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Cell protein degradation in cultured rat embryo fibroblasts suppression by vinblastine of the enhanced proteolysis by serum-deficient media
51i
Biochimica et Biophysica Acta, 451 (1976) 511--516 © Elsevier/North-Holland Biomedical Press
BBA 28114
CELL PROTEIN D E G R A D A T I O N IN C U L T U R E D RAT EMBRYO FIBROBLASTS SUPPRESSION BY VINBLASTINE OF THE ENHANCED PROTEOLYSIS BY SERUM-DEFICIENT MEDIA
J. S. AMENTA a, F. M. BACCINO b and M. J. SARGUS a
a Department o f Pathology, University o f Pittsburgh School o f Medicine, Pittsburgh, Pa. 15261 (U.S.A.) and b Instituto di Patologia Generale, Torino (Italy) (Received June 9th, 1976)
Summary Rat embryo fibroblasts grown in Eagle's minimal essential medium with 10% serum were labeled with L-[14C]leucine. After a 24 h cold chase, rates of proteolysis were evaluated by measuring the appearance of trichloroacetic acid-soluble 14C in the media. Cells remaining in minimal essential medium with 10% serum (basal) showed a proteolysis rate of 1% per h, whereas cells placed in minimal essential medium alone (serum-deficient) showed a stimulation of proteolysis to 3--4% per h. This enhanced proteolysis was transitory, occurring only for the first 4--8 h after cells were placed in the serum
The role of lysosomes in the general degradation of cell protein remains speculative. Bacteria, which do not contain lysosomes, offer an obvious example of protein turnover occurring through a non-lysosomal mechanism [1]. Alternatively, the degradation of cell proteins in eukaryotes can be inhibited with agents that both accumulate in lysosomes in high concentrations and inhibit at least one acid proteinase [2,3]. Experiments with liver perfusion have also provided support for a lysosomal role in cell proteolysis; perfusion conditions which increase proteolysis concomitantly activate cell autophagy [4].
512 Functioning microtubules appear to be necessary for optimal activity of the vacuolar system. Extracellular proteins can be incorporated into the vacuolar system of the cell by endocytosis, producing heterophagic vacuoles which, after fusing with lysosomes, hydrolyze the exogenous proteins. A number of workers have reported that, by affecting the directional flow of endocytic vacuoles [ 5], inhibitors of microtubules interfere with their merger with lysosomes [6,7] and thereby decrease the rate of degradation of the carried proteins [8--10] {although contrasting views have been expressed [11,12]). These studies suggested to us that microtubular inhibitors might have an effect in reducing the rate of hydrolysis of cell proteins, at least to the extent that the vacuolar system is involved in this process. The present report demonstrates that these inhibitors do inhibit the degradation of endogenous cell proteins, but only under conditions in which cell proteolysis has been previously augmented by a sudden change in medium composition. Methods Stock fibroblast cultures were derived from 18-day rat embryos, Fisher Strain F-344 (Hilltop Laboratories, Scottdale, Pa., U.S.A.). Fibroblasts subcultured from frozen stock cultures were grown in 75 cm: Falcon flasks in Eagle's minimum essential medium supplemented with 10% fetal calf serum. L-[~4C]Leucine (0.8 pCi/flask) was added 4 days prior to each experiment at the time of subculture. Cultures were then chased in fresh medium for 24 h prior to testing. On the day of the experiment, cultures were generally in a sub-confluent stage with 200-500 pg of protein per flask. For each experiment, cells were washed twice with phosphate-buffered saline and placed in 10 ml of fresh unlabeled medium containing either 10% serum (basal) or no serum (serum deficient). Vinblastine was added at concentrations of 10-5--10-~M as indicated in each experiment. At intervals of 0, 1, 2, 4 and 24 h, 0.5 ml aliquots of media were removed and proteins precipitated by adding 0.5 ml of cold 16% (w/v) trichloroacetic acid. Before trichloroacetic acid, 10 mg of bovine serum albumin were added to the aliquots taken from the serum-deficient media to precipitate the protein more effectively. After centrifugation, acid-soluble ~4C was measured in the clear supernatant by liquid scintillation counting. The acid-insoluble fraction was measured by determining the difference between the total radioactivity in an aliquot of media and the acid-soluble fraction. At the end of each experiment, the cell layers were washed twice with phosphate-buffered saline and solubilized in 4 ml of 0.1 M NaOH/0.4% (w/v) sodium desoxycholate. Total cell ~4C was determined on a 0.5 ml aliquot; acid-soluble ~4C was also determined on an aliquot precipitated with trichloroacetic acid as described for the serumdeficient medium. Total cell protein was determined by the method of Lowry et al. [13]. All results were reported as percentages of total radioactivity. Results Prelabeled rat embryo fibroblasts were incubated for four hours with 10 -s M vinblastine in both basal and serum-deficient media. When the incubation was
513 maintained in minimal essential medium with 10% serum (basal), vinblastine had no effect upon the rate of cell proteolysis, proteolysis averaging in these experiments 1% per hour. When cells were placed in minimal essential medium without added serum {serum-deficient), the rate of cell proteolysis in the 4 h period was stimulated three- to fourfold, achieving a rate of 4% per h in the experiment. Under serum-deficient conditions, 10-SM vinblastine had a marked effect, reducing proteolysis to a rate of 2.8% per h. Thus, vinblastine at this concentration effects a 40% decrease in the induced proteolysis. A lesser, b u t still definite, inhibitory effect was also noted with 10-6M vinblastine, while concentrations of 10 -7 M were ineffective. At concentrations higher than 10-SM vinblastine, toxic effects were manifested by loss of cells into the medium as well as increase in cell permeability to trypan blue. Studying the phenomenon over a 24 h period, we found the increase in proteolytic rate occurred primarily during the first few hours after the transition of the cells from basal to serum
EFFECT OF VINBLASTINE ON CELL PROTEOLYSIS 10% SERUM
0% SERUM
40OCONTROL eVB
820-
I0-
0
J
I"2
hrs.
° CONTROL
2'4
0
i2 hrs.
/
/
24
Fig. 1. E f f e c t o f v i n b l a s t i n e o n cell p r o t e o l y s i s . T r i c h l o r o a c e t i c a c i d - s o l u b l e 14C in m e d i a w i t h 10% a n d 0% s e r u m p l o t t e d against i n c u b a t i o n t i m e . e , v i n b l a s t i n e (VB) 1 0 -5 M a d d e d at 0 h. E a c h p o i n t r e p r e s e n t s t h e average o f a t l e a s t f o u r e x p e r i m e n t s .
514
ments were higher in serum
-3.2 -28
-24
i
20
16 ~
~8
12
08
-04
0
2 4 6 8 SERUM CONGENTRATION
10%
Fig. 2. F f f e c t of s e r u m c o n c e n t r a t i o n in m e d i u m on p r o t e o l y s i s and p r o t e i n loss f r o m ceils. I4C in m e d i a a f t e r 4 h i n c u b a t i o n in m i n i m a l essential m e d i u m with a d d e d s e r u m as i n d i c a t e d o n abscissa, t r i c h l o r o -
acetic acid-soluble 14C i n d i c a t e d on left o r d i n a t e , t r i c h l o r o a c e t i c acid-insoluble on r i g h t o r d i n a t e . Each p o i n t r e p r e s e n t s the m e a n of t w o e x p e r i m e n t s .
515
Additional data supporting the concept that the stimulated proteolysis induced by serum
Cell protein turnover clearly involves more than simple hydrolysis of peptide bonds. Continuing protein degradation in the cell requires energy [14] and, at least under some conditions, protein synthesis [14,15], while more recent studies have demonstrated that intact cellular structure is an important facet of protein turnover [ 16]. Undoubtedly autophagy involving lysosomes and other elements of the vacuolar system results in the hydrolysis of some cell protein. Unfortunately, most of the data on this phenomenon are based on morphologic studies and the quantitative significance of this process remains speculative. Mortimore and Mondin [17] using a liver perfusion system, have found an increase in proteolysis induced after the first 30 min of perfusion under the usual conditions. This induced proteolysis, associated with an increase in the number of autophagic vacuoles and secondary lysosomes, can "be largely inhibited by adding amino acids and insulin to the perfusion medium [4], and can be restored by adding glucagon. In these experiments the contribution of the induced degradation to overall protein turnover accounts for at least 50% of total protein degraded in the time intervals studied. Using hepatoma cells in cultures, Tomkins and co-workers [18,19] have observed a similar t w o - t o threefold augmentation of cell protein degradation under conditions of serum deficiency in the media. Morphologic studies on HeLa cells placed in a serumamino acid-deficient medium have demonstrated a striking increase in the number of autophagic vacuoles within 3 h [20]. We have extended the foregoing observations to non-neoplastic fibroblast cultures. Our results show that serum-deficient medium increases the rate of proteolysis two- to threefold over the 4 h interval and appears qualitatively and quantitatively similar to the induced proteolysis reported by other workers
516
[4,19--22]. We have also observed retraction of cytoplasmic processes and rounding of cells at the same time. Vinblastine, as well as vincristine and colchicine (unpublished data), partially inhibit both the serum-deficientinduced proteolysis and the morphologic changes. In view of the known effect of serum and amino acid deprivation on induced autophagy and increased proteolysis in cultured cells, it appears that at least under serum
G o l d b e r g , A . L . and Dice. J . F . ( 1 9 7 4 ) A n n . Rev. B i o c h e m . 4 3 , 8 3 5 - - 8 6 9 W i b o , M. a n d P o o l e , B. ( 1 9 7 4 ) J. Cell Biol. 6 3 , 4 3 0 - - 4 4 0 Dean, R.T. (1975) Nature 257,414--416 N e e l y , A . N . , N e l s o n , P.B. a n d M o r t i m o r e , G . E . ( 1 9 7 4 ) B i o c h i m . Biophys. Acta 3 3 8 , 4 5 8 - - 4 7 2 S i l v e r b l a t t , F . J . , T y s o n , G . E . and Bulger, R . E . ( 1 9 7 4 ) Lab Invest. 3 1 , 1 7 0 - - 1 8 0 M a l a w i s t a , S.E, ( 1 9 7 1 ) B l o o d 3 7 , 5 1 9 - - 5 2 9 M a l a w i s t a , S.E. ( 1 9 7 5 ) A n n . N.Y. A c a d . Sci. 2 5 3 , 7 3 8 - - 7 4 3 E k h o l m , R., E r i c s o n , L . E . , J o s e f s s o n , J . - O . and Melander, A. ( 1 9 7 4 ) E n d o c r i n o l o g y 9 4 , 6 4 1 - - 6 4 9 G o r d o n , A . H . ( 1 9 7 3 ) in L y s o s o m e s in Biology and Pathology ( D i n g l e , J . T . a n d Fell, H., eds.), Part 3, p p . 8 9 - - 1 3 7 , N o r t h - H o l l a n d P u b l i s h i n g Co., A m s t e r d a m W e i s s m a n n , G., D u k o r , P. and Sessa, G. ( 1 9 7 1 ) in I m m u n o p a t h o l o g y of I n f l a m m a t i o n (Forscher, B.K. a n d H o u c k , J . C . , eds.), p p . 1 0 7 - - 1 1 7 , E x c e r p t a M e d i c a , Amsterdam P e s a n t i , E.L. a n d A x l l n e , S.G. ( 1 9 7 5 ) J. E x p . Meal. 1 4 2 , 9 0 3 - - 9 1 3 B h i s e y , A . N . a n d F r e e d , J . J . ( 1 9 7 1 ) E x p t l . Cell Res. 6 4 , 4 3 0 - - 4 3 8 L o w r y , D . H . , R o s e b r o u g h , N.J., F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 S i m p s o n , M.V, ( 1 9 5 3 ) J. Biol. C h e m . 2 0 1 , 1 4 3 - - 1 5 4 S t e i n b e r g , D. and V a u g h a n , M. ( 1 9 5 6 ) A r c h . B i o c h e m . B i o p h y s . 6 5 , 9 5 - - 1 6 5 B r o s t r o m , C.O. and Jeffay, H. ( 1 9 7 0 ) J. Biol. C h e m . 2 4 5 , 4 0 0 1 - - 4 0 0 8 M o r t i m o r e , G . E . a n d M o n d i n , C.E. ( 1 9 7 0 ) J. Biol. C h e m . 2 4 5 , 2 3 7 5 - - 2 3 8 3 A u r i e c h i o , F., M a r t i n , J r . , D. a n d T o m k i n s , G. ( 1 9 6 9 ) N a t u r e 2 4 4 , 8 0 6 - - 8 0 8 H e r s h k o , A. a n d T o m k i n s , G.M. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 7 1 0 - - 7 1 4 M i t c h e n e r , J.S., S h e l b u r n e , J . D . , B r a d f o r d , W.D. and Hawkins, H . K . ( 1 9 7 6 ) A m . J. of P a t h . 8 3 , 4 8 5 - 491 P o o l e , B. a n d W i b o , M. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 6 2 2 1 - - 6 2 2 6 Cast(Jr, L . N . ( 1 9 7 5 ) J. Cell Biol. 6 7 , 5 8 a
Biochimica et Biophysica Acta, 451 (1976) 511--516 © Elsevier/North-Holland Biomedical Press
BBA 28114
CELL PROTEIN D E G R A D A T I O N IN C U L T U R E D RAT EMBRYO FIBROBLASTS SUPPRESSION BY VINBLASTINE OF THE ENHANCED PROTEOLYSIS BY SERUM-DEFICIENT MEDIA
J. S. AMENTA a, F. M. BACCINO b and M. J. SARGUS a
a Department o f Pathology, University o f Pittsburgh School o f Medicine, Pittsburgh, Pa. 15261 (U.S.A.) and b Instituto di Patologia Generale, Torino (Italy) (Received June 9th, 1976)
Summary Rat embryo fibroblasts grown in Eagle's minimal essential medium with 10% serum were labeled with L-[14C]leucine. After a 24 h cold chase, rates of proteolysis were evaluated by measuring the appearance of trichloroacetic acid-soluble 14C in the media. Cells remaining in minimal essential medium with 10% serum (basal) showed a proteolysis rate of 1% per h, whereas cells placed in minimal essential medium alone (serum-deficient) showed a stimulation of proteolysis to 3--4% per h. This enhanced proteolysis was transitory, occurring only for the first 4--8 h after cells were placed in the serum
The role of lysosomes in the general degradation of cell protein remains speculative. Bacteria, which do not contain lysosomes, offer an obvious example of protein turnover occurring through a non-lysosomal mechanism [1]. Alternatively, the degradation of cell proteins in eukaryotes can be inhibited with agents that both accumulate in lysosomes in high concentrations and inhibit at least one acid proteinase [2,3]. Experiments with liver perfusion have also provided support for a lysosomal role in cell proteolysis; perfusion conditions which increase proteolysis concomitantly activate cell autophagy [4].
512 Functioning microtubules appear to be necessary for optimal activity of the vacuolar system. Extracellular proteins can be incorporated into the vacuolar system of the cell by endocytosis, producing heterophagic vacuoles which, after fusing with lysosomes, hydrolyze the exogenous proteins. A number of workers have reported that, by affecting the directional flow of endocytic vacuoles [ 5], inhibitors of microtubules interfere with their merger with lysosomes [6,7] and thereby decrease the rate of degradation of the carried proteins [8--10] {although contrasting views have been expressed [11,12]). These studies suggested to us that microtubular inhibitors might have an effect in reducing the rate of hydrolysis of cell proteins, at least to the extent that the vacuolar system is involved in this process. The present report demonstrates that these inhibitors do inhibit the degradation of endogenous cell proteins, but only under conditions in which cell proteolysis has been previously augmented by a sudden change in medium composition. Methods Stock fibroblast cultures were derived from 18-day rat embryos, Fisher Strain F-344 (Hilltop Laboratories, Scottdale, Pa., U.S.A.). Fibroblasts subcultured from frozen stock cultures were grown in 75 cm: Falcon flasks in Eagle's minimum essential medium supplemented with 10% fetal calf serum. L-[~4C]Leucine (0.8 pCi/flask) was added 4 days prior to each experiment at the time of subculture. Cultures were then chased in fresh medium for 24 h prior to testing. On the day of the experiment, cultures were generally in a sub-confluent stage with 200-500 pg of protein per flask. For each experiment, cells were washed twice with phosphate-buffered saline and placed in 10 ml of fresh unlabeled medium containing either 10% serum (basal) or no serum (serum deficient). Vinblastine was added at concentrations of 10-5--10-~M as indicated in each experiment. At intervals of 0, 1, 2, 4 and 24 h, 0.5 ml aliquots of media were removed and proteins precipitated by adding 0.5 ml of cold 16% (w/v) trichloroacetic acid. Before trichloroacetic acid, 10 mg of bovine serum albumin were added to the aliquots taken from the serum-deficient media to precipitate the protein more effectively. After centrifugation, acid-soluble ~4C was measured in the clear supernatant by liquid scintillation counting. The acid-insoluble fraction was measured by determining the difference between the total radioactivity in an aliquot of media and the acid-soluble fraction. At the end of each experiment, the cell layers were washed twice with phosphate-buffered saline and solubilized in 4 ml of 0.1 M NaOH/0.4% (w/v) sodium desoxycholate. Total cell ~4C was determined on a 0.5 ml aliquot; acid-soluble ~4C was also determined on an aliquot precipitated with trichloroacetic acid as described for the serumdeficient medium. Total cell protein was determined by the method of Lowry et al. [13]. All results were reported as percentages of total radioactivity. Results Prelabeled rat embryo fibroblasts were incubated for four hours with 10 -s M vinblastine in both basal and serum-deficient media. When the incubation was
513 maintained in minimal essential medium with 10% serum (basal), vinblastine had no effect upon the rate of cell proteolysis, proteolysis averaging in these experiments 1% per hour. When cells were placed in minimal essential medium without added serum {serum-deficient), the rate of cell proteolysis in the 4 h period was stimulated three- to fourfold, achieving a rate of 4% per h in the experiment. Under serum-deficient conditions, 10-SM vinblastine had a marked effect, reducing proteolysis to a rate of 2.8% per h. Thus, vinblastine at this concentration effects a 40% decrease in the induced proteolysis. A lesser, b u t still definite, inhibitory effect was also noted with 10-6M vinblastine, while concentrations of 10 -7 M were ineffective. At concentrations higher than 10-SM vinblastine, toxic effects were manifested by loss of cells into the medium as well as increase in cell permeability to trypan blue. Studying the phenomenon over a 24 h period, we found the increase in proteolytic rate occurred primarily during the first few hours after the transition of the cells from basal to serum
EFFECT OF VINBLASTINE ON CELL PROTEOLYSIS 10% SERUM
0% SERUM
40OCONTROL eVB
820-
I0-
0
J
I"2
hrs.
° CONTROL
2'4
0
i2 hrs.
/
/
24
Fig. 1. E f f e c t o f v i n b l a s t i n e o n cell p r o t e o l y s i s . T r i c h l o r o a c e t i c a c i d - s o l u b l e 14C in m e d i a w i t h 10% a n d 0% s e r u m p l o t t e d against i n c u b a t i o n t i m e . e , v i n b l a s t i n e (VB) 1 0 -5 M a d d e d at 0 h. E a c h p o i n t r e p r e s e n t s t h e average o f a t l e a s t f o u r e x p e r i m e n t s .
514
ments were higher in serum
-3.2 -28
-24
i
20
16 ~
~8
12
08
-04
0
2 4 6 8 SERUM CONGENTRATION
10%
Fig. 2. F f f e c t of s e r u m c o n c e n t r a t i o n in m e d i u m on p r o t e o l y s i s and p r o t e i n loss f r o m ceils. I4C in m e d i a a f t e r 4 h i n c u b a t i o n in m i n i m a l essential m e d i u m with a d d e d s e r u m as i n d i c a t e d o n abscissa, t r i c h l o r o -
acetic acid-soluble 14C i n d i c a t e d on left o r d i n a t e , t r i c h l o r o a c e t i c acid-insoluble on r i g h t o r d i n a t e . Each p o i n t r e p r e s e n t s the m e a n of t w o e x p e r i m e n t s .
515
Additional data supporting the concept that the stimulated proteolysis induced by serum
Cell protein turnover clearly involves more than simple hydrolysis of peptide bonds. Continuing protein degradation in the cell requires energy [14] and, at least under some conditions, protein synthesis [14,15], while more recent studies have demonstrated that intact cellular structure is an important facet of protein turnover [ 16]. Undoubtedly autophagy involving lysosomes and other elements of the vacuolar system results in the hydrolysis of some cell protein. Unfortunately, most of the data on this phenomenon are based on morphologic studies and the quantitative significance of this process remains speculative. Mortimore and Mondin [17] using a liver perfusion system, have found an increase in proteolysis induced after the first 30 min of perfusion under the usual conditions. This induced proteolysis, associated with an increase in the number of autophagic vacuoles and secondary lysosomes, can "be largely inhibited by adding amino acids and insulin to the perfusion medium [4], and can be restored by adding glucagon. In these experiments the contribution of the induced degradation to overall protein turnover accounts for at least 50% of total protein degraded in the time intervals studied. Using hepatoma cells in cultures, Tomkins and co-workers [18,19] have observed a similar t w o - t o threefold augmentation of cell protein degradation under conditions of serum deficiency in the media. Morphologic studies on HeLa cells placed in a serumamino acid-deficient medium have demonstrated a striking increase in the number of autophagic vacuoles within 3 h [20]. We have extended the foregoing observations to non-neoplastic fibroblast cultures. Our results show that serum-deficient medium increases the rate of proteolysis two- to threefold over the 4 h interval and appears qualitatively and quantitatively similar to the induced proteolysis reported by other workers
516
[4,19--22]. We have also observed retraction of cytoplasmic processes and rounding of cells at the same time. Vinblastine, as well as vincristine and colchicine (unpublished data), partially inhibit both the serum-deficientinduced proteolysis and the morphologic changes. In view of the known effect of serum and amino acid deprivation on induced autophagy and increased proteolysis in cultured cells, it appears that at least under serum
G o l d b e r g , A . L . and Dice. J . F . ( 1 9 7 4 ) A n n . Rev. B i o c h e m . 4 3 , 8 3 5 - - 8 6 9 W i b o , M. a n d P o o l e , B. ( 1 9 7 4 ) J. Cell Biol. 6 3 , 4 3 0 - - 4 4 0 Dean, R.T. (1975) Nature 257,414--416 N e e l y , A . N . , N e l s o n , P.B. a n d M o r t i m o r e , G . E . ( 1 9 7 4 ) B i o c h i m . Biophys. Acta 3 3 8 , 4 5 8 - - 4 7 2 S i l v e r b l a t t , F . J . , T y s o n , G . E . and Bulger, R . E . ( 1 9 7 4 ) Lab Invest. 3 1 , 1 7 0 - - 1 8 0 M a l a w i s t a , S.E, ( 1 9 7 1 ) B l o o d 3 7 , 5 1 9 - - 5 2 9 M a l a w i s t a , S.E. ( 1 9 7 5 ) A n n . N.Y. A c a d . Sci. 2 5 3 , 7 3 8 - - 7 4 3 E k h o l m , R., E r i c s o n , L . E . , J o s e f s s o n , J . - O . and Melander, A. ( 1 9 7 4 ) E n d o c r i n o l o g y 9 4 , 6 4 1 - - 6 4 9 G o r d o n , A . H . ( 1 9 7 3 ) in L y s o s o m e s in Biology and Pathology ( D i n g l e , J . T . a n d Fell, H., eds.), Part 3, p p . 8 9 - - 1 3 7 , N o r t h - H o l l a n d P u b l i s h i n g Co., A m s t e r d a m W e i s s m a n n , G., D u k o r , P. and Sessa, G. ( 1 9 7 1 ) in I m m u n o p a t h o l o g y of I n f l a m m a t i o n (Forscher, B.K. a n d H o u c k , J . C . , eds.), p p . 1 0 7 - - 1 1 7 , E x c e r p t a M e d i c a , Amsterdam P e s a n t i , E.L. a n d A x l l n e , S.G. ( 1 9 7 5 ) J. E x p . Meal. 1 4 2 , 9 0 3 - - 9 1 3 B h i s e y , A . N . a n d F r e e d , J . J . ( 1 9 7 1 ) E x p t l . Cell Res. 6 4 , 4 3 0 - - 4 3 8 L o w r y , D . H . , R o s e b r o u g h , N.J., F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 S i m p s o n , M.V, ( 1 9 5 3 ) J. Biol. C h e m . 2 0 1 , 1 4 3 - - 1 5 4 S t e i n b e r g , D. and V a u g h a n , M. ( 1 9 5 6 ) A r c h . B i o c h e m . B i o p h y s . 6 5 , 9 5 - - 1 6 5 B r o s t r o m , C.O. and Jeffay, H. ( 1 9 7 0 ) J. Biol. C h e m . 2 4 5 , 4 0 0 1 - - 4 0 0 8 M o r t i m o r e , G . E . a n d M o n d i n , C.E. ( 1 9 7 0 ) J. Biol. C h e m . 2 4 5 , 2 3 7 5 - - 2 3 8 3 A u r i e c h i o , F., M a r t i n , J r . , D. a n d T o m k i n s , G. ( 1 9 6 9 ) N a t u r e 2 4 4 , 8 0 6 - - 8 0 8 H e r s h k o , A. a n d T o m k i n s , G.M. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 7 1 0 - - 7 1 4 M i t c h e n e r , J.S., S h e l b u r n e , J . D . , B r a d f o r d , W.D. and Hawkins, H . K . ( 1 9 7 6 ) A m . J. of P a t h . 8 3 , 4 8 5 - 491 P o o l e , B. a n d W i b o , M. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 6 2 2 1 - - 6 2 2 6 Cast(Jr, L . N . ( 1 9 7 5 ) J. Cell Biol. 6 7 , 5 8 a