o d e r a t e l y (1.8 and 2.3 days) when c o m p a r e d to media supplemented with only 10% serum (1). The q u a n t i t y o f lipid/million cells c u l t u r e d on media K and L was ca. the same as m e d i u m C. Quantitation of Neutral Lipid Classes
The percentage distribution of lipid classes in the neutral lipid fraction o f cells grown on the various media is given in Table lI. Free sterol, shown to be cholesterol by GLC and TLC analyses, and triglycerides were the m a j o r neutral lipids u n d e r most g r o w t h conditions.
HEPATOMA CELL NEUTRAL
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982
The removal of bovine serum from the medium had little effect upon the cellular neutral lipid composition (Table II); however, a change was observed when fetal calf serum was decreased 5-2.5% or by the use of lipid-free serum. Cells grown on medium containing only bovine serum showed more than an 85% reduction in triglycerides; this reduction resulted in an apparent rise in the level of the other classes. Cells grown on medium containing lipid-free serum contained elevated percentages of free fatty acids and diglycerides and decreased amounts of triglyceride. The addition of free fatty acids to medium containing lipid-free serum resulted in a decrease of cellular cholesterol and cholesterol esters. The replacement of dextrose with maltose, addition of sterculia foetida oil (triglycerides), and the addition of insulin to media containing serum had little effect upon the hepatoma neutral lipid composition, except for a moderate increase in free fatty acids. The netural lipid composition of the media from the last half of the incubation period also is given in Table II. Sterol ester, the major neutral lipid of bovine and fetal calf serums, remained high in the media lipids. The growth medium obtained from cells grown in medium prepared with lipid-free serum contained elevated percentages of free sterol and free fatty acids and a decreased percentage of sterol ester. The addition of linoleic acid to medium containing lipid-free serum reduced the percentage of sterol ester growth as was observed in the cells. The increase in free fatty acids of this growth medium, probably resulted from unmetabolized linoleic acid, accounted for some of the apparent decrease in sterol ester percentage. Trigiycerides, the major cell neutral lipid class, were present only in minor amounts in all the media after cell growth, regardless of media supplementation.
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The fatty acid composition of triglycerides and sterol esters derived from cells grown on various media is compared in Table III. Remarkably, the composition of the triglycerides remained relatively unchanged, except for two or three media. IAnoleic acid replaced 18:1 in the triglycerides from cells cultured on medium supplemented with lipid-free serum plus linoleic acid. The other significant change observed was the increase of octadecenoic acids in cells cultured on medium supplemented with lipid-free serum or 2.5% fetal calf serum. Cellular sterol esters, shown to be cholesterol esters by GLC and TLC, exhibited only slight changes in fatty acid composition, except
983
H E P A T O M A CELL N E U T R A L LIPIDS TABLE IV Calculated and Determined Carbon N u m b e r Distribution of Triglyeerides Derived f r o m Minimal Deviation H e p a t o m a 7288C Cells Cultured on Variety of Media
Medium b A B
C D E F G H I
j K L
Origin of data c Deter. Calc. Deter. Calc. Deter. Calc. Deter. Calc. Deter. Calc. Deter. Calc. Deter. Calc. Deter. Calc. Deter. Calc. Deter. Calc. Deter. Calc. Deter. Calc.
Carbon n u m b e r percentages a 48
50
52
54
56
58
60
0.2 0.9 2.8 1.1 3.0 1.7 3.8 2.4
9.6 6.5 14.3 7.4 15.3 10.4 17.g 11.2 19.0 7.0 12.2 10.9 12.6 10.9 11.4 8.4 6.5 4.2 14.8 8.9 11.6 8.4 10.6 8.2
26.7 26.2 28.7 2'7.2 31.9 32.8 31.6 31.6 23.4 27.5 28.5 31.9 31.0 31.9 31.0 30.1 19.6 18.2 28.2 26.6 26.5 25.8 27.6 25.1
4.5.8 51.5 36.8 45.8 38.1 43.4 34.5 43.9 3"7.6 50.5 40.0 4'7.2 42.8 4"/.2 43.5 47.9 40.'/ 43.8 38.2 43.6 41.0 45.5 42.1 43.8
13.0 11.8 12.2 13.2 8.9 9.0 9.0 9.8 12.6 12.7 12.8 7.0 10.4 7.0 9.1 9.6 22.1 22.2 11.4 11.9 12.5 13.1 13.0 12.7
3.4 2.7 4.2 4.5 2.1 2.2 2.4 0.8 4.6 1.1 3.8 T 2.6 T 2.3 2.4 7.6 8.6 4.8 5.8 4.4 4.5 4.1 4.4
1.3 T 1.0 0.7 0.7 T 0.9 0 2.8 0 1.2 0 0.5 0 2.0 T 2.3 1.9 1.1 0.8 1.9 0.7 1.4 0.7
1.0 1.5 2.2 2.2 0.8 1.3 1.4 T 1.4 1.8 1.6 1.7 1.1 1.6
aA carbon n u m b e r represents the s u m o f carbon a t o m s in the three fatty acids esterilied to glycerol. bRefer to Table I for complete description of growth media. CDeter. = Determined a n d Calc. = Calculated. The calculated values are for a r a n d o m distribution of fatty acids a m o n g all three glycerol positions. Only the quantity of C-16, C-18, etc., acids (Table 1II) and t h e n u m b e r of combinations of acids to give a particular carbon n u m b e r dictate the value of t h e calculated carbon percentages. w h e n cells w e r e g r o w n o n m e d i u m s u p p l e mented with lipid-flee serum plus linoleic acid. Linoleic acid replaced 18:1 when linoleic acid was added to the medium. Cholesterol esters f r o m cells g r o w n o n m e d i u m s u p p l e m e n t e d with only bovine serum contained the next highest percentage of linoleic acid. The percentage o f l i n o l e i c a c i d i n t h e c h o l e s t e r o l e s t e r s d e c l i n e d as t h e level o f b o v i n e s e r u m in t h e m e d i a was r e d u c e d . Generally, the percentage of m o n o e n o i c a c i d s i n c r e a s e d as t h e l e v e l o f s e r u m in t h e m e d i u m d e c r e a s e d . Determined triglyceride carbon number percentages are compared with calculated percenta g e s f o r all g r o w t h c o n d i t i o n s i n T a b l e I V . T h e calculated random distribution percentages g e n e r a l l y a g r e e d v e r y well w i t h t h e d e t e r m i n e d values. The most notable exception was triglycerides from cells grown on medium supplemented with only bovine serum. Some moderate differences also were noted in carbon numb e r s 50 a n d 5 4 w h e n b o v i n e s e r u m s u p p l e m e n t e d t h e m e d i a at t h e 10 a n d 2 0 % levels.
Media Triglycerides and Sterol Esters
Triglycerides derived from growth media a f t e r h a r v e s t o f H T C cells d i f f e r e d d r a m a t i c a l l y in f a t t y a c i d c o m p o s i t i o n ( T a b l e V ) f r o m t h e cellular triglyceride composition ( T a b l e III). Palmitate and octadecenoate percentages were ca. e q u a l , w h e r e a s 1 8 : 1 c o n c e n t r a t i o n s w e r e 6 t i m e s g r e a t e r t h a n 1 6 : 0 p e r c e n t a g e s in c e l l u l a r triglycerides. Media triglycerides contained a h i g h level o f l i n o l e i c a c i d w h e n f r e e l i n o l e i c a c i d w a s a d d e d to t h e m e d i u m . G e n e r a l l y , t h e c o m position of the harvest media triglycerides a g r e e d v e r y well w i t h t h e f a t t y a c i d c o m p o sition of triglycerides derived from bovine and f e t a l c a l f s e r u m s s h o w n at t h e b o t t o m o f T a b l e V. The fatty acid composition of sterol esters derived from media after the growth of HTC cells is s h o w n in T a b l e V a l o n g w i t h t h e c o m p o sition of sterol esters from bovine and fetal calf serums. When the media contained bovine serum in any proportion, the sterol esters exhibited a high percentage of linoleic acid similar to bovine serum sterol esters. The composition LIPIDS, VOL. 9, NO. 12
984
R. W O O D
A N D J. F A L C H
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HEPATOMA CELL NEUTRAL LIPIDS of sterol esters isolated from the media containing only fetal calf serum resembled the composition of fetal calf serum sterol esters. Unlike the effect upon cellular triglycerides, media triglycerides and cellular cholesterol esters, linoleic acid added to medium supplemented with liNd-free serum had no effect upon the composition of media sterol esters. DISCUSSI ON
The substitution of maltose for glucose in the medium greatly inhibited the growth rate of HTC cells, presumably due to the lack of maltase, but had little effect upon the composition of cellular or media neutral lipids. Insulin, reported to stimulate cell growth (9) and fatty acid biosynthesis from glucose in fat cells (10,11), had no significant effect upon the growth rate or the neutral lipids of these hepatoma cells under the culture conditions employed. Likewise, the addition of sterculia foetida oil to the medium (a triglyceride that contains cyclopropene fatty acids known to alter oleic acid biosynthesis in the rat liver [12,131) had little or no effect upon neutral lipid class composition. The specific effects of the various growth medium upon monoene biosynthesis in HTC cells are reported in a companion article (14). A number of investigators (I5-23) have shown with a variety of cultured cells that, when serum lipids were present in the media, de novo lipid biosynthesis was minimal and most cellular lipids were derived from the serum. A previous report from this laboratory demonstrated that the harvest media contained as much, and in most cases more, total neutral lipid and total phospholipid as was present originally in the media (1). The harvest media from the three additional growth conditions described in this report likewise contained more lipid than the media before cell cultivation. Analyses of individual phospholipid classes (2) and neutral lipid classes (this study) have shown that the fatty acid composition of cellular and media lipid classes differed dramatically and that the growth of HTC cells in the various media did not change its composition significantly. These data indicate that HTC cells, unlike most cultured cells, utilize serum cholesterol esters, triglycerides, and phosphoglycerides sparingly, if at all. On the other hand, free fatty acids (palmitic and linoleic) added to the medium were taken up and incorporated into neutral glycerides and phosphoglycerides. M e d i u m triglycerides, but not cholesterol esters, contained an elevated percentage of linoleic acid when linoleic acid was added to the
985
medium, indicating a release of triglycerides from the cell. The uptake of free fatty acids, incorporation into cellular neutral glycerides and phosphoglycerides, and release into tile medium, by these cells also have been demonstrated with radioactive fatty acid (24). The deletion of all bovine serum from the media resulted in only marginal changes in neutral lipid class and fatty acid compositions. However, bovine serum alone reduced cellular triglyceride content dramatically. Since bovine serum did not inhibit triglyceride synthesis in the presence of fetal calf serum, the latter must contain a factor that counteracts the inhibitory effect of bovine serum. The effect of bovine serum also was observed in the comparison of determined and calculated triglyceride carbon number percentages (Table IV). Calculated ran: dom distribution percentages showed less agreei ment with determined values when the triglycerides were from cells grown on media containing more bovine serum than fetal calf serum, or bovine serum alone. However, the degree of randomness in the distribution of fatty acids among the triglyceride positions in HTC cells is much greater than shown to occur in normal rat l i v e r triglu (25). Ehrlich ascites cells show positional specificity of triglyceride fatty acids but do not exhibit the same preferential pairing of acids in the glyceride (5) as rat liver triglycerides exhibit (25). The loss of the ability to synthesize specific molecular species of glycerides, as opposed to all possible species, may be a metabolic defect of neoplasms. Linoleic acid in the growth medium reduced the percentage of monoenoic acids, especially 18: I, in cellular and medium triglycerides and cellular cholesterol esters 50-75%, whereas palmitic acid failed to show significant accumulation or effect. A similar effect of linoleic acid upon phospholipid monoenes has been reported. Media containing more bovine serum than fetal calf serum or only bovine serum, a rich source of esterified linoleic acid, gave rise to cellular triglycerides and cholesterol esters with a higher percentage of linoleic acid and lower percentage of monoenoic acids compared to cells grown on medium containing 2.5% fetal calf serum or liNd-free serum. These results suggest that the level of linoleic acid may regulate fatty acid biosynthesis, particularly monoene acid syn-thesis in these cultured hepatoma cells. This conclusion is consistent with data that suggests rat and mouse hepatic lipogenesis is influenced more by dietary linoleate than other acids (26-28). The specific effects of serum lipids and lipid-free serum upon the isomeric monoene fatty acid content of individual neutral lipids and phospholipids are reported in a companion LIPIDS, VOL. 9, NO. 12
986
R. WOOD AND J. FALCH
article (14). T w o a d d i t i o n a l p o i n t s s h o u l d be m a d e regarding the c o m p o s i t i o n o f the n e u t r a l lipids. First, n e i t h e r t h e triglycerides n o r t h e cholesterol esters f r o m the cells c u l t u r e d on t h e lipidfree s e r u m m e d i u m c o n t a i n e d m o r e t h a n trace levels of 2 0 : 3 or 22:3 acids, f a t t y acids t h a t b e c o m e e l e v a t e d in m a n y cell lines grown u n d e r c o n d i t i o n s w h e r e essential f a t t y acid deficiencies exist (29). This o b s e r v a t i o n is in k e e p i n g w i t h t h e l o w level o f t h e s e acids f o u n d in t h e p h o s p h o l i p i d s o f cells g r o w n on this m e d i u m and s u p p o r t s t h e idea p r o p o s e d earlier t h a t these cells m a y lack t h e e n z y m e s y s t e m req u i r e d t o d e s a t u r a t e f u r t h e r m o n o e n o i c acids effectively (2). S e c o n d l y , t h e f a t t y acid c o m p o sition o f cellular triglycerides s h o w s r e m a r k a b l y close a g r e e m e n t w i t h t h e c o m p o s i t i o n o f t h e c h o l e s t e r o l esters (Table III). This is in c o n t r a s t to n o r m a l rat liver triglycerides and c h o l e s t e r o l esters, w h i c h e x h i b i t c h a r a c t e r i s t i c and readily d i s t i n g u i s h a b l e c o m p o s i t i o n s (30-32). This p r o b a b l y r e p r e s e n t s a f u r t h e r lack o f s p e c i f i c i t y in lipid m e t a b o l i s m o f n e o p l a s t i c cells at t h e class level. The inability o f this n e o p l a s t i c cell t o s y n t h e s i z e specific c h o l e s t e r o l e s t e r species could be r e l a t e d t o t h e a p p a r e n t inability to s y n t h e s i z e specific species o f triglycerides discussed earlier. ACKNOWLEDGMENTS This work was supported by a grant from the National Cancer Institute (USPH 5-RO1 CA 12973). REFERENCES 1. Wood, R., Lipids 8:690 (1973). 2. Wood, R., and J. Falch, Ibid. 8:702 (1973). 3. Kuksis, A., L. Marai, and D.G. Gornall, J. Lipid Res. 8:352 (1967). 4. Kuksis, A., O. Stachnyk, and B.J. Holub, Ibid. 10:660 (1969). 5. Wot, d, R., and F. Snyder, Arch. Biochem. Bio-
LIPIDS, VOL. 9, NO. 12
phys. 131:478 (1969). 6. Radin, N.S., J. C_hromatog. 20:392 (1965). 7. Litchfield, C., R.D. Harlow, and R. Reiser, Lipids 2:363 (1967). 8. Wood, R., and R.D. Harlow, Ibid. 5:776 (1970). 9. Otten, J., G.S. Johnson, and I. Pastan, J. Biol. Chem. 247:7082 (1972). 10. Jungas, R.L., Endocrinology 86:1368 (1970). 11. Coore, H.G., R.M. Denton, B.R. Martin, and P.J. Randle, Biochem. J. 125:115 (1971). 12. Raju, P.K., and R. Reiser, J. Biol. Chem. 242:379 (1967). 13. Raju, P.K., and R. Reiser, Biochim. Biophys. Acta 176:48 (1969). 14. Wood, R., J. Falch, and R.D. Wiegand, Lipids 9:987 (1974). 15. Watson, J.A., Ibid. 7:146 (1972). 16. Howard, B.V., and D. Kritchevsky, Biochim. Biophys. Acta 187:293 (1969). 17. Bailey, J.M., Ibid. 125:226 (1966). 18. MacKenzie, C.G., J.B. MacKenzie, O.K. Reiss, and J.A. Wisneski, J. Lipid Res. 11:571 (1970). 19. MacKenzie, C.G., J.B. MacKenzie, and O.K. Reiss, in "Lipid Merabolism in Tissue Culture," Edited by G.H. Rothblat and D. Kritchevsky, Wistar Institute Symposium Monograph No. 6, Philadelphia, Pa., 1967, p. 63. 20. Moskowitz, M.S., Ibid. 1967, p. 49. 21. Geyer, R.P., Ibid. 1967, p. 33. 22. Bailey, J.M., G.O. Gey, and M.K. Gey, Proc. Soc. Expt. Biol. Med. 100:~86 (1959). 23. Jacobs, R.A., W.S. Sly, and P.W. Majerus, J. Biol. Chem. 248:1268 (1973). 24. Wiegand, R.D., and R. Wood, Lipids 9:141 (1974). 25. Wood, R., and R.D. Harlow, Arch. Biochem. Biophys. 131:495 (1969). 26. Ailmann, D.W., and D.M. Gibson, J. Lipid Res. 6:51 (1965). 27. Abraham, S., Amer. J. Clin. Nutr. 23:1120 (1970). 28. Bartley, J.C., and S. Abraham, Biochim. Biophys. Acta 280:258 (1972). 29. Bailey, J.M., B.V. Howard, L.M. Dunbar, and S.F. Tillman, Lipids 7:125 (1972). 30. Dittmer, J.C., and D.J. Hanahan, J. Biol. Chem. 234:1976 (1959). 31. Getz, G.S., W. Bartley, F. Stirpe, B.M. Notton, and A. Renshaw, Biochem. J. 80:176 (1961). 32. Wood, R., Arch. Biochem. Biophys. 141:174 (1970). [ R e c e i v e d J u n e 11, 1974]