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The mechanism of enzyme secretion by the cell
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS ~-04, 6 7 - 7 2
(1964)
The Mechanism of Enzyme Secretion by the Cell III. Intermediate Stages in Amylase Transport as Revealed by Pulse Labeling of Slices of Parotid Gland I M I C H A E L S C H R A M M AND A V N E R B D O L A H From the Department of Biological Chemistry, The Hebrew University, Jerusalem, Israel
Received June 3, 1963 Rat parotid gland slices incubated for 5 minutes with C14-amino acids incorporated radioactivity mainly into exportable proteins such as amylase. Excess labeled amino acids were effectively removed. It was thus possible to study the subeellular distribution, transport, and secretion of a constant amount of radioactive amylase. The specific radioactivity of amylase was initially highest in the microsomes and lowest in the zymogen granules. Two intermediate fractions, sedimented at 15,000g and 4000g, showed a specific amylase radioactivity many times higher than that of the zymogen granules. These two fractions contained a relatively large proportion of the newly synthesized labeled amylase. It is therefore suggested that the intermediate fractions represent transitory steps in the development of the zymogen granule. Amylase secreted by the labeled slice was at first of low specific radioactivity which rose steeply with time to a maximum. Within 3 hours the slice secreted about 70% of the labeled protein and amylase. During secretion the specific amylase radioactivity declined steeply in the microsomes and in the 15,000g fraction but increased in the 4000g fraction and in the zymogen granules. INTRODUCTION The studies on chymotrypsinogen (1) and R N a s e (2) in the pancreas and on a-amylase in the parotid (3) strongly indicate t h a t these exportable proteins are synthesized in the ribosomes and are finally accumulated in the zymogen granules. However, the intermediate stages in the transport of these enzymes between the microsomes and the fully packed zymogen granules remain essentially unidentified. I t would seem most likely t h a t the zymogen granule passes several stages of maturation, b u t these have not been isolated as yet. Whether the exportable proteins pass through a soluble phase before accumulation in the membrane bound granules is also not This work was supported by grants from the National Science Foundation (G-22153) and the National Institutes of Health, Public Health Service (AI-03426-03).
established. Furthermore, one wonders whether the exocrine gland cell maintains rigid order in the sequence of transport so t h a t enzyme is not secreted before it has reached the zymogen granules. These problems had been previously approached by studying the distribution of radioactive enzyme in subcellular fractions of the glands after injecting C14-amino acids into whole animals (1-3). I t seemed to us t h a t slices of rat parotid gland, demonstrating specific secretion of enzymes in a well-defined medium (4), would be a more suitable system for the following reasons: I t is possible to label the slice for a short period and then remove excess C1~-amino acids so t h a t the transport of a constant a m o u n t of Clt-protein can be subsequently followed. The secretion of labeled enzyme into the medium is readily measured in con]unction with the changes in its subcellular distribu67
68
SCHRAMM AND BDOLAH
tion. T h e effect of m e d i u m c o m p o n e n t s a n d s t i m u l a n t s of secretion on t h e r a t e of C 14 i n c o r p o r a t i o n into e x p o r t a b l e e n z y m e a n d on its subcellular d i s t r i b u t i o n is also a m e n a b l e to i n v e s t i g a t i o n . T h e p r e s e n t c o m m u n i c a t i o n p r e s e n t s evidence to i n d i c a t e t h a t i n t e r m e d i a t e stages in the d e v e l o p m e n t o f t h e z y m o g e n g r a n u le do exist a n d can be isolated. I t is f u r t h e r show n t h a t a m y l a s e a l r e a d y p r e s e n t in t h e gla nd slice prior to labeling is secreted prefere n t i a l l y before t h e n e w l y s y n t h e s i z e d radioa c t i v e e n z y m e g r a d u a l l y a p p e a r s in th e medium. MATERIALS AND METHODS Rat parotid glands were obtained from animals starved for 24 hours (5). The preparation and incubation of slices as well as the methods for study of enzyme secretion were described in a preceding paper (4). PROCEDURE FOR LABELING OF PAROTID GLAND SLICES Slices were incubated for 5 minutes in a KrebsRinger-bicarbonate (KRB) medium which contained 5 mM sodium pyruvate and 6 #g. per milliliter uniformly labeled C14-protein hydrolysate (300 ~c. per milligram). To stop incorporation of labeled amino acids, slices were washed with KRB medium on filter paper in a Buchner funnel and subsequently incubated with a large excess of nonradioactive protein hydrolysate. Incubation was for 15 minutes in a KRB medium containing 2 mg. per milliliter casein hydrolysate, 30 gg. per milliliter L-tryptophan, and 5 mM sodium pyruvate. The medium was removed by filtration and the slices were washed as above. When the slices were used subsequently to study the secretion of labeled proteins, the incubation was carried out in the KRB- casein hydrolysate medium described above. Further incorporation of the small amount of labeled amino acids still remaining in the slice could thus be inhibited. Although incubation with amino acids was often continued for 3 hours, the net synthesis of amylase did not exceed 10% of the amount present at zero time. Secretion of radioactive enzyme could thus be conveniently Studied in the absence of appreciable changes in the total amount of enzyme in the system.
Triton x-100. After 5 minutes the suspension was further diluted in 0.02 M phosphate buffer, pH 6.9, to yield a protein concentration of 0.5-2 mg. per milliliter. Triton was not added to soluble fractions. Protein was precipitated by addition of one volume of 10% triehloroacetic acid. The suspension was kept in ice for 15 minutes; the precipitate was then isolated by centrifugation at room temperature and washed once with 5% trichloroacetie acid. The precipitate was suspended in 90% formic acid, transferred to a planehet, dried, and counted. Control experiments showed that further extractions of the trichloroacetic acid precipitate by hot trichloroacetic acid and organic solvents did not detectably reduce the specific radioactivity of the protein. To isolate amylase, an aliquot of 1 ml. containing 500 enzyme units was fractionated with ethanol and the enzyme was specifically precipitated with glycogen (6). The washed enzymeglycogen complex was dissolved in 1 ml. 0.02 M phosphate buffer, pH 6.9, containing 7 mM NaC1. An aliquot was transferred to a planchet, dried, and counted. Enzyme activity and protein content of the purified amylase were also determined. The enzyme isolated as the glycogen-amylase complex was obtained with an over-all yield of about 70%. The specific enzymic activity was 2000-2500 units per milligram protein, which is about equal to that of the crystalline enzyme (6). Subcellular fractions such as the microsomes were poor in amylase (5); nonradioactive enzyme was therefore added before ethanol fractionation. If a large excess of pure amylase were added as carrier, the specific enzymic activity of the mixture would be very high even before purification and one could not test whether foreign labeled protein would indeed be removed by the purification procedure. It therefore seemed preferable to use as carrier a fraction rich in amylase rathel than pure enzyme. To this purpose the contentE of zymogen granules (5) was chosen as the carrier. Its specific amylase activity was about 800 unit,~ per milligram protein. An aliquot of the radioac rive fraction containing a known amount of amy lase units was supplemented with nonradioactive zymogen granule contents to give a final concentration of 500 enzyme units per milliliter. In the most extreme cases 480 units carrier were adde( to 20 units of the radioactive fraction. On th( basis of protein, however, the carrier representee about 50% of the mixture.
ISOLATION OF PROTEIN AND AMYLASE FOR MEASUREMENTS OF RADIOACTIVITY
CALCULATIONS OF RADIOACTIVITY
The washed pellets of subcellular fractions were suspended in a minimal volume of 0.1%
The specific radioactivity of amylase is ex pressed as counts/min./mg, enzyme protein an(
INTERMEDIATE
STAGES IN AMYLASE TRANSPORT
is corrected according to t h e dilution factor when carrier amylase was added. T o t a l counts ia t h e amylase of a n y fraction are calculated from t h e counts found in the purified enzyme corrected for losses during purification. Corrections for self-absorption were unnecessary because of t h e small weight of t h e dried sample. A t h i n window gas flow counter (Nuclear Chicago) was used. ~SOLATION OF SUBCELLULAR FRACTIONS P r e p a r a t i o n of the homogenate and differential c e n t r i f u g a t i o n were carried out essentially as described previously (4, 5). T h e centrifugal force a n d centrifugation time were varied according to t h e specific requirements of the experiment and are given u n d e r Results. MATERIALS Uniformly labeled Cl~-a/gal protein hydrolysate was obtained from t h e Radiochemical Centre, Amersham, E n g l a n d . Casein hydrolysate (by hydrochloric acid, salt-free) was a p r o d u c t of M a n n Research Laboratories. O t h e r chemicals were commercial preparations of highest p u r i t y available. RESULTS
When slices were labeled for 5 minutes with C14-protein hydrolysate and subsequently washed free of excess radioactive n~terial, 80 % of the counts were found in protein. It was to be expected that within
69
short periods of labeling, incorporation would be mainly confined to those proteins which are produced in large amounts for secretion. It was indeed found that about 35 % of the protein counts were located in a-amylase, an enzyme which had been calculated to represent roughly 40 % of the secretory proteins of the parotid gland (5). As shown in Table I the ratio of C ~4 in amylase to C ~4in total protein varies for all subcellular fractions only in the range of 23-40%. This observation is again in line with the assumption that incorporation was mainly limited to exportable proteins. It is highly significant that a relatively large portion of the counts in protein and amylase were located in the fractions between the heavy zymogen granules (1000 g) and the microsomes (105,000 g). Moreover, these intermediate fractions had a specific amylase radioactivity many times higher than that of the typical zymogen granules. It would therefore seem most likely that the fractions sedimented at 4000 and 16,000 g contain immature zymogen granules, partly packed with highly labeled radioactive amylase which had been released by the microsomes. Table I further shows that the final supernatant, while containing the largest relative amount of total counts, has a specific
TABLE I DISTRIBUTION OF C14-PRoTEIN AND C14-AMYLASE IN THE SLICE LABELED WITH AMINO ACIDSa Fraction Centrifugal force (g.) I II III IV V Supernatant
250 1000 4000 15,000 105,000
Relative disCI4 in amylase Specific activityradioof Durationof tribution of amy- Relative distribution of as per cent amylase centrifugation lase activity b C14Pr~ of C14in protein (counts/ (rain.) (%) (%) min,/mg.) 5 l0 15 15 30
7.0 21.0 3.0 0.7 0.5 72.0
2.5 7.0 1 3 . 0 + (3.9) 8.5 + (7.9) 8 . 0 + (1.6) 30.0
23 29 30 40 30 33
1370 1900 26,000 87,000 100,000 2200
Slices from 6 glands were labeled, washed, a n d i n c u b a t e d for 15 m i n u t e s to remove radioactive amino acids as described u n d e r Materials a n d Methods. T h e homogenate s u b s e q u e n t l y prepared in 0.25 M sucrose contained 35 mg. protein, 22,000 amylase units, and 164,000 counts per m i n u t e in protein. T h e outline of fractionation is given in the table. All fractions were washed once. The s u p e r n a t a n t a f t e r washing fraction I was added to t h e rest of t h e homogenate. The s u p e r n a t a n t s o b t a i n e d a f t e r washing fractions I I , I l I , a n d IV c o n t a i n e d a relatively large a m o u n t of r a d i o a c t i v e p r o t e i n which was almost completely sedimented by c e n t r i f u g a t i o n at 105,000g for 30 minutes. T h e values of p r o t e i n counts in these s e d i m e n t s are w r i t t e n in b r a c k e t s alongside those of t h e next heavier fraction. b T h e a m o u n t in the t o t a l homogenate is defined as 100k.
70
SCHRAMM AND BDOLAH
granule fractions I 4- II and III was at the end of incubation higher than at zero time. a~.~o 432 Moreover, the specific amylase radioactivity of the microsomal fraction V which was ,, .% highest at zero time (100 times higher than fractions I 4- II and 10 times higher than ~.~c~ It" ," -o ~-12o o~ / / ," -12~, t fraction III) dropped at the end of 3 hours .~ ~ / /r . ~ ~ incubation to a value below that of fractions IV and III. A comparison of the specific radioactivities ~ / // ~ -I8 ~. .__.~ / g," .~ ~ / E of amylase with those of total protein shows .~ looc , / / # "~,/4 < that the latter are much lower in most subcellular fractions, both at zero time and at . . 60. . . . 120 0 J,-' O 180 0 ._]oZ the end of 3 hours. In contrast, total protein Time f rnin) secreted into the extracellular medium had FIG. l. Secretion of radioactive amylase after pulse labeling of the slice. The data were obtained almost the same specific radioactivity as the from the same experiment as described in Tables amylase secreted (Table II). One could II and III. Zero minutes designates addition of therefore assume that exportable proteins epinephrine (marked with arrow). 9 specific are all synthesized and secreted at similar radioactivity of amylase; O, total counts/rain high rates while constitutive proteins of the in amylase; ~, amylase units. The experimental subcellular fractions are produced at a low points indicate the time at which slices were rate and consequently have a low specific transferred to fresh medium. It should be noted radioactivity. During the last hour of that the values for specific amylase radioactivity incubation the specific radioactivity of the represent mean values for the enzyme secreted amylase secreted is already declining and is during each incubation interval. much lower than that still remaining in the subcellular fractions I I I - - V (Table II). amylase radioactivity which is many times These findings suggest that individual cells lower than that of the intermediate fractions in the slice do not act in complete synIII and IV. When labeled slices were incubated in chronization (cf. ref. 8). Some of the cells KRB medium and stimulated by epineph- might rapidly synthesize radioactive enzyme rine, the amylase secreted initially had a but secrete it only sluggishly during subselow specific radioactivity, which rose steeply quent incubation periods. Table III shows the decrease of amylase with time to a maximum and then started to fall off (Fig. 1). It is also demonstrated activity, radioactive amylase, and total that the maximal secretion rate of total labeled protein in the subcellular fractions enzyme is attained during the first 30 min- during the 3 hour period of secretion by the utes after epinephrine addition, whereas slice. About 70 % of the amylase and labeled radioactive enzyme is secreted at a maximal protein were secreted during the experiment. rate only later, during the period of 30-60 The relative amounts of labeled amylase minutes incubation (cf. ref. 7). It appears and labeled protein remaining in each fracmost likely that nonradioactive amylase tion after 3 hours are quite similar, with the which was stored in the zymogen granules exception of the microsomal fraction V. before labeling of the gland is secreted first Table III further demonstrates that fracand that newly formed radioactive enzyme tions IV and V retained 76 % of their initial cannot be exported directly after being amylase activity although only 12 and 11%, released from the microsomes. These conclu- respectively, of the radioactive enzyme sions are further supported by the results remained. Apparently the labeled enzyme presented in Table II. The specific radio- present in these fractions at zero time was activity of amylase in fractions V (micro- transported to the heavier zymogen granule somal) and IV decreased by about 80% fractions while being replaced by newly during 3 hours, while that of the zymogen formed amylase, synthesized from the non,
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INTERMEDIATE STAGES IN AMYLASE TRANSPORT
71
TABLE II S 'ECIFIC RADIOACTIVITY OF AMYLASE AND PROTEIN
D U R I N G S E C R E T I O N BY T H E S L I C E a
Subcellular fractions
Protein secreted in incubation medium
Counts/min./mg. a m y l a s e
Homogenate I -4- II III IV V Supernatant
Counts/min./mg. total protein
At 0 time
After 3 hr.
At 0 time
After 3 hr.
-940 10,300 85,000 90,000 1170
-2700 12,500 15,000 11,500 1700
1600 1260 3100 5300 2000 960
1440 1300 1460 1120 1200 1390
Incubation interval (rain.)
Counts/ min./mg, amylase
Counts/ min./mg. total protein
0-30 30~}0 60-120 120-180
1200 4200 5900 4200
1300 3400 4500 3500
a Slices from 18 glands were labeled and washed. After 15 minutes incubation with excess nonradioactive amino acids, a third of the slices was homogenized and fractionated to determine values before addition of epinephrine (0 time). The other portion of slices was incubated in medium containing the stimulant. The slices were transferred to fresh medium at the end of each incubation interval indicated in the table. The used media were saved for analysis of secreted proteins. At the end of 3 hours, slices were homogenized and fractionated. The designation of subcellular fractions is identical with that given in Table 1. However, the first fraction was isolated by centrifugation at 1000g for 10 minutes. Values for total amylase activity and radioactivity are given in Table III. TABLE I I I CHANGES
IN DISTRIBUTION
AND T O T A L A M O U N T OF C 1 4 - A M Y L A S E AND C 1 4 - P R O T E I N
DURING
SECRETION a Amylase activity At 0 time (units)
Homogenate I + II III IV V Supernatant
17,700 5100 670 100 65 11,300
C la in protein
C ~4 in amylase
After 3 hr. seAfter 3 hr. seAfter 3 hr. secretion (as % of At 0 time cretion (as % of At 0 time cretion (as % of amount in (counts/rain.) counts in (counts/rain.) counts in fraction at fraction at fraction at 0 time) 0 time) 0 time)
32 7 19 76 76 40
56,000 10,100 10,500 9000 4200 19,200
36 17 17 19 45 56
19,290b 2300 3500 4300 2300 6900
30b 19 23 12 11 58
a Data in the table were obtained from the experiment described under Table II. The values after 3 hours secretion were calculated for the same amount of slices as used at zero time. b Amylase was not isolated from the homogenate. The value given represents the arithmetical sum of individual fractions. radioactive a m i n o acids of the i n c u b a t i o n medium. DISCUSSION T h e work presented shows t h a t slices of r a t p a r o t i d g l a n d readily i n c o r p o r a t e labeled a m i n o acids into a m y l a s e a n d other proteins. T h e highest specific r a d i o a c t i v i t y is i n i t i a l l y f o u n d in the microsomal fraction. T h i s f r a c t i o n also shows the m o s t precipitous decline in specific a m y l a s e r a d i o a c t i v i t y with time, d u r i n g secretion of the enzyme.
T h e r e is therefore little d o u b t t h a t a m y l a s e a n d other exportable p r o t e i n s are s y n t h e sized in the ribosomes, as a l r e a d y s h o w n b y o t h e r workers s t u d y i n g the d i s t r i b u t i o n of labeled e n z y m e s after i n j e c t i n g CI4-amino acids into whole a n i m a l s (1-3). I t m i g h t r e a s o n a b l y be a s s u m e d t h a t there are several i n t e r m e d i a t e stages b e t w e e n the release of a m y l a s e from the ribosomes a n d the final a c c u m u l a t i o n of the e n z y m e in the m a t u r e z y m o g e n granules. While there is some s u p p o r t for this a s s u m p t i o n from
72
SCHRAMM AND BDOLAH
electron microscope studies (8, 9), direct evidence has been lacking. In the present work, subcellular fractions which show characteristics expected of intermediate stages in the formation of zymogen granules were successfully isolated. These fractions contained a high concentration of radioactive amylase after pulse labeling. The centrifugal force and centrifugation time required also indicate that the fractions might contain amylase-bearing particles which are different from microsomes or mature zymogen granules. The stage in the sequence of transport in which enzyme accumulates in the soluble fraction is less clear. Previous studies suggested that the soluble fraction represents enzyme released from the microsomes to be packed into zymogen granules (3, 10). Our results show, however, that the specific radioactivity of amylase in the soluble fraction is many times lower than that of the intermediate granule fractions. Yet the supernatant contained the largest absolute amount of labeled amylase found in any single fraction. It would seem quite possible that the enzyme isolated as the supernatant fraction represents an artificial mixture of highly labeled enzyme released from the microsomes and a large amount of nonradioactive enzyme already secreted by the zymogen granules and partly present in the ducts of the gland at the time of labeling. Part of the radioactivity in the supernatant might also have been contributed by the highly labeled particulate fractions damaged during isolation. It should be emphasized that the parotid gland slice used in these studies was obtained from 24-hour starved animals and was
therefore fully loaded with exportable enzymes. The rate of enzyme synthesis and transport in such a gland preparation might be much slower than in a gland depleted of its exportable proteins before labeling. It is therefore possible that intermediate stages in enzyme transport could not be as readily distinguished in previous studies employing animals depleted of exportable proteins prior to injection of labeled amino acids
(1-3). ACKNOWLEDGMENT The authors are most grateful to Mrs. Sarah Eldar for her excellent assistance. REFERENCES 1. SIEKEVITZ,P., AND PALADE,G. E., J. Biophys, Biochem. Cytol. 7, 619 (1960). 2. MORRIS, A. J., AND DICMAN, S. R., J. Biol. Chem. 235, 1404 (1960). 3. GROMET-ELttANAN, Z., AND WINNICK, W., Biochim. Biophys. Acta 69, 85 (1963). 4. BDOLAH, A., BEN-ZvI, R., AND SCHRAMM,M., Arch. Biochem. Biophys., 104, 58-66 (1963). 5. SCHRAMM,M., AND DANON, D., Biochim. Bio. phys. Acta 50, 102 (1961). 6. LOYTER, A., AND SCHRAMM, M., Biochim. Biophy~. Acta 65, 200 (1962).
7. JVNQEEIaA, L. C. U., HIRSCH, G. C., ANb ROTHSCHILD, H. A., Biochem. J. 61, 275 (1955). 8. SJOSTRAND,F. S. in "Ciba Foundation Symposium, The Exocrine Pancreas, London, 1961," p. 15. Churchill, London and New York, 1962. 9. PALADE, G. E., SIEKEVITZ,P., AND CARO, L. G. in "Ciba Foundation Symposium, The Exocrine Pancreas, London, 1961," p. 23. Churchill, London and New York, 1962. 10. LAIRD, A. K., AND BARTON, A. D., Biochim. Biophys. Acta 27, 12 (1958).
(1964)
The Mechanism of Enzyme Secretion by the Cell III. Intermediate Stages in Amylase Transport as Revealed by Pulse Labeling of Slices of Parotid Gland I M I C H A E L S C H R A M M AND A V N E R B D O L A H From the Department of Biological Chemistry, The Hebrew University, Jerusalem, Israel
Received June 3, 1963 Rat parotid gland slices incubated for 5 minutes with C14-amino acids incorporated radioactivity mainly into exportable proteins such as amylase. Excess labeled amino acids were effectively removed. It was thus possible to study the subeellular distribution, transport, and secretion of a constant amount of radioactive amylase. The specific radioactivity of amylase was initially highest in the microsomes and lowest in the zymogen granules. Two intermediate fractions, sedimented at 15,000g and 4000g, showed a specific amylase radioactivity many times higher than that of the zymogen granules. These two fractions contained a relatively large proportion of the newly synthesized labeled amylase. It is therefore suggested that the intermediate fractions represent transitory steps in the development of the zymogen granule. Amylase secreted by the labeled slice was at first of low specific radioactivity which rose steeply with time to a maximum. Within 3 hours the slice secreted about 70% of the labeled protein and amylase. During secretion the specific amylase radioactivity declined steeply in the microsomes and in the 15,000g fraction but increased in the 4000g fraction and in the zymogen granules. INTRODUCTION The studies on chymotrypsinogen (1) and R N a s e (2) in the pancreas and on a-amylase in the parotid (3) strongly indicate t h a t these exportable proteins are synthesized in the ribosomes and are finally accumulated in the zymogen granules. However, the intermediate stages in the transport of these enzymes between the microsomes and the fully packed zymogen granules remain essentially unidentified. I t would seem most likely t h a t the zymogen granule passes several stages of maturation, b u t these have not been isolated as yet. Whether the exportable proteins pass through a soluble phase before accumulation in the membrane bound granules is also not This work was supported by grants from the National Science Foundation (G-22153) and the National Institutes of Health, Public Health Service (AI-03426-03).
established. Furthermore, one wonders whether the exocrine gland cell maintains rigid order in the sequence of transport so t h a t enzyme is not secreted before it has reached the zymogen granules. These problems had been previously approached by studying the distribution of radioactive enzyme in subcellular fractions of the glands after injecting C14-amino acids into whole animals (1-3). I t seemed to us t h a t slices of rat parotid gland, demonstrating specific secretion of enzymes in a well-defined medium (4), would be a more suitable system for the following reasons: I t is possible to label the slice for a short period and then remove excess C1~-amino acids so t h a t the transport of a constant a m o u n t of Clt-protein can be subsequently followed. The secretion of labeled enzyme into the medium is readily measured in con]unction with the changes in its subcellular distribu67
68
SCHRAMM AND BDOLAH
tion. T h e effect of m e d i u m c o m p o n e n t s a n d s t i m u l a n t s of secretion on t h e r a t e of C 14 i n c o r p o r a t i o n into e x p o r t a b l e e n z y m e a n d on its subcellular d i s t r i b u t i o n is also a m e n a b l e to i n v e s t i g a t i o n . T h e p r e s e n t c o m m u n i c a t i o n p r e s e n t s evidence to i n d i c a t e t h a t i n t e r m e d i a t e stages in the d e v e l o p m e n t o f t h e z y m o g e n g r a n u le do exist a n d can be isolated. I t is f u r t h e r show n t h a t a m y l a s e a l r e a d y p r e s e n t in t h e gla nd slice prior to labeling is secreted prefere n t i a l l y before t h e n e w l y s y n t h e s i z e d radioa c t i v e e n z y m e g r a d u a l l y a p p e a r s in th e medium. MATERIALS AND METHODS Rat parotid glands were obtained from animals starved for 24 hours (5). The preparation and incubation of slices as well as the methods for study of enzyme secretion were described in a preceding paper (4). PROCEDURE FOR LABELING OF PAROTID GLAND SLICES Slices were incubated for 5 minutes in a KrebsRinger-bicarbonate (KRB) medium which contained 5 mM sodium pyruvate and 6 #g. per milliliter uniformly labeled C14-protein hydrolysate (300 ~c. per milligram). To stop incorporation of labeled amino acids, slices were washed with KRB medium on filter paper in a Buchner funnel and subsequently incubated with a large excess of nonradioactive protein hydrolysate. Incubation was for 15 minutes in a KRB medium containing 2 mg. per milliliter casein hydrolysate, 30 gg. per milliliter L-tryptophan, and 5 mM sodium pyruvate. The medium was removed by filtration and the slices were washed as above. When the slices were used subsequently to study the secretion of labeled proteins, the incubation was carried out in the KRB- casein hydrolysate medium described above. Further incorporation of the small amount of labeled amino acids still remaining in the slice could thus be inhibited. Although incubation with amino acids was often continued for 3 hours, the net synthesis of amylase did not exceed 10% of the amount present at zero time. Secretion of radioactive enzyme could thus be conveniently Studied in the absence of appreciable changes in the total amount of enzyme in the system.
Triton x-100. After 5 minutes the suspension was further diluted in 0.02 M phosphate buffer, pH 6.9, to yield a protein concentration of 0.5-2 mg. per milliliter. Triton was not added to soluble fractions. Protein was precipitated by addition of one volume of 10% triehloroacetic acid. The suspension was kept in ice for 15 minutes; the precipitate was then isolated by centrifugation at room temperature and washed once with 5% trichloroacetie acid. The precipitate was suspended in 90% formic acid, transferred to a planehet, dried, and counted. Control experiments showed that further extractions of the trichloroacetic acid precipitate by hot trichloroacetic acid and organic solvents did not detectably reduce the specific radioactivity of the protein. To isolate amylase, an aliquot of 1 ml. containing 500 enzyme units was fractionated with ethanol and the enzyme was specifically precipitated with glycogen (6). The washed enzymeglycogen complex was dissolved in 1 ml. 0.02 M phosphate buffer, pH 6.9, containing 7 mM NaC1. An aliquot was transferred to a planchet, dried, and counted. Enzyme activity and protein content of the purified amylase were also determined. The enzyme isolated as the glycogen-amylase complex was obtained with an over-all yield of about 70%. The specific enzymic activity was 2000-2500 units per milligram protein, which is about equal to that of the crystalline enzyme (6). Subcellular fractions such as the microsomes were poor in amylase (5); nonradioactive enzyme was therefore added before ethanol fractionation. If a large excess of pure amylase were added as carrier, the specific enzymic activity of the mixture would be very high even before purification and one could not test whether foreign labeled protein would indeed be removed by the purification procedure. It therefore seemed preferable to use as carrier a fraction rich in amylase rathel than pure enzyme. To this purpose the contentE of zymogen granules (5) was chosen as the carrier. Its specific amylase activity was about 800 unit,~ per milligram protein. An aliquot of the radioac rive fraction containing a known amount of amy lase units was supplemented with nonradioactive zymogen granule contents to give a final concentration of 500 enzyme units per milliliter. In the most extreme cases 480 units carrier were adde( to 20 units of the radioactive fraction. On th( basis of protein, however, the carrier representee about 50% of the mixture.
ISOLATION OF PROTEIN AND AMYLASE FOR MEASUREMENTS OF RADIOACTIVITY
CALCULATIONS OF RADIOACTIVITY
The washed pellets of subcellular fractions were suspended in a minimal volume of 0.1%
The specific radioactivity of amylase is ex pressed as counts/min./mg, enzyme protein an(
INTERMEDIATE
STAGES IN AMYLASE TRANSPORT
is corrected according to t h e dilution factor when carrier amylase was added. T o t a l counts ia t h e amylase of a n y fraction are calculated from t h e counts found in the purified enzyme corrected for losses during purification. Corrections for self-absorption were unnecessary because of t h e small weight of t h e dried sample. A t h i n window gas flow counter (Nuclear Chicago) was used. ~SOLATION OF SUBCELLULAR FRACTIONS P r e p a r a t i o n of the homogenate and differential c e n t r i f u g a t i o n were carried out essentially as described previously (4, 5). T h e centrifugal force a n d centrifugation time were varied according to t h e specific requirements of the experiment and are given u n d e r Results. MATERIALS Uniformly labeled Cl~-a/gal protein hydrolysate was obtained from t h e Radiochemical Centre, Amersham, E n g l a n d . Casein hydrolysate (by hydrochloric acid, salt-free) was a p r o d u c t of M a n n Research Laboratories. O t h e r chemicals were commercial preparations of highest p u r i t y available. RESULTS
When slices were labeled for 5 minutes with C14-protein hydrolysate and subsequently washed free of excess radioactive n~terial, 80 % of the counts were found in protein. It was to be expected that within
69
short periods of labeling, incorporation would be mainly confined to those proteins which are produced in large amounts for secretion. It was indeed found that about 35 % of the protein counts were located in a-amylase, an enzyme which had been calculated to represent roughly 40 % of the secretory proteins of the parotid gland (5). As shown in Table I the ratio of C ~4 in amylase to C ~4in total protein varies for all subcellular fractions only in the range of 23-40%. This observation is again in line with the assumption that incorporation was mainly limited to exportable proteins. It is highly significant that a relatively large portion of the counts in protein and amylase were located in the fractions between the heavy zymogen granules (1000 g) and the microsomes (105,000 g). Moreover, these intermediate fractions had a specific amylase radioactivity many times higher than that of the typical zymogen granules. It would therefore seem most likely that the fractions sedimented at 4000 and 16,000 g contain immature zymogen granules, partly packed with highly labeled radioactive amylase which had been released by the microsomes. Table I further shows that the final supernatant, while containing the largest relative amount of total counts, has a specific
TABLE I DISTRIBUTION OF C14-PRoTEIN AND C14-AMYLASE IN THE SLICE LABELED WITH AMINO ACIDSa Fraction Centrifugal force (g.) I II III IV V Supernatant
250 1000 4000 15,000 105,000
Relative disCI4 in amylase Specific activityradioof Durationof tribution of amy- Relative distribution of as per cent amylase centrifugation lase activity b C14Pr~ of C14in protein (counts/ (rain.) (%) (%) min,/mg.) 5 l0 15 15 30
7.0 21.0 3.0 0.7 0.5 72.0
2.5 7.0 1 3 . 0 + (3.9) 8.5 + (7.9) 8 . 0 + (1.6) 30.0
23 29 30 40 30 33
1370 1900 26,000 87,000 100,000 2200
Slices from 6 glands were labeled, washed, a n d i n c u b a t e d for 15 m i n u t e s to remove radioactive amino acids as described u n d e r Materials a n d Methods. T h e homogenate s u b s e q u e n t l y prepared in 0.25 M sucrose contained 35 mg. protein, 22,000 amylase units, and 164,000 counts per m i n u t e in protein. T h e outline of fractionation is given in the table. All fractions were washed once. The s u p e r n a t a n t a f t e r washing fraction I was added to t h e rest of t h e homogenate. The s u p e r n a t a n t s o b t a i n e d a f t e r washing fractions I I , I l I , a n d IV c o n t a i n e d a relatively large a m o u n t of r a d i o a c t i v e p r o t e i n which was almost completely sedimented by c e n t r i f u g a t i o n at 105,000g for 30 minutes. T h e values of p r o t e i n counts in these s e d i m e n t s are w r i t t e n in b r a c k e t s alongside those of t h e next heavier fraction. b T h e a m o u n t in the t o t a l homogenate is defined as 100k.
70
SCHRAMM AND BDOLAH
granule fractions I 4- II and III was at the end of incubation higher than at zero time. a~.~o 432 Moreover, the specific amylase radioactivity of the microsomal fraction V which was ,, .% highest at zero time (100 times higher than fractions I 4- II and 10 times higher than ~.~c~ It" ," -o ~-12o o~ / / ," -12~, t fraction III) dropped at the end of 3 hours .~ ~ / /r . ~ ~ incubation to a value below that of fractions IV and III. A comparison of the specific radioactivities ~ / // ~ -I8 ~. .__.~ / g," .~ ~ / E of amylase with those of total protein shows .~ looc , / / # "~,/4 < that the latter are much lower in most subcellular fractions, both at zero time and at . . 60. . . . 120 0 J,-' O 180 0 ._]oZ the end of 3 hours. In contrast, total protein Time f rnin) secreted into the extracellular medium had FIG. l. Secretion of radioactive amylase after pulse labeling of the slice. The data were obtained almost the same specific radioactivity as the from the same experiment as described in Tables amylase secreted (Table II). One could II and III. Zero minutes designates addition of therefore assume that exportable proteins epinephrine (marked with arrow). 9 specific are all synthesized and secreted at similar radioactivity of amylase; O, total counts/rain high rates while constitutive proteins of the in amylase; ~, amylase units. The experimental subcellular fractions are produced at a low points indicate the time at which slices were rate and consequently have a low specific transferred to fresh medium. It should be noted radioactivity. During the last hour of that the values for specific amylase radioactivity incubation the specific radioactivity of the represent mean values for the enzyme secreted amylase secreted is already declining and is during each incubation interval. much lower than that still remaining in the subcellular fractions I I I - - V (Table II). amylase radioactivity which is many times These findings suggest that individual cells lower than that of the intermediate fractions in the slice do not act in complete synIII and IV. When labeled slices were incubated in chronization (cf. ref. 8). Some of the cells KRB medium and stimulated by epineph- might rapidly synthesize radioactive enzyme rine, the amylase secreted initially had a but secrete it only sluggishly during subselow specific radioactivity, which rose steeply quent incubation periods. Table III shows the decrease of amylase with time to a maximum and then started to fall off (Fig. 1). It is also demonstrated activity, radioactive amylase, and total that the maximal secretion rate of total labeled protein in the subcellular fractions enzyme is attained during the first 30 min- during the 3 hour period of secretion by the utes after epinephrine addition, whereas slice. About 70 % of the amylase and labeled radioactive enzyme is secreted at a maximal protein were secreted during the experiment. rate only later, during the period of 30-60 The relative amounts of labeled amylase minutes incubation (cf. ref. 7). It appears and labeled protein remaining in each fracmost likely that nonradioactive amylase tion after 3 hours are quite similar, with the which was stored in the zymogen granules exception of the microsomal fraction V. before labeling of the gland is secreted first Table III further demonstrates that fracand that newly formed radioactive enzyme tions IV and V retained 76 % of their initial cannot be exported directly after being amylase activity although only 12 and 11%, released from the microsomes. These conclu- respectively, of the radioactive enzyme sions are further supported by the results remained. Apparently the labeled enzyme presented in Table II. The specific radio- present in these fractions at zero time was activity of amylase in fractions V (micro- transported to the heavier zymogen granule somal) and IV decreased by about 80% fractions while being replaced by newly during 3 hours, while that of the zymogen formed amylase, synthesized from the non,
u
~
i
' /~-
"-t3 6
!
c)
aJ
INTERMEDIATE STAGES IN AMYLASE TRANSPORT
71
TABLE II S 'ECIFIC RADIOACTIVITY OF AMYLASE AND PROTEIN
D U R I N G S E C R E T I O N BY T H E S L I C E a
Subcellular fractions
Protein secreted in incubation medium
Counts/min./mg. a m y l a s e
Homogenate I -4- II III IV V Supernatant
Counts/min./mg. total protein
At 0 time
After 3 hr.
At 0 time
After 3 hr.
-940 10,300 85,000 90,000 1170
-2700 12,500 15,000 11,500 1700
1600 1260 3100 5300 2000 960
1440 1300 1460 1120 1200 1390
Incubation interval (rain.)
Counts/ min./mg, amylase
Counts/ min./mg. total protein
0-30 30~}0 60-120 120-180
1200 4200 5900 4200
1300 3400 4500 3500
a Slices from 18 glands were labeled and washed. After 15 minutes incubation with excess nonradioactive amino acids, a third of the slices was homogenized and fractionated to determine values before addition of epinephrine (0 time). The other portion of slices was incubated in medium containing the stimulant. The slices were transferred to fresh medium at the end of each incubation interval indicated in the table. The used media were saved for analysis of secreted proteins. At the end of 3 hours, slices were homogenized and fractionated. The designation of subcellular fractions is identical with that given in Table 1. However, the first fraction was isolated by centrifugation at 1000g for 10 minutes. Values for total amylase activity and radioactivity are given in Table III. TABLE I I I CHANGES
IN DISTRIBUTION
AND T O T A L A M O U N T OF C 1 4 - A M Y L A S E AND C 1 4 - P R O T E I N
DURING
SECRETION a Amylase activity At 0 time (units)
Homogenate I + II III IV V Supernatant
17,700 5100 670 100 65 11,300
C la in protein
C ~4 in amylase
After 3 hr. seAfter 3 hr. seAfter 3 hr. secretion (as % of At 0 time cretion (as % of At 0 time cretion (as % of amount in (counts/rain.) counts in (counts/rain.) counts in fraction at fraction at fraction at 0 time) 0 time) 0 time)
32 7 19 76 76 40
56,000 10,100 10,500 9000 4200 19,200
36 17 17 19 45 56
19,290b 2300 3500 4300 2300 6900
30b 19 23 12 11 58
a Data in the table were obtained from the experiment described under Table II. The values after 3 hours secretion were calculated for the same amount of slices as used at zero time. b Amylase was not isolated from the homogenate. The value given represents the arithmetical sum of individual fractions. radioactive a m i n o acids of the i n c u b a t i o n medium. DISCUSSION T h e work presented shows t h a t slices of r a t p a r o t i d g l a n d readily i n c o r p o r a t e labeled a m i n o acids into a m y l a s e a n d other proteins. T h e highest specific r a d i o a c t i v i t y is i n i t i a l l y f o u n d in the microsomal fraction. T h i s f r a c t i o n also shows the m o s t precipitous decline in specific a m y l a s e r a d i o a c t i v i t y with time, d u r i n g secretion of the enzyme.
T h e r e is therefore little d o u b t t h a t a m y l a s e a n d other exportable p r o t e i n s are s y n t h e sized in the ribosomes, as a l r e a d y s h o w n b y o t h e r workers s t u d y i n g the d i s t r i b u t i o n of labeled e n z y m e s after i n j e c t i n g CI4-amino acids into whole a n i m a l s (1-3). I t m i g h t r e a s o n a b l y be a s s u m e d t h a t there are several i n t e r m e d i a t e stages b e t w e e n the release of a m y l a s e from the ribosomes a n d the final a c c u m u l a t i o n of the e n z y m e in the m a t u r e z y m o g e n granules. While there is some s u p p o r t for this a s s u m p t i o n from
72
SCHRAMM AND BDOLAH
electron microscope studies (8, 9), direct evidence has been lacking. In the present work, subcellular fractions which show characteristics expected of intermediate stages in the formation of zymogen granules were successfully isolated. These fractions contained a high concentration of radioactive amylase after pulse labeling. The centrifugal force and centrifugation time required also indicate that the fractions might contain amylase-bearing particles which are different from microsomes or mature zymogen granules. The stage in the sequence of transport in which enzyme accumulates in the soluble fraction is less clear. Previous studies suggested that the soluble fraction represents enzyme released from the microsomes to be packed into zymogen granules (3, 10). Our results show, however, that the specific radioactivity of amylase in the soluble fraction is many times lower than that of the intermediate granule fractions. Yet the supernatant contained the largest absolute amount of labeled amylase found in any single fraction. It would seem quite possible that the enzyme isolated as the supernatant fraction represents an artificial mixture of highly labeled enzyme released from the microsomes and a large amount of nonradioactive enzyme already secreted by the zymogen granules and partly present in the ducts of the gland at the time of labeling. Part of the radioactivity in the supernatant might also have been contributed by the highly labeled particulate fractions damaged during isolation. It should be emphasized that the parotid gland slice used in these studies was obtained from 24-hour starved animals and was
therefore fully loaded with exportable enzymes. The rate of enzyme synthesis and transport in such a gland preparation might be much slower than in a gland depleted of its exportable proteins before labeling. It is therefore possible that intermediate stages in enzyme transport could not be as readily distinguished in previous studies employing animals depleted of exportable proteins prior to injection of labeled amino acids
(1-3). ACKNOWLEDGMENT The authors are most grateful to Mrs. Sarah Eldar for her excellent assistance. REFERENCES 1. SIEKEVITZ,P., AND PALADE,G. E., J. Biophys, Biochem. Cytol. 7, 619 (1960). 2. MORRIS, A. J., AND DICMAN, S. R., J. Biol. Chem. 235, 1404 (1960). 3. GROMET-ELttANAN, Z., AND WINNICK, W., Biochim. Biophys. Acta 69, 85 (1963). 4. BDOLAH, A., BEN-ZvI, R., AND SCHRAMM,M., Arch. Biochem. Biophys., 104, 58-66 (1963). 5. SCHRAMM,M., AND DANON, D., Biochim. Bio. phys. Acta 50, 102 (1961). 6. LOYTER, A., AND SCHRAMM, M., Biochim. Biophy~. Acta 65, 200 (1962).
7. JVNQEEIaA, L. C. U., HIRSCH, G. C., ANb ROTHSCHILD, H. A., Biochem. J. 61, 275 (1955). 8. SJOSTRAND,F. S. in "Ciba Foundation Symposium, The Exocrine Pancreas, London, 1961," p. 15. Churchill, London and New York, 1962. 9. PALADE, G. E., SIEKEVITZ,P., AND CARO, L. G. in "Ciba Foundation Symposium, The Exocrine Pancreas, London, 1961," p. 23. Churchill, London and New York, 1962. 10. LAIRD, A. K., AND BARTON, A. D., Biochim. Biophys. Acta 27, 12 (1958).