Comparisons of the Effects of BaciZZus subtilis Protease, Type VIII (Subtilopeptidase A), and Insulin on Isolated Adipose Cells

Transcription

1 THE Jourmar. OF B~LOGICAL CHEMISTRY Vol. 212, No. 16, Issue of August 25, PP , 1967 Pentea in U.S.A. Comparisons of the Effects of BaciZZus subtilis Protease, Type VIII (Subtilopeptidase A), and Insulin on Isolated Adipose Cells I. GLUCOSE AND PALMITIC ACID METABOLISM J. F. Kuo, I. K. DILL, AND C. E. H~LMLXTND (Received for publication, March 15, 1967) From the Chemotherapy Research Section, Lederle Laboratories, American Cyanamid Company, Pearl River, New York SUMMARY The effects of insulin and Bacillus subtilis protease, Type VIII (Subtilopeptidase A, EC ) on the conversion by isolated adipose cells of extracellular glucose to COz, fatty acids, and glyceride-glycerol, and of extracellular palmitic acid to CO2 and cell triglyceride have been compared. The protease was found to simulate insulin action by (a) stimulation of glucose uptake and of oxidation of glucose carbon 1 via the pentose pathway; (b) stimulation of fatty acid synthesis from glucose; (c) causing still greater utilization of glucose (oxidation and lipogenesis) in the presence of palmitic acid; (d) enhancement of the carrier-mediated, stereospecific transport for glucose; and by (e) causing decreased oxidation and increased esterification of extracellular palmitic acid in the presence of glucose. Similar effects were observed with trypsin, a-chymotrypsin, and Strefitomyces griseus protease Type VI. It is suggested that these insulin-like effects may in part be due to a limited modification of plasma membranes caused by proteolytic processes. Plasma membranes of mammalian cells have been postulated to be the site of hormonal action on cell surfaces (l-3). The significance of the phospholipid constituent of lipoprotein membranes with regard to glucose utilization by adipose cells has been well demonstrated by the effects caused by phospholipases (3-5). The insulin-like effects of several proteinases on isolated adipose cells reported elsewhere (6,7) may be due to a limited hydrolysis of a protein moiety of the membrane structure. In the present study another proteinase, Bacillus subtilis protease, Type VIII (Subtilopeptidase A, EC ), is shown to possess insulinlike activity. Its effects on conversion of glucose to COz, to glyceride-glycerol, and to fatty acids are compared with those of insulin and other proteases. The problem concerning whether or not the facilitated utilization mediated by protease action is specific for glucose has also been examined by studying the effects on the oxidation and esterification of medium palmitic acid. An abstract of this investigation has appeared (8). EXPERIMENTAL PROCEDURE The epididymal fat pads of male Sprague-Dawley rats, weighing 100 to 150 g, have been used in this study. The procedures for preparing and incubating isolated adipose cells and determining radioactivity in COz and total lipid were essentially the same as described by Rodbell (9), with some modification (6). To determine radioactivity in triglyceride fatty acids, an aliquot of total lipid was saponified according to the method of Rodbell (9). Unless otherwise stated, the incubation mixture for glucose metabolism consisted of 1 ml of Krebs-Ringer bicarbonate buffer, ph 7.4, containing 4% bovine serum albumin, free adipocytes ranging from 31 to 60 mg, or 0.20 PC of uniformly labeled n-glucose-r4c (or n-glucose-lj4c) and sufficient unlabeled n-glucose to make a concentration of 1 mm. For studies of palmitic acid metabolism, 0.2 PC of palmitic acid-1-14c (made to 1 pmole by supplementing with sufficient unlabeled palmitic acid) was dissolved in hexane and added to the incubation vial. The solvent was removed under reduced pressure prior to the addition of the cell suspension and additives. WOZ produced from palmitic acid-l-14c was absorbed with watersaturated phenylethylamine in the usual way after the incubation mixture was acidified by adding 0.2 ml of 2 N HzS04. The amount of palmitic acid esterified into cell triglyceride was determined by immediately re-extracting the total lipid organic layer at a basic ph after adding 0.3 ml of 2 N NaOH. This step removes nonesterified fatty acid from the organic layer. In order to minimize the error caused by palmitic acid-l-14c extraction into the organic layer at basic ph (usually less than 5% of total counts under the experimental conditions), the radioactivity in the triglyceride fraction was corrected for controls in which the incubation mixture was extracted at zero time. 3659

2 3660 Efects of Bacillus subtilis Protease on Isolated Adipose Cells. I Vol. 242, No. 16 In all experiments two to four replicate samples were used, and each experiment was carried out two to three times in order to insure its reproducibility. Collagenase was purchased from Worthington; norepinephrine (nn-arterenol-bcl, Lot 52666)) a-chymotrypsin (crystalline, Lot 53251), trypsin (crystalline, Lot 44370), pahnitic acid-l-14c 2.5!A I-- Insulin Insulin - B _.- subtilis protease ~ X----X S.qriseus pdease Protease (&ml) I 1 I I 600 I I b9 /ml> FIG. 1. Insulin-like effects of B. subtilis protease on the utilization of uniformly labeled glucose-w and glucose-lj4c by isolated adipose cells. Free adipocytes (39 mg per ml) were incubated for 2 hours at 37, with shaking, in 1 ml of Krebs- Ringer bicarbonate buffer, ph 7.4, containing 4% dialyzed bovine serum albumin (Fraction V) and PC of either uniformly labeled glucose-w or glucose-l-w which were all made to 1 mole by supplementing with sufficient unlabeled glucose. Insulin and S. griseus protease were incubated for comparison. Each point given in the figure is the mean of three determinations. For further experimental details see Experimental Procedure. (29.6 mc per mmole, Lot 65098), and n-glucose-l-14c (10.0 mc per mmole, Lot 68003) were from Calbiochem; insulin (24 i.u. per mg, Lot 74B-1020), bovine serum albumin (Fraction V, Lot 15B-1520), protease, Type VI (Pronase, from Streptomyces griseus, repurified, Lot 85B-2360), and protease, Type VIII (Subtilopeptidase A, crystalline, Lot 66B-0380, from Bacillus subtilis, special strain) were from Sigma. Uniformly labeled n-glucoser4c (14.3 mc per mmole) was a product of new England Nuclear. RESULTS The insulin-like effects of B. subtilis protease on glucose utilization by isolated adipose cells are presented in Fig. 1. As observed for X. griseus protease, Type VI (6), and trypsin and chymotrypsin (7), the enhanced glucose utilization was reduced at a high concentration (20 pg per ml) of the enzyme. Proteases from Bacillus and Xtreptomyces, like insulin, stimulated preferential oxidation of carbon 1 of glucose, as shown by the comparable production of i4c02 from either glucose-lj4c (Fig. 1B) or uniformly labeled glucosej4c (Fig. 1D). The stimulatory effect of insulin on the oxidation of glucose carbon 1 via the pentose cycle in adipose tissue has been reported by Flatt and Ball (10). In contrast to the above parameter, the conversion of uniformly labeled glucosej4c to total lipid (Fig. 1C) was found to be much greater than that of glucose-l-14c (Fig. IA) in the absence and presence of either insulin or the enzymes. It is desirable to know whether the enhanced lipogenesis caused by proteolytic enzymes resembles insulin by effecting a preponderantly greater synthesis of fatty acid than of glycerideglycerol (10). The results presented in Table I indicate this is indeed the case. B. subtilis protease, X. griseus protease, a-chymotrypsin, and trypsin all increased fatty acid synthesis from either uniformly labeled glucose-r4c or glucose-l-14c by about 7- to lo- and 6- to g-fold, respectively, while the glyceride-glycerol synthesis was enhanced by only 2-fold. Insulin caused a a still greater stimulation in fatty acid synthesis from glucose (9- and H-fold higher than control from uniformly labeled glucase-r4c and glucose-l-14c, respectively), but the stimulation in glycerol synthesis was comparable with that of proteases (approximately 2.5-fold). For comparison, norepinephrine showed about a 2- to 3-fold stimulation in both fatty acid and glycerideglycerol synthesis (Line 7, Table I). In order to compare the insulin-like effects with the proteolytic effect of Bacillus enzyme on free adipocytes, several concen- TABLE I Effects of insulin, proteinases, and norepinephrine on conversion of uniformly labeled glucose-w and glucose-l J4C to fatty acid and glyceride-glycerol by isolated adipose cells Experimental conditions were the same as in Fig. 1 except that free adipocytes (44 mg per ml) were incubated with 0.2 MC (1 pmole) of either uniformly labeled glucose-i4c or glucose-lj4c. Each value given is the mean (&S.E.) of three determinations. Additives Conversion of uniformly labeled glucose-w to Conversion of glucose-l-w to Fatty acid Glyceride-glycerol Fatty acid Glyceride-glycerol mpmoles/g cells/z hrs None... Insulin (1000 &ml).... B. subtilis protease (5 pg/ml) S. griseus protease (4 rg/ml). &Jhymotrypsin (10 pg/ml). Trypsin (10 rg/ml).. Norepinephrine (1 pg/ml).. 40 f f f f f f f f f f f f f f f f f f f f f f f f f f f f 9

3 Issue of August 25, 1967 J. F. Kuo, I. K. Dill, and C. E. Holmlund 3661 trations of protease were incubated with cells in the presence of different concentrations of bovine serum albumin, an alternative substrate of the enzyme. As shown in Fig. 2, glucose metabolism was greatly impaired (32 $$ of control) by a high concentration of protease (30 pg per ml) in the absence of bovine serum albumin, although a marked stimulation of lipid synthesis (more than 220% of the control lacking bovine serum albumin) was observed in an incubation medium containing bovine serum albumin. Moreover, this stimulation increased with increasing bovine serum albumin concentration. At a concentration of enzyme of 6 pg per ml, a significant stimulation (127%) was observed in the absence of bovine serum albumin, and in the presence of bovine serum albumin (1.2% to 6%) lipid synthesis was increased to 240% of the control. At a lower enzyme concentration (1 pg per ml), an even higher stimulation (160%) was obtained in the absence of bovine serum albumin, and this effect was maximum at 1.2yo bovine serum albumin, and then was reduced somewhat when the bovine serum albumin concentration was 3.6% or higher. Based on the above observations, the B. subtilis enzyme at a concentration of approximately 0.5 pg/ml, or lower, will give a maximal insulin-like response in the absence of bovine serum albumin. The protective effect of bovine serum albumin on the insulin-like activity of S. g&ens protease at higher concentration has been previously reported (6). Rodbell (3) found that the basal as well as insulin-stimulated glucose utilization by free adipocytes was competitively inhibited by 3-O-methylglucose, but not inhibited by L-glucose. He concluded, therefore, that insulin stimulated the glucose transport by a carrier-mediated, stereospecific process. Blecher (5) has reported that 3-O-methylglucose competitively inhibited glucose utilization stimulated by phospholipase A. In the present study also, 3-0-methylglucose was found to be a competitive inhibitor &g (M- X IO ~34 FIG. 3. Lineweaver-Burk plots of rates of (A) basal, (B) (Ychymotrypsin-stimulated, and (C) protease (B. sumis)-stimulated glucose utilization by free adipocytes in the presence and absence of 3-0-methylglucose (S-O-MG). Experimental conditions were essentially the same as Fig. 1 except that the free adipocytes (31 mg per ml) were incubated with 0.2 pc (1 pmole) of uniformly labeled glucose-w. Velocity (ZJ) is expressed as micromoles of glucose converted to total lipid per gram of cells per a-hour incubation period. The concentrations of a-chymotrypsin and B. subtilis protease were 10 pg per ml and 6 rg per ml, respectively. Each point given in the figure is the mean of four determinations. L 0 Contd q 3-O-MG(i5mk.4) I L-Gluwse(i5mM) 7 i Additive, Number FIG. 4. Effects of 3-O-methylglucose (9-0~MG) and n-glucose on the basal and insulin- or protease-stimulated glucose utilization by isolated adipose cells. Experimental conditions were essentially the same as Fig. 1 except that l-ml cell suspensions were incubated for 2 hours in 0.2 PC (1 Mmole) of uniformly labeled glucose-w (glucose-u-w), in the presence and absence of either 3-O-methylglucose, 15 mm, or n-glucose, 15 mm. Additives: (I) represents none (basal) ; (2) insulin, 1000 pu; (3) B. subtilis protease 5 pg; (4) S. griseus protease, 5 pg; (6) oc-chymotrypsin, 10 pg; (6) trypsin, 10 pg. Each value as given in the figure is the mean (fs.e.) of four determinations. FIG. 2. Effect of concentration of bovine serum albumin (B&4) on the insulin-like activity of B. subtilis protease. Experimental conditions were essentially the same as Fig. 1 except that 0.2 PC (1 pmole) of uniformly labeled glucose-w (glucose- U-W) was used. Each point given in the figure is the mean of four determinations. of basal glucose utilization, as well as of glucose utilization stimulated by either a-chymotrypsin or B. subtilis protease (Fig. 3). Based on these findings, it is evident that the basal glucose transport system was not altered by either insulin or enzymes, e.g. phospholipase A, phospholipase C (3), ar-chymotrypsin, and B. subtilis protease. This similarity in the response of basal and protease-stimulated glucose utilization to phlorizin inhibition has been previously reported (11). The apparent K, of glucose

4 3662 Efects of Bacillus subtilis Protease on Isolated Adipose Cells. I Vol. 242, No. 16,4 I B) C-Triglyceride I I I I I I I, Incubation Time (min.) FIG. 5. Utilization of palmitic acid-l-w by isolated adipose cells in the presence and absence of insulin and B. subtilis protease. Free adipocytes (50 mg per ml) were incubated for 2 hours at 37, with shaking, in 1 ml of Krebs-Ringer bicarbonate buffer, ph 7.4, containing 3% dialyzed bovine serum albumin, 1 pmole of glucose, and 0.5 PC of palmitic acid (1 pmole). The amount of palmitic acid-l-l% esterified into cell triglyceride was determined by immediately re-extracting the total lipid organic layer (extracted at acidic ph) at alkaline ph after adding 0.3 ml of 2 N NaOH. The values reported were corrected for controls in which the incubation mixtures were extracted at zero time. Each point given in the figure is the mean (&S.E.) of four determinations. For further experimental details see Experimental Procedure. ) fi 100 I I 1 B) 4C-Tviglycevide 1 B.subtilis protease (,ug/ml) -- FIG. 6. Comparison of the effects of insulin and B. subtilis protease on the utilization of palmitic acid-l-l% and uniformly labeled glucose-w (glucose-u-w) by isolated adipose cells. Experimental conditions wereessentially thesame as Fig. 5 except that the free adipocytes (50 mg per ml) were incubated in either 0.2 PC of palmitic acid-l-14c (1 pmole) or 0.2 PC of uniformly labeled glucose-w. The incubation mixtures were always made to 1 mm with respect to glucose. The scale for WO2 production from palmitic acid-l-w has been magnified IO-fold. Each point given in the figure is the mean (&S.E.) of four determinations. utilization by isolated adipose cells has been shown to be lower in the presence of insulin (3), but not in the presence of phospholipase A (5), even though the latter, like insulin (3), increased considerably the V,,, of glucose uptake. In the present study, the apparent Km of enzyme-stimulated glucose utilization, either by a-chymotrypsin (Fig. 3B) or by B. subtilis protease (Fig. 3C), was not affected. Moreover, the K; of 3-O-methylglucose was essentially the same in the presence of enzyme. Insulin was also found to be without significant effect on the Ki of phlorizin on glucose utilization. Although 3-O-methylglucose inhibited the basal as well as the elevated glucose utilization stimulated by either insulin or proteolytic enzymes, L-glucose had no effect in any instance (Fig. 4). It is concluded, therefore, that the glucose transport stimulated by Bacillus protease, Streptomyces protease, a-chymotrypsin, and trypsin, like insulin and phospholipase C (3), is also a carriermediated stereospecific process. Furthermore, the process stimulated by these proteinases may be very similar to that stimulated by insulin. Fig. 5 presents the results of palmitic acid-l-% oxidation (A) and esterification (B) by isolated adipose cells incubated in 1 mm glucose medium. Both basal oxidation and esterification of palmitic acid-l-14c were linear during the at-hour incubation period. A change in the slope of the rate of both oxidation and esterification, however, was observed at 30 min in the presence of insulin or BuciZZus enzyme. The reason for the termination of ester&cation after 90 min of incubation time in the presence of protease may be due to excess damage of the cell membrane by the enzyme at a concentration of 8 pg per ml in 3% bovine serum albumin medium. In contrast to the enhancement of the ester& cation process by either insulin or Bacillus protease, the oxidation of extracellular fatty acid was found to be greatly reduced in the presence of these agent,s. This is due to the fact that glucose is a more direct energy source than palmitic acid for free adipocytes or because in the presence of glucose more fatty acid can be esterified, or both. ATP and glycerol phosphate produced by the higher glucose metabolism in the presence of insulin or BucilZus enzyme are required for the esterification process. Comparison of the effects of insulin and B. subtilis protease on the metabolism of glucose and palmitic acid by adipose cells is presented in Fig. 6. As the concentration of insulin increased, the oxidation of glucose was progressively stimulated, while the oxidation of palmitate was reduced. B. subtilis protease up to a concentration of about 4 pg per ml acted like insulin. However, as the enzyme concentration increased beyond this level, the oxidation of glucose was gradually inhibited while the oxidation of palmitate was stimulated. Insulin stimulated the conversion of glucose and palmitate to triglyceride, as did the B. subtih protease, up to a concentration of 4 to 5 c(g per ml. Higher concentrations of enzyme caused a reduction in the synthesis of triglyceride from both precursors. The results presented in Table II disclose the relationship of glucose to palmitate metabolism. In the absence of glucose, neither insulin nor proteases (enzymes from Bacillus and Streptomyces, cy-chymotrypsin, and trypsin) decreased palmitic acid- 1-W oxidation or increased its esterification. Furthermore, such processes, in general, were not significantly affected by inhibitors of glucose utilization, e.g. phlorizin or 3-0-methylglucose. On the other hand, in the presence of glucose, insulin as well as proteases reduced palmitic acid oxidation below the basal value. This reduction is not observed when either phlorizin or 3-0-

5 Issue of August 25, 1967 J. F. Kuo, I. K. Dill, and C. E. Holmlund 3663 TABLE Effects of insulin, proteinases, and norepinephrine on palmitate-1-w utilization by isolated adipose cells incubated in presence and absence of glucose, phlorizin, and 3-0-methylglucose, singly or in combination Free adipocytes (31 mg per ml) were incubated with 0.25 PC of palmitic acid-l-w (1 rmole) with or without glucose (1 rmole) at 37 for 1; hours. The concentrations of phlorizin and 3-O-methylglucose were 9.17 X lo+ M and 1.5 X low2 M. II Conversion of p&&ate-l-w to Triglyceride-W Additives + Glucose - Glucose + Glucose None None None f3 f2 f2 fl f5 Insulin (1000 &ml) f5 fl f2 f3 f8 B. subtilis protease ( rdml) f7 f5 fl f3 f8 S. griseus protease ( fig/ml) f2 fl f4 f9 f2 oc-chymotrypsin (lorg/ ml) fl fl f2.f5 fl Trypsin (10 rg/ml) f3 f2 f4 f4 fl Norepinephrine(lpg/ml) f2 f4 f3 f5 fl mwnoles/~ cells/l 5 hrs 72 1,279 1,132 f7 f 92 f ,485 1,808 f 10 f 277 f ,398 2,371 f8 f 106 f 17t 51 1,377 1,759 f5 f 136 f 60( 66 1,005 2,390 f7 f 87 f ,368 1,251 f2 f 62 f ,261 f2 f 255 f 61 1,625 3,124 2,592 2,541 f 121 f 598 f 75: f54 1,420 10,973 4,684 4,014 f 130 f 3,066 f 561 f 389 2,173 6,325 2,536 2,169 f 299 f 199 f 26: f ,653 3,369 2,582 f 178 f 278 f 27( f ,351 4,185 3,575 f 205 f 877 f 79; f 80 1,057 6,072 2,354 2,623 f 105 f 453 f 30 f 268 1,283 1,846 2,117 1,228 f 87 f 214 f 61 f TABLE Effect of palmitic acid on glucose utilization by isolated adipose cells in presence and absence of insulin and proteolytic enzymes Free adipocytes (40 mg per ml) were incubated with 0.2 PC (1 pmole) of uniformly labeled glucose-y! for 2 hours in the absence and presence of palmitic acid (2 pmoles) in 1 ml of bicarbonatebovine serum albumin medium. Each value given in the table is the mean trs.e.) of four determinations. III 8) Total Lipid Palm&c Acid (,u moles/ml) FIG. 7. Effect of palmitic acid on the utilization of glucose by isolated adipose cells in the presence and absence of insulin and B. subtilis protease. Free adipocytes (60 mg per ml) were incubated with 0.2 PC of uniformly labeled glucose-w (1 pmole) for 2 hours in 1 ml of Krebs-Ringer bicarbonate buffer, ph 7.4, containing 4y0 bovine serum albumin in the presence of palmitic acid at the concentrations indicated. Each point given in the figure is the mean (rts.e.) of four determinations. methylglucose is also present. The basal rate of palmitic acid esterification was markedly stimulated (Z&fold) in the presence of glucose, while still greater stimulation w&s observed with insulin (7-fold) or proteases (4- to g-fold). Phlorizin and 3-O- Additives Conversion Minu;c;;lmitic of glucose to total lipid Plus 2 rmqles of palmititd per pm&s/g cellsjz hrs None f & 0.07 Insulin (1000 &ml) f f 0.02 S. griseus protease (5 pg/ml) f f 0.03 B. subtilis protease (6 pg/ml) f f 0.03 cw-chymotrypsin (10 pg/ml) f f 0.03 Trypsin (10 rg/ml) f f 0.03 methylglucose reduced the elevated rate of esterification. It is evident that increased palmitate esterification is related to elevated glucose uptake. A lipolytic hormone, e.g. norepinephrine, inhibited both oxidation and esterification of medium palmitic acid. It is also desirable to know the effect of concentration of medium palmitic acid on the glucose utilization by adipose cells. As shown in Fig. 7, in the presence of insulin or Bacillus enzyme, palmitic acid markedly stimulated both oxidation and lipogenesis of glucose, the latter being more pronounced. The effect of palmitic acid on the basal rate of glucose utilization was found to

6 E$ects of Bacillus subtilis Protease on Isolated Adipose Cells. I Vol. 242, No. 16 be insignificant. Other proteases, such as Streptomyces protease, a-chymotrypsin, and trypsin, were all shown to stimulate further the conversion of glucose to cell lipid by 1.3 to 1.5-fold in the presence of 2 mm palmitic acid (Table III). DISCUSSION Hydrolytic modification of the protein moiety of lipoprotein plasma membranes of isolated adipose cells has been suggested to account for the insulin-like behavior of several proteinases (6,7). The stimulatory effect of chymotrypsin (a-, /3-, and y-) and trypsin on glucose uptake may be interpreted by assuming that these enzymes preferentially cleave peptide linkages according to their rather well defined specificity. If so, then the peptide bonds involving the carbonyl function of aromatic amino acids (cleaved by chymotrypsin) or of basic ammo acids (cleaved by trypsin) may be the possible site of their primary action. Limited cleavage may be sufficient to bring about a favorable state of transport without impairing membrane structure, whereas enzymes at high concentrations have been found to be destructive (7). The insulin-lie effects of S. griseus protease, Type VI, (6), an enzyme with unusually broad specificity, as well as of B. subtilis protease, in the present study, may also be explained on the basis of their capacity to cleave, among others, the above mentioned peptide bonds. However, the possibility that an interaction on the cell membranes other than hydrolytic modification for the insulin-like response of adipose cells is not entirely excluded. Acid (ph 1.6)-treated X. griseus protease, which lost 95% of its original proteolytic activity, has been shown to retain the full insulin-like activity of unmodified, native enzyme (6). Leakage of ultraviolet-absorbing materials from free adi- pocytes into incubation medium in the presence of native Xtreptomyces enzyme at higher concentration was found to be accompanied by a concomitant reduction in its insulin-like activity (6). If the insulin-like effects of proteases are due to their enzymatic activities, their actions on plasma membranes may be to initiate the transformation of the membrane from a laminated to a micellar (globular) configuration. The latter form has been proposed to result in the formation of interglobular channels, which thereby promote passage of solute (12, 13). A modification of either constituent of the membrane-lipoprotein complex, protein moiety by proteases or phospholipid by phospholipase C as suggested by Rodbell (3), could induce such a transformation. REFERENCES 1. KRAHL, M. E., The action of insulin on cells, Academic Press, New York, 1961, p HECHTER, O., AND LESTER, G., Recent Progr. Hormone Res., 16, 139 (1960). 3. RODBELL, M., J. Biol. Chem., 241, 130 (1966). 4. BLECHER; M., Biochem. Biopiys. Res. &mm&, 21, 202 (1965). 5. BLECHER, M., Biochem. Biophys. Res. Commun., 23, 68 (1966). 6. Kuo,J. F., HOLMLUND, C.E., DILL, I.K., AND BOHONOS, N., Arch. Biochem. Biophys., 117,269 (1966). 7. Kuo. J. F.. HOLMLUND. C. E.. AND DILL. I. K.., Life Sci.., &7 (1966). Kuo, J. F., DILL, I. K., AND HOLMLUND, C. E., Fed. Proc., 26, 858 (1967). RODBELL, M., J. Biol. Chem., 239, 373 (1964). FLATT, J. P., AND BALL, E. G., J. Biol. Chem., 239, 675 (1964). Kuo, J. F., DILL, I. K., AND HOLMLUND, C. E., Biochim. Biophys. Acta, in press. LUCY, J. A., J. Theor. Biol., 7, 360 (1964). SJ~STRAND, F. S., in G. H. BOURNE (Editor), Cytology and cell physiology, Academic Press, New York, 1964, p. 311.