- Email: [email protected]
[8] Secretory membranes of the rat parotid gland: Preparation and comparative characterization
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RAT PAROTID SECRETORY MEMBRANES
75
directly recaptured shortly after exocytosis. Moreover, the molecular intermixing that might possibly have occurred after fusion between ZG and plasma membranes could be eliminated by the coated vesicle mechanism, whose molecular filtration function has been recognized,zz Since coated pits and vesicles have been found also in the Golgi area, in apposition or continuity to cisternae and condensing vacuoles, it is possible that this mechanism operates also at the proximal terminal of the ZG pathway, to sort out patches of ZG membrane from Golgi membrane components. The scheme of membrane recycling that we have briefly summarized is certainly compatible with the available evidence. However, at the present time it should be considered only hypothetical. Direct investigations in this field would require the study of specific ZG membrane proteins, to be identified in intact cells and isolated in pure form from homogenates or cell fractions. Taking into account the low concentration of these proteins, these studies could be carried out only by using specific antibodies (immunochemistry and immunocytochemistry at the electron microscope level). Unfortunately, however, the only available antibodies (obtained in the rabbit by injection of either the pure protein or entire ZG membranes) are directed against GP2, 6,15,16 which, because of its yet dubious association with the membrane, and concomitant localization in the ZG content, does not appear to be suitable for these studies. 22 B. M. F. Pierce and M. S. Bretscher, Anna. Rev. Biochem. 50, 85 (1981).
[8] S e c r e t o r y M e m b r a n e s o f t h e R a t P a r o t i d G l a n d : Preparation
and Comparative Characterization I
By PETER ARVAN,
RICHARD S. CAMERON,
and J. DAVID CASTLE
Exocrine secretory glands consist of functionally polarized epithelial cells having an organization such that synthesis of macromolecules (especially proteins) destined for export begins near the base of the cell, packaging into membrane-bounded containers and intracellular storage as secretion granules occur near the apex of the cell, and discharge takes place selectively at the apical cellular surface. This region, designated the apical plasma membrane, represents a discrete fraction of the cell surface that is segregated from the basolateral domain by junctional complexes. Release of secretory products by exocytosis involves the specific interaction of secretion granule and apical plasma membranes, focal rearrangement ofbilayer structures in These studies were supported in part by USPHS Grants GM 26524 and AM 29868. METHODS IN ENZYMOLOGY, VOL. 98
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181998-1
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SPECIALIZED METHODS
[8]
regions of contact, and eventual coalescence of the two membranes with direct extracellular deposition of secretory products. Although exocytosis may be initiated by the fusion of these two membrane types, secreting cells of many tissues (including nonexocrine varieties) can exhibit compound exocytosis in which further release takes place by fusion of granules with the membranes of previously discharged granules that are already continuous with the apical plasma membrane. In this case, granule membranes are equivalent to plasma membranes as acceptors for further granule discharge. Consequently, granule and apical plasma membranes may contain common constituents that facilitate the specific events of the release process in stimulated cells. As initial steps in undertaking a comparative compositional analysis of membranes that serve as partners in exocytosis, we have obtained purified fractions of secretion granule membranes and plasma membranes containing the apical domain from rat parotid gland. This tissue was chosen for study because a single secretory cell type constitutes 85- 90% of the cellular volume of the gland and because it is known to contain especially low levels of lipolytic and proteolytic activities2 that potentially could elicit alterations of membrane components during isolation. Preparation of a Plasma Membrane Fraction Containing the Apical Domain Since enzymic marker activities exclusive to the apical surface ofacinar cells of parotid are not known, we sought an isolation procedure that could at least partially preserve morphologically recognizable regions constituting the apical surface. Two important features of the procedure developed are that (a) it selects for large sheets of membrane and (b) very hypoosmotic media are used at the outset, as in the case of plasma membrane purification from rat liver, 3 so that osmotically sensitive organelles (especially secretion granules and mitochondria) are either damaged or disrupted during homogenization. A flow diagram of the procedure is shown in Fig. I.
Reagents Medium A: 0.5 mMMgClz, 1 mMNaHCO3, pH 7.4 Medium B: 0.5 m M MgCI2, 1 mMNaHCO3, 0.7 m M E D T A , pH 7.4 Medium C: 0.5 m M MgCI2, 1 mMNaHCO3, 1.7 m M E D T A , pH 7.4 Procedure. The plasma membrane preparation is carried out entirely at 4 °. Parotid tissue (5.5 g), obtained from 16 male Sprague-Dawley rats (100- 125 g) starved overnight, is cleaned of connective tissue, minced with
2 M. Schramm and D. Danon, Biochim. Biophys. Acta 50, 102 (1961). 3 T. K. Ray, Biochim. Biophys. Acta 196, 1 (1970).
[8]
RAT PAROTIDSECRETORYMEMBRANES
77
HOMOGENIZEDTISSUE, 20% (W/V) A SPIN 70G X 30 SEC/ ~ PI Sl REPEATA P2 $2 POOL$I_3, DILUTETO 1%
REPEATA P3 $3"'"
FI LTER
P4 RESUSPENDED
SPIN AS
S4
BEFORE ~
THROUGH0.3M SUCRO~J
P5 RESUSPENDED
SPIN 82,500G X 2HR
S5
IADJUST SUCROSE ~.~CONC.TO 1.3~ . . . .
x
INTERFACEBAND SPIN
~
PLASMAMEMBRANE PELLET
\ REST (SUP)
FIG. 1.~OWdiagramofplasmamembranepreparation. razor blades, and homogenized (15 sec at 1900 rpm with a Polytron homogenizer followed by three strokes with a Brendler Teflon pestle homogenizer at 1300 rpm) in medium A at 20% w/v. The resulting suspension is spun at 70 g for 0.5 min to yield the first supernatant and a pellet that is rehomogenized with the Brendler homogenizer in the same volume of medium A. The procedure is repeated again, the final pellet is discarded, and the three supernatants are pooled and diluted to 1% (original tissue weight per volume) with medium B. Dilution and net EDTA serve to reduce organelle aggregation. The suspension is filtered through four layers of cheesecloth plus one layer of 105-#m nylon mesh to produce the homogen-
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SPECIALIZED METHODS
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ate, the initial fraction on which recoveries of enzyme activities are based. Differential centrifugation of the homogenate (825 gav × 15 min) is used to obtain the fourtb supernatant ($4, saved for assays) and the fourth pellet (P4) consisting of nuclei, acinar debris (especially basement membrane-containing elements), aggregated microsomes, and plasma membrane sheets. P4 is resuspended (three strokes in a Dounce homogenizer with tight pestle), diluted to one-half the volume of $1_3 with medium B, layered above 5-ml 0.3 M sucrose in medium B in 30-ml Corex (Coming Glass) tubes, and subjected to recentrifugation under identical conditions. The 0.3 M sucrose layer minimizes contamination of the pellet (Ps) by microsomal elements retained in the supernatant ($5). P5 is resuspended in ~ 4 ml of sucrose concentration of 1.38 M, and diluted to 125 ml using 1.38 M sucrose in medium B. The diluted suspension is loaded into centrifuge tubes, overlaid with 0.3 M sucrose in medium B, and subjected to centrifugation (9.9 × 106gav × rain) in a Beckman SW 27 rotor in order to float plasma membrane sheets to the 0.3 - 1.38 M interface away from the other particulates noted in P4 (and Ps) that pellet under these conditions. The interface band collected in a small volume is supplemented with EDTA to give a net concentration of 1.2 mM, diluted to 0.35 M sucrose with medium C, thoroughly dispersed with three strokes in a tight-fitting Dounce homogenizer (to reduce membrane aggregation), and subjected to centrifugation (3.3 × 106 gav× rain) in a Beckman SW 41. Plasma membrane pellets and the overlying supernatant are used for assays. Electron Microscopic Characterization of t h e P l a s m a Membrane Fraction
Since morphological appearance constitutes an important initial evaluation for the purity and integrity of isolated membranes containing distinct regions, aliquots of the fraction are fixed in aldehydes,4 postfixed in 1% osmium tetroxide, and prepared for microscopy according to standard procedures. Figure 2 shows a representative low-power electron micrograph of the plasmalemmal fraction. The fraction predominantly consists of large membrane sheets that show in part regions retaining the organization of the apical surface in situ. Apical membranes are joined to one another by elements of the junctional complex, often forming closed profiles, the former cytoplasmic aspects of which are oriented outwardly. Extracted or collapsed microvilli invaginate from the surface into the space bounded by the apical membranes; basolateral membranes extend beyond the junctional complexes either to contact other apical compartments or to terminate as free ends. Frequently several lumenal profiles, mostly with cross-sec4 R. C. Graham and M. J. Karnovsky, J. Histochem. Cytochem. 14, 291 (1966).
[8]
RAT PAROTID SECRETORY MEMBRANES
79
FIG. 2. Representative electron micrograph of the purified parotid plasma membrane fraction. Individual plasmalemmal sheets are quite extended and contorted and contain multiple profiles of apical membrane (A) enclosing the former lumenal space. Junctional complexes (jc) adjoin the apical surfaces of neighboring cells, and the contiguous lateral membrane (L) is often multiply studded with desmosomes. Lumenal compartments contain remnants of microvilli often appearing as vesicular profiles (in favorable sections, continuous with the apical surface) and less frequently as apparently highly flattened vesicles (arrows at lower right). In addition, the fraction can be seen to contain unidentified smooth membrane elements (arrowhead) possibly deriving from the plasma membrane sheets, microsomes (m), and filamentous material especially in the vicinity of junctional elements and the apical surface. Bar = 1/tm.
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tional diameters comparable to those of secretion granules, are observed in individual sheets of plasma membrane. Both the small diameters and multiplicity of profiles suggest that the membranes originate from acinar secretory cells and that the organization corresponds to that of the tubular secretory canaliculi constituting the luminal surface in situP This interpretation is favored further since an estimated 85-90% of the gland volume is composed of acinar cells. Morphological characterization additionally indicates that the plasmalemmal fraction is contaminated by low levels of rough microsomes and ribosomal aggregates; occasionally, damaged mitochondria and, rarely, centrioles and extracted nuclei are observed in the fraction. Cytoplasmic filaments, emanating from elements of the junctional complex, frequently can be seen in association with the membrane sheets.
Plasma Membrane Yield and Analysis for Organelle Contaminants Using Marker Enzyme Activities
Typically ~ 0.15 mg of protein is recovered in the purified plasma membrane fraction per gram wet weight of tissue. This yield is unusually low in comparison to that of preparations from liver, where 5 - 10 times as much protein is obtained from a comparable amount of tissue. 6 The reduced yield probably reflects a decreased amount of large membrane sheets as a result of the vigorous homogenization required to disrupt connective tissue and basement membranes surrounding acini. The low level of contamination of the plasma membrane fraction observed by electron microscopy is confirmed by assays of enzymes considered to mark specific organelles selectively. Membrane-associated enzymes are assayed except for secretion granules and lysosomes, where unique markers have not been identified. In these cases it is possible to examine only the extent of contamination by species present in the organelle content. Table I presents, for the purified plasmalemmal fraction, data that have been extracted from comprehensive studies of activity distributions throughout the fractionation procedure. Generally, recoveries of enzyme activities at each step of fractionation are very good; the exception is UDPgalactosyltransferase (EC 2.4.1.22), where recovery of activity at the final step of plasma membrane purification is incomplete. As can be seen in Table I, 0.4% or less of total homogenate activity for each marker is recovered in the final plasma membrane fraction, with at most a low level of enrichment for any contaminant. 5 j. F. Parks, Am. J. Anat. 108, 303 (1961). 6 A. L. Hubbard, D. A. Wall, and A. Ma, J. CellBiol. 96, 217 (1983).
[8]
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81
TABLE I ORGANELLE CONTAMINANT ANALYSIS OF THE PLASMALEMMAL FRACTION a
Enzyme
Organdie
Cytochrome c oxidased NADH-cytochrome c reductase ~ (rotenone-insensitive) UDPgalactosyltransferasef fl-N-Acetyl-D-glucosaminidaseg a-Amylase h
% Recoveryb
Relative specific activityc
Mitochondria Microsomes
0.27 0.40
1.17 1.76
Golgi complex Lysosomal content Granule content
0.39 0.29 0.008
1.70 1.26 0.04
Values presented are averages for a minimum of three experiments. t, Percentage recovery is based on 100% activity in the homogenate. , Relative specific activity = specific activity ratio, plasmalemmal fraction: homogenate. Protein was assayed according to Markwell et al.~2 d Cytochrome c oxidase [T. J. Peters, M. Muller, and C. de Duve, J. Exp. Med. 136, 1117 (1972)]. The first-order rate constant is used as a measure of activity [E. E. Max, D. B. P. Goodman, and H. Rasmussen, Biochim. Biophys. Acta 511, 224 (1978)]. Analysis of amine oxidase indicates a distribution very similar to that reported for cytochrome c oxidase. e G. L. Sottocasa, B. Kuylenstierna, L. Ernster, and A. Bergstrand, J. Cell Biol. 32, 415 (1967). This activity is indicated as a microsomal marker, although in other systems it has been shown to mark mitocfiondriai outer membranes, Golgi membranes [N. Borgese and J. Meldolesi, J. Cell Biol. 85, 501 (1980)], and even plasma membranes [E. D. Jarasch, J. Kartenbeck, G. Bruder, D. J. Morr6, and W. W. Franke, J. CellBiol. 80, 37 (1979)]. Thus the recovery and purification probably reflect the sum of contamination by a variety of organelles. fUDPgalactosyltransferase: B. Fleischer, this series, Vol. 31, p. 180. The assay uses [3H]UDPgalactose in the presence of 2 m M ATP and 2 mg of asialogalactofetuin per milliliter. g fl-N-Acetyl-D-glucosaminidase[J. Findlay, G. A. Levvy, and C. A. Marsh,Biochem. J. 669, 467 (1958)]. h a-Amylase. P. Bernfeld, this series, Vol. 1, p. 149. Preparation
of Secretion
Granule Membranes
Precursor Fraction: Secretion Granules. As starting material for granule membrane purification, secretion granules have been isolated by a procedure that capitalizes on their high density in hyperosmotic sucrose solutions. Routinely, 4.5 - 5.0 g of tissue (corresponding to the parotid glands of 14- 16 rats starved overnight) thoroughly cleaned of lymph nodes and surrounding connective tissue are used in a procedure modified from that developed by Castle el al. 7 for purification of granules from rabbit parotid glands. Processing involved the following steps carried out at 4 o: (a) mincing of tissue with razor blades and homogenization--10 sec with a Polyton (1900 rpm) followed by five strokes ( 1300 rpm) with a Brendler homogenizer-- in 0.3 M 7 j. D. Castle, J. D. Jamieson, and G. E. Palade, J. CellBiol. 64, 182 (1975).
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sucrose, 10 m m imidazole, 0.5 m M MgClz, pH 7.1; (b) sedimentation (70 g X 30 sec) of large particulates and their rehomogenization (Brendler mortar and pestle only) in the same volume to yield a total homogenate, 5% tissue weight per volume; (c) low-speed centrifugation (7000 gay × min) to remove nuclei and cellular debris; (d) filtration of the supernatant through 20-gm nylon mesh and addition of EDTA to a final concentration of 5 mM; (e) recentrifugation (1.3 X 105 gav× min) of the supernatant on a sucrose step gradient having two layers containing 1.0 M and 2.0 M sucrose, respectively, each supplemented with 4% Ficol1400, 10 mMimidazole, and 5 m M EDTA; ( f ) collection of a crude granule fraction at the 1.0/2.0 M sucrose interface and adjustment of the suspension to contain 1.5 M sucrose, 4% Ficoll 400, 10 m M imidazole, 5 m M EDTA; (g) centrifugation (1.24 × 107 gav× min) in a sucrose step gradient having underlayers of 1.55 M and 2.0 M sucrose and an overlayer of 0.8 M sucrose (each containing the same additives as the crude granule load); (h) collection of the purified granules from the 1.55 - 2.0 M interface. Using the secretory protein a-amylase as an approximation of a granule-specific marker for parotid tissue, we routinely recover ~ 22% of the total activity of the homogenate in the purified fraction. Mitochondria constitute the only significant organelle contaminant both morphologically and biochemically; 1.7% of the amine oxidase (flavin-containing) activity (an outer membrane marker8) of the homogenate is located in the granule fraction? To prepare purified granules for lysis, sucrose is diluted to 0.9 M using 0.2 M sucrose, 10 m M imidazole, and 5 m M EDTA, and the granules are pelleted by centrifugation (1.6 × 106 gay X min). The dilution typically results in a 15 -20% loss of granules by lysis, which is thus far unavoidable.
Reagents for Granule L ysis and Membrane Purification Lysis medium: 0.19 M KCI, 10 m M imidazole, 5 m M EDTA, pH 7.1 Buffered sucroses: 2.0 M, 0.9 M sucrose each containing 0.19 M KCI, 10 m M imidazole, 5 m M EDTA, pH 7.1 Procedure. The protocol developed for lysis takes advantage of the known lability of parotid secretion granules in KCl-containing media. ~° Purified granules are resuspended and diluted to 30 ml in lysis medium and maintained at 0 ° for 12 hr; clearing of the suspension indicates extensive lysis. In order to separate the low-density granule membranes from organelle contaminants and soluble content proteins, the lysate is adjusted to a 8 M. R. Castro Costa, S. Edelstein, C. M. Castiglione, H. Chao, and X. O. Breakfield, Biochem. Genet. 18, 577 (1980). 9 We are currently modifying the granule purification procedure to reduce the level of mitochondrial contamination further. ~oM. Sehramm, R. Ben-Zvi, and A. Bdolah, Biochem. Biophys. Res. Commun. 18, 446 (1965).
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RAT PAROTID SECRETORY MEMBRANES
83
final sucrose concentration of 1 M (using buffered 2 M sucrose), overlaid with buffered 0.9 M sucrose and lysis medium, respectively, and subjected to centrifugation (1.5 × 107 gav× min) in a Beckman SW 27.1 rotor. Granule membranes band at the lysis medium-0.9 M interface, whereas residual contaminants (mitochondria) and unlysed granules sediment into a pellet. The membranes are collected, diluted to 0.07 M sucrose with lysis medium, and pelleted by centrifugation (1.5 X 107 gay × min). Resuspended membranes are used for assays and morphology. Electron Microscopic Observation of the Granule Membrane Fraction Processing of secretion granule membranes for electron microscopy employs the same procedure already outlined for plasma membranes. The fraction is very homogeneous in appearance, consisting of closed, smoothsurfaced vesicles 0 . 3 - 0 . 8 / t m in diameter with no evidence of residual granule content (see Fig. 3). The diameters are smaller than those of intact granules ( ~ l / t m ) indicating that the vesicles represent fragments of the original membranes. Estimation of Mitochondrial and Soluble Secretory Contaminants Since mitochondria constitute the principal organelle contaminant of the granule fraction, the distribution of amine oxidase activity is followed during purification of granule membranes. With 90- 100% recovery of total activity, 3- 4% of that originally present in the granule lysate is detected in the membrane fraction. On the basis of the specific activity (units of amine oxidase per micromole of lipid phosphorus 1~and per milligram of protein ~2) of a purified parotid mitochondrial fraction, we estimate that a maximum ~3 of 1% of the lipid and 3% of the protein of the granule membrane fraction could be contributed by mitochondrial contamination. Contamination of granule membranes by secretory proteins is estimated by two means: (a) by following routinely the distribution of a-amylase during subfraction~tion of granule lysates; (b) for a more comprehensive examination, by adding biosynthetically labeled rat parotid secretory proteins (prepared in the manner of Castle et aL7)to granules during lysis, since this reagent is a more representative marker of total granule content. 1~G. R. Bartlett. J. Biol. Chem. 234, 446 (1959). ~2 M. A. K. Markwell, S. M. Hass, L. L. Bieber, and N. E. Tolbert, Anal. Biochem. 37, 206 (1978). ~3The estimated levels of contamination constitute upper limits because the most probable contaminants of the granule membrane fraction are outer mitochondrial membranes rather than intact mitochondria. The former will have much higher amine oxidase specific activities, hence will contribute less lipid and protein per unit of activity as contaminants.
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FIG. 3. Electron micrograph of the secretion granule membrane fraction. Membranes appear as smooth-surfaced, mostly closed vesicles, in some cases multivesicular, apparently owing to resealing of larger membrane pieces around smaller membrane fragments. Bar = 1 /tm.
[8]
RAT PAROTID SECRETORY MEMBRANES
85
Radioactive protein found in the final fraction (detected by scintillation counting and by fluorography 14 of 3H-labeled polypeptides resolved in SDS-gel electrophoretograms) is thought to compete and hence is used to approximate the extent of trapping and/or adsorption of residual content to the purified granule membranes. In both cases the membranes are freed of more than 99.99% of the contaminant markers. Although these levels of contaminant removal suggest that the granule membranes are highly purified, residual amylase in the membrane fraction is estimated to represent 5% of the total protein of this fraction (based on the specific activity of purified rat parotid amylase: 2780 units per milligram ofproteinlS). Consequently, we estimate that, taken together, mitochondrial protein and residual secretory protein constitute at most 10-15% of the protein of the granule membrane fraction. Enzyme Activities Associated with the Plasmalemma and Secretion Granule Membranes
Assays for several enzymes known to mark plasma membranes of other cell types indicate that our parotid plasmalemmal fraction is not significantly enriched in alkaline phosphatase and leucyl aminopeptidase (l A-fold and 1.2-fold higher specific activities than the homogenate, respectively) and is only modestly enriched in 5'-nucleotidase (3.8-fold over the homogenate). As shown in Table II, two enzymes, sodium-potassium adenosine triphosphatase (Na+,K+-ATPase) and 7-glutamyltransferase (7-glutamyltranspeptidase; GGTPase) exhibit large enrichments. Although distributions of these markers are discussed in detail elsewhere,16 several features are worthy of mention here. I. The low enrichments for alkaline phosphatase and 5'-nucleotidase favor an origin of the plasma membrane sheets in acinar cells, since histochemical studies have suggested that these activities are associated with other parotid cell types. 17,18 Consequently, these markers are of little use where the isolation of membranes involved in exocytosis in parotid tissue is intended; morphological observations provide the key means of analysis for apical secretory surface. 2. Na+,K+-ATPase is a classical marker of the basolateral domain in polarized epithelia. Although the specific activity increases substantially in discontinuous gradient centrifugation, ~ 70% of the activity of P5 pellets 14 R. A. Laskey and A. D. Mills, Eur. J. Biochem. 56, 335 (1975). 15 T. G. Sanders and W. J. Rutter, Biochemistry 11, 130 (1972). 16 p. Arvan and J. D. Castle, J. Cell Biol. 95, 8 (1982). ~7j. R. Garrett and P. A. Parsons, Histochem. J. 5, 463 (1973). ~8S. Yamashina and K. Kawai, Histochemistry 60, 255 (1979).
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TABLE II ENZYMES MARKING PAROTID PLASMALEMMA AND SECRETION GRANULE MEMBRANESa
Relative Enzyme Na+,K+-ATPase b
Fraction Homogenate
Plasma membranes Homogenate
Secretion granules (23% o f total amylase) Granule membranes GGTPase a
Homogenate
Plasma membranes Homogenate
Secretion granules (23% o f total amylase) Granule membranes
Specific activity (per/tmol lipid
Recovery (%)
specific activity
100 4.6 100 0.4
1.0 20. l 1.0 0.07
-----
ND ~
--
--
100 5.9 100 4.4
1.0 25.6 1.0 0.3
-0.68 ---
2.8
30.6
0.21
phosphate)
a With the exception of data reported for Na+,K+-ATPase recovery in granules, which represent the average o f two experiments, all data reported constitute the averages for at least six different experiments. t, Na+,K+_ATPase has been shown to be equivalent in rat parotid tissue to potassium-stimulated p-nitrophenylphosphatase and is quantitated by measuring p-nitrophenol formation in an assay mixture containing 5 mMp-nitrophenylphosphate, 0.1 M imidazole, pH 7.5, 0.01 M MgCIz, 0.75 m M EGTA, 0.01 M NaCI, and 0.01 M either KCI or choline chloride. " N D , not detected. a y-Glutamyltransferase [S. S. Tate and A. Meister, J. Biol. Chem. 249, 7593 ( 1974)].
rather than floats to the interface band. This activity is likely to be associated with large plasma membrane-containing structures intimately contacting basement membranes. We are anticipating that for the purified fraction the Na+,K+-ATPase activity resides in the lateral membrane segments emanating from the junctional complexes in the plasmalemmal sheets. 3. 7-Glutamyltransferase has been shown to be highly enriched on the free surface of epithelial cells, notably those specialized for secretion and absorption.~9 We favor the notion of a similar localization in parotid acinar cells, especially since, in contrast to Na+,K+-ATPase, ~ 70% of the GGTPase activity of P5 is found in the interface band. Consequently, these two enzymes may constitute markers for distinct plasmalemmal domains in parotid acinar cells and in the isolated membrane sheets. 4. 7-Glutamyltransferase is present in secretion granule fractions at an average yield (six separate preparations) of 4.4% of the total homogenate ~9 A. Meister aod S. S. Tate, Annu. Rev. Biochem. 45 559 (1976).
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RAT PAROTID SECRETORY MEMBRANES
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activity. We estimate that ~ 17% of the total tissue GGTPase is associated with secretion granules according to the assumption that amylase approximates a granule-specific marker and that we recover about one-quarter (23%) of the total tissue amylase activity in the purified granule fraction. Since granule-associated protein represents a large percentage of the total protein of parotid, GGTPase is actually depurified (threefold in Table II) in preparing a secretion granule fraction. The recovery of GGTPase activity in the steps following granule lysis is 98%. After taking into account the loss of purified granules during sucrose dilution and centrifugation prior to lysis, we find that 80% of the GGTPase activity of the granule fraction is recovered in the purified membranes. Most of the remaining activity is found in the load and pellet fractions of the discontinuous gradient. 5. The presence of GGTPase activity in granule membranes (and the apparent absence of Na+,K+-ATPase) suggests that there is a compositional overlap of membranes that serve as fusion partners during exocytosis. Further, the overlap is qualitative but apparently not quantitative, since the specific activity of GGTPase relative to lipid phosphorus (as a reliable normalization for this membrane-associated enzyme) for the granule membrane fraction is 30% that of the plasmalemmal fraction. Considering that the plasma membrane-associated activity may be concentrated in regions comprising the apical surface, the quantitative difference in surface concentration may be as much as 10-fold. 2° These studies provide the rationale for a detailed immunocytochemical study of GGTPase localization. Further, they form the basis for future detailed investigations of compositional similarities and differences between membranes that attain functional continuity and of the resulting implications to the mechanisms of both exocytosis and membrane recycling.
20 At this point 10-fold clearly represents an estimate for two reasons: ( 1) The value is based on data obtained with purified fractions, rough microsomes; and mitochondria (which are low-level contaminants of the plasmalemmal fraction) contain very low activities of GGTPase. Hence their contribution of lipid phosphorus exceeds their contribution of GGTPase to the plasmalemmal fraction. (2) At present we do not have quantitative estimates of the fraction of surface area of isolated plasma membrane that constitutes apical region.
RAT PAROTID SECRETORY MEMBRANES
75
directly recaptured shortly after exocytosis. Moreover, the molecular intermixing that might possibly have occurred after fusion between ZG and plasma membranes could be eliminated by the coated vesicle mechanism, whose molecular filtration function has been recognized,zz Since coated pits and vesicles have been found also in the Golgi area, in apposition or continuity to cisternae and condensing vacuoles, it is possible that this mechanism operates also at the proximal terminal of the ZG pathway, to sort out patches of ZG membrane from Golgi membrane components. The scheme of membrane recycling that we have briefly summarized is certainly compatible with the available evidence. However, at the present time it should be considered only hypothetical. Direct investigations in this field would require the study of specific ZG membrane proteins, to be identified in intact cells and isolated in pure form from homogenates or cell fractions. Taking into account the low concentration of these proteins, these studies could be carried out only by using specific antibodies (immunochemistry and immunocytochemistry at the electron microscope level). Unfortunately, however, the only available antibodies (obtained in the rabbit by injection of either the pure protein or entire ZG membranes) are directed against GP2, 6,15,16 which, because of its yet dubious association with the membrane, and concomitant localization in the ZG content, does not appear to be suitable for these studies. 22 B. M. F. Pierce and M. S. Bretscher, Anna. Rev. Biochem. 50, 85 (1981).
[8] S e c r e t o r y M e m b r a n e s o f t h e R a t P a r o t i d G l a n d : Preparation
and Comparative Characterization I
By PETER ARVAN,
RICHARD S. CAMERON,
and J. DAVID CASTLE
Exocrine secretory glands consist of functionally polarized epithelial cells having an organization such that synthesis of macromolecules (especially proteins) destined for export begins near the base of the cell, packaging into membrane-bounded containers and intracellular storage as secretion granules occur near the apex of the cell, and discharge takes place selectively at the apical cellular surface. This region, designated the apical plasma membrane, represents a discrete fraction of the cell surface that is segregated from the basolateral domain by junctional complexes. Release of secretory products by exocytosis involves the specific interaction of secretion granule and apical plasma membranes, focal rearrangement ofbilayer structures in These studies were supported in part by USPHS Grants GM 26524 and AM 29868. METHODS IN ENZYMOLOGY, VOL. 98
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181998-1
76
SPECIALIZED METHODS
[8]
regions of contact, and eventual coalescence of the two membranes with direct extracellular deposition of secretory products. Although exocytosis may be initiated by the fusion of these two membrane types, secreting cells of many tissues (including nonexocrine varieties) can exhibit compound exocytosis in which further release takes place by fusion of granules with the membranes of previously discharged granules that are already continuous with the apical plasma membrane. In this case, granule membranes are equivalent to plasma membranes as acceptors for further granule discharge. Consequently, granule and apical plasma membranes may contain common constituents that facilitate the specific events of the release process in stimulated cells. As initial steps in undertaking a comparative compositional analysis of membranes that serve as partners in exocytosis, we have obtained purified fractions of secretion granule membranes and plasma membranes containing the apical domain from rat parotid gland. This tissue was chosen for study because a single secretory cell type constitutes 85- 90% of the cellular volume of the gland and because it is known to contain especially low levels of lipolytic and proteolytic activities2 that potentially could elicit alterations of membrane components during isolation. Preparation of a Plasma Membrane Fraction Containing the Apical Domain Since enzymic marker activities exclusive to the apical surface ofacinar cells of parotid are not known, we sought an isolation procedure that could at least partially preserve morphologically recognizable regions constituting the apical surface. Two important features of the procedure developed are that (a) it selects for large sheets of membrane and (b) very hypoosmotic media are used at the outset, as in the case of plasma membrane purification from rat liver, 3 so that osmotically sensitive organelles (especially secretion granules and mitochondria) are either damaged or disrupted during homogenization. A flow diagram of the procedure is shown in Fig. I.
Reagents Medium A: 0.5 mMMgClz, 1 mMNaHCO3, pH 7.4 Medium B: 0.5 m M MgCI2, 1 mMNaHCO3, 0.7 m M E D T A , pH 7.4 Medium C: 0.5 m M MgCI2, 1 mMNaHCO3, 1.7 m M E D T A , pH 7.4 Procedure. The plasma membrane preparation is carried out entirely at 4 °. Parotid tissue (5.5 g), obtained from 16 male Sprague-Dawley rats (100- 125 g) starved overnight, is cleaned of connective tissue, minced with
2 M. Schramm and D. Danon, Biochim. Biophys. Acta 50, 102 (1961). 3 T. K. Ray, Biochim. Biophys. Acta 196, 1 (1970).
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RAT PAROTIDSECRETORYMEMBRANES
77
HOMOGENIZEDTISSUE, 20% (W/V) A SPIN 70G X 30 SEC/ ~ PI Sl REPEATA P2 $2 POOL$I_3, DILUTETO 1%
REPEATA P3 $3"'"
FI LTER
P4 RESUSPENDED
SPIN AS
S4
BEFORE ~
THROUGH0.3M SUCRO~J
P5 RESUSPENDED
SPIN 82,500G X 2HR
S5
IADJUST SUCROSE ~.~CONC.TO 1.3~ . . . .
x
INTERFACEBAND SPIN
~
PLASMAMEMBRANE PELLET
\ REST (SUP)
FIG. 1.~OWdiagramofplasmamembranepreparation. razor blades, and homogenized (15 sec at 1900 rpm with a Polytron homogenizer followed by three strokes with a Brendler Teflon pestle homogenizer at 1300 rpm) in medium A at 20% w/v. The resulting suspension is spun at 70 g for 0.5 min to yield the first supernatant and a pellet that is rehomogenized with the Brendler homogenizer in the same volume of medium A. The procedure is repeated again, the final pellet is discarded, and the three supernatants are pooled and diluted to 1% (original tissue weight per volume) with medium B. Dilution and net EDTA serve to reduce organelle aggregation. The suspension is filtered through four layers of cheesecloth plus one layer of 105-#m nylon mesh to produce the homogen-
78
SPECIALIZED METHODS
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ate, the initial fraction on which recoveries of enzyme activities are based. Differential centrifugation of the homogenate (825 gav × 15 min) is used to obtain the fourtb supernatant ($4, saved for assays) and the fourth pellet (P4) consisting of nuclei, acinar debris (especially basement membrane-containing elements), aggregated microsomes, and plasma membrane sheets. P4 is resuspended (three strokes in a Dounce homogenizer with tight pestle), diluted to one-half the volume of $1_3 with medium B, layered above 5-ml 0.3 M sucrose in medium B in 30-ml Corex (Coming Glass) tubes, and subjected to recentrifugation under identical conditions. The 0.3 M sucrose layer minimizes contamination of the pellet (Ps) by microsomal elements retained in the supernatant ($5). P5 is resuspended in ~ 4 ml of sucrose concentration of 1.38 M, and diluted to 125 ml using 1.38 M sucrose in medium B. The diluted suspension is loaded into centrifuge tubes, overlaid with 0.3 M sucrose in medium B, and subjected to centrifugation (9.9 × 106gav × rain) in a Beckman SW 27 rotor in order to float plasma membrane sheets to the 0.3 - 1.38 M interface away from the other particulates noted in P4 (and Ps) that pellet under these conditions. The interface band collected in a small volume is supplemented with EDTA to give a net concentration of 1.2 mM, diluted to 0.35 M sucrose with medium C, thoroughly dispersed with three strokes in a tight-fitting Dounce homogenizer (to reduce membrane aggregation), and subjected to centrifugation (3.3 × 106 gav× rain) in a Beckman SW 41. Plasma membrane pellets and the overlying supernatant are used for assays. Electron Microscopic Characterization of t h e P l a s m a Membrane Fraction
Since morphological appearance constitutes an important initial evaluation for the purity and integrity of isolated membranes containing distinct regions, aliquots of the fraction are fixed in aldehydes,4 postfixed in 1% osmium tetroxide, and prepared for microscopy according to standard procedures. Figure 2 shows a representative low-power electron micrograph of the plasmalemmal fraction. The fraction predominantly consists of large membrane sheets that show in part regions retaining the organization of the apical surface in situ. Apical membranes are joined to one another by elements of the junctional complex, often forming closed profiles, the former cytoplasmic aspects of which are oriented outwardly. Extracted or collapsed microvilli invaginate from the surface into the space bounded by the apical membranes; basolateral membranes extend beyond the junctional complexes either to contact other apical compartments or to terminate as free ends. Frequently several lumenal profiles, mostly with cross-sec4 R. C. Graham and M. J. Karnovsky, J. Histochem. Cytochem. 14, 291 (1966).
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RAT PAROTID SECRETORY MEMBRANES
79
FIG. 2. Representative electron micrograph of the purified parotid plasma membrane fraction. Individual plasmalemmal sheets are quite extended and contorted and contain multiple profiles of apical membrane (A) enclosing the former lumenal space. Junctional complexes (jc) adjoin the apical surfaces of neighboring cells, and the contiguous lateral membrane (L) is often multiply studded with desmosomes. Lumenal compartments contain remnants of microvilli often appearing as vesicular profiles (in favorable sections, continuous with the apical surface) and less frequently as apparently highly flattened vesicles (arrows at lower right). In addition, the fraction can be seen to contain unidentified smooth membrane elements (arrowhead) possibly deriving from the plasma membrane sheets, microsomes (m), and filamentous material especially in the vicinity of junctional elements and the apical surface. Bar = 1/tm.
80
SPECIALIZED METHODS
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tional diameters comparable to those of secretion granules, are observed in individual sheets of plasma membrane. Both the small diameters and multiplicity of profiles suggest that the membranes originate from acinar secretory cells and that the organization corresponds to that of the tubular secretory canaliculi constituting the luminal surface in situP This interpretation is favored further since an estimated 85-90% of the gland volume is composed of acinar cells. Morphological characterization additionally indicates that the plasmalemmal fraction is contaminated by low levels of rough microsomes and ribosomal aggregates; occasionally, damaged mitochondria and, rarely, centrioles and extracted nuclei are observed in the fraction. Cytoplasmic filaments, emanating from elements of the junctional complex, frequently can be seen in association with the membrane sheets.
Plasma Membrane Yield and Analysis for Organelle Contaminants Using Marker Enzyme Activities
Typically ~ 0.15 mg of protein is recovered in the purified plasma membrane fraction per gram wet weight of tissue. This yield is unusually low in comparison to that of preparations from liver, where 5 - 10 times as much protein is obtained from a comparable amount of tissue. 6 The reduced yield probably reflects a decreased amount of large membrane sheets as a result of the vigorous homogenization required to disrupt connective tissue and basement membranes surrounding acini. The low level of contamination of the plasma membrane fraction observed by electron microscopy is confirmed by assays of enzymes considered to mark specific organelles selectively. Membrane-associated enzymes are assayed except for secretion granules and lysosomes, where unique markers have not been identified. In these cases it is possible to examine only the extent of contamination by species present in the organelle content. Table I presents, for the purified plasmalemmal fraction, data that have been extracted from comprehensive studies of activity distributions throughout the fractionation procedure. Generally, recoveries of enzyme activities at each step of fractionation are very good; the exception is UDPgalactosyltransferase (EC 2.4.1.22), where recovery of activity at the final step of plasma membrane purification is incomplete. As can be seen in Table I, 0.4% or less of total homogenate activity for each marker is recovered in the final plasma membrane fraction, with at most a low level of enrichment for any contaminant. 5 j. F. Parks, Am. J. Anat. 108, 303 (1961). 6 A. L. Hubbard, D. A. Wall, and A. Ma, J. CellBiol. 96, 217 (1983).
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RAT PAROTID SECRETORY MEMBRANES
81
TABLE I ORGANELLE CONTAMINANT ANALYSIS OF THE PLASMALEMMAL FRACTION a
Enzyme
Organdie
Cytochrome c oxidased NADH-cytochrome c reductase ~ (rotenone-insensitive) UDPgalactosyltransferasef fl-N-Acetyl-D-glucosaminidaseg a-Amylase h
% Recoveryb
Relative specific activityc
Mitochondria Microsomes
0.27 0.40
1.17 1.76
Golgi complex Lysosomal content Granule content
0.39 0.29 0.008
1.70 1.26 0.04
Values presented are averages for a minimum of three experiments. t, Percentage recovery is based on 100% activity in the homogenate. , Relative specific activity = specific activity ratio, plasmalemmal fraction: homogenate. Protein was assayed according to Markwell et al.~2 d Cytochrome c oxidase [T. J. Peters, M. Muller, and C. de Duve, J. Exp. Med. 136, 1117 (1972)]. The first-order rate constant is used as a measure of activity [E. E. Max, D. B. P. Goodman, and H. Rasmussen, Biochim. Biophys. Acta 511, 224 (1978)]. Analysis of amine oxidase indicates a distribution very similar to that reported for cytochrome c oxidase. e G. L. Sottocasa, B. Kuylenstierna, L. Ernster, and A. Bergstrand, J. Cell Biol. 32, 415 (1967). This activity is indicated as a microsomal marker, although in other systems it has been shown to mark mitocfiondriai outer membranes, Golgi membranes [N. Borgese and J. Meldolesi, J. Cell Biol. 85, 501 (1980)], and even plasma membranes [E. D. Jarasch, J. Kartenbeck, G. Bruder, D. J. Morr6, and W. W. Franke, J. CellBiol. 80, 37 (1979)]. Thus the recovery and purification probably reflect the sum of contamination by a variety of organelles. fUDPgalactosyltransferase: B. Fleischer, this series, Vol. 31, p. 180. The assay uses [3H]UDPgalactose in the presence of 2 m M ATP and 2 mg of asialogalactofetuin per milliliter. g fl-N-Acetyl-D-glucosaminidase[J. Findlay, G. A. Levvy, and C. A. Marsh,Biochem. J. 669, 467 (1958)]. h a-Amylase. P. Bernfeld, this series, Vol. 1, p. 149. Preparation
of Secretion
Granule Membranes
Precursor Fraction: Secretion Granules. As starting material for granule membrane purification, secretion granules have been isolated by a procedure that capitalizes on their high density in hyperosmotic sucrose solutions. Routinely, 4.5 - 5.0 g of tissue (corresponding to the parotid glands of 14- 16 rats starved overnight) thoroughly cleaned of lymph nodes and surrounding connective tissue are used in a procedure modified from that developed by Castle el al. 7 for purification of granules from rabbit parotid glands. Processing involved the following steps carried out at 4 o: (a) mincing of tissue with razor blades and homogenization--10 sec with a Polyton (1900 rpm) followed by five strokes ( 1300 rpm) with a Brendler homogenizer-- in 0.3 M 7 j. D. Castle, J. D. Jamieson, and G. E. Palade, J. CellBiol. 64, 182 (1975).
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SPECIALIZED METHODS
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sucrose, 10 m m imidazole, 0.5 m M MgClz, pH 7.1; (b) sedimentation (70 g X 30 sec) of large particulates and their rehomogenization (Brendler mortar and pestle only) in the same volume to yield a total homogenate, 5% tissue weight per volume; (c) low-speed centrifugation (7000 gay × min) to remove nuclei and cellular debris; (d) filtration of the supernatant through 20-gm nylon mesh and addition of EDTA to a final concentration of 5 mM; (e) recentrifugation (1.3 X 105 gav× min) of the supernatant on a sucrose step gradient having two layers containing 1.0 M and 2.0 M sucrose, respectively, each supplemented with 4% Ficol1400, 10 mMimidazole, and 5 m M EDTA; ( f ) collection of a crude granule fraction at the 1.0/2.0 M sucrose interface and adjustment of the suspension to contain 1.5 M sucrose, 4% Ficoll 400, 10 m M imidazole, 5 m M EDTA; (g) centrifugation (1.24 × 107 gav× min) in a sucrose step gradient having underlayers of 1.55 M and 2.0 M sucrose and an overlayer of 0.8 M sucrose (each containing the same additives as the crude granule load); (h) collection of the purified granules from the 1.55 - 2.0 M interface. Using the secretory protein a-amylase as an approximation of a granule-specific marker for parotid tissue, we routinely recover ~ 22% of the total activity of the homogenate in the purified fraction. Mitochondria constitute the only significant organelle contaminant both morphologically and biochemically; 1.7% of the amine oxidase (flavin-containing) activity (an outer membrane marker8) of the homogenate is located in the granule fraction? To prepare purified granules for lysis, sucrose is diluted to 0.9 M using 0.2 M sucrose, 10 m M imidazole, and 5 m M EDTA, and the granules are pelleted by centrifugation (1.6 × 106 gay X min). The dilution typically results in a 15 -20% loss of granules by lysis, which is thus far unavoidable.
Reagents for Granule L ysis and Membrane Purification Lysis medium: 0.19 M KCI, 10 m M imidazole, 5 m M EDTA, pH 7.1 Buffered sucroses: 2.0 M, 0.9 M sucrose each containing 0.19 M KCI, 10 m M imidazole, 5 m M EDTA, pH 7.1 Procedure. The protocol developed for lysis takes advantage of the known lability of parotid secretion granules in KCl-containing media. ~° Purified granules are resuspended and diluted to 30 ml in lysis medium and maintained at 0 ° for 12 hr; clearing of the suspension indicates extensive lysis. In order to separate the low-density granule membranes from organelle contaminants and soluble content proteins, the lysate is adjusted to a 8 M. R. Castro Costa, S. Edelstein, C. M. Castiglione, H. Chao, and X. O. Breakfield, Biochem. Genet. 18, 577 (1980). 9 We are currently modifying the granule purification procedure to reduce the level of mitochondrial contamination further. ~oM. Sehramm, R. Ben-Zvi, and A. Bdolah, Biochem. Biophys. Res. Commun. 18, 446 (1965).
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RAT PAROTID SECRETORY MEMBRANES
83
final sucrose concentration of 1 M (using buffered 2 M sucrose), overlaid with buffered 0.9 M sucrose and lysis medium, respectively, and subjected to centrifugation (1.5 × 107 gav× min) in a Beckman SW 27.1 rotor. Granule membranes band at the lysis medium-0.9 M interface, whereas residual contaminants (mitochondria) and unlysed granules sediment into a pellet. The membranes are collected, diluted to 0.07 M sucrose with lysis medium, and pelleted by centrifugation (1.5 X 107 gay × min). Resuspended membranes are used for assays and morphology. Electron Microscopic Observation of the Granule Membrane Fraction Processing of secretion granule membranes for electron microscopy employs the same procedure already outlined for plasma membranes. The fraction is very homogeneous in appearance, consisting of closed, smoothsurfaced vesicles 0 . 3 - 0 . 8 / t m in diameter with no evidence of residual granule content (see Fig. 3). The diameters are smaller than those of intact granules ( ~ l / t m ) indicating that the vesicles represent fragments of the original membranes. Estimation of Mitochondrial and Soluble Secretory Contaminants Since mitochondria constitute the principal organelle contaminant of the granule fraction, the distribution of amine oxidase activity is followed during purification of granule membranes. With 90- 100% recovery of total activity, 3- 4% of that originally present in the granule lysate is detected in the membrane fraction. On the basis of the specific activity (units of amine oxidase per micromole of lipid phosphorus 1~and per milligram of protein ~2) of a purified parotid mitochondrial fraction, we estimate that a maximum ~3 of 1% of the lipid and 3% of the protein of the granule membrane fraction could be contributed by mitochondrial contamination. Contamination of granule membranes by secretory proteins is estimated by two means: (a) by following routinely the distribution of a-amylase during subfraction~tion of granule lysates; (b) for a more comprehensive examination, by adding biosynthetically labeled rat parotid secretory proteins (prepared in the manner of Castle et aL7)to granules during lysis, since this reagent is a more representative marker of total granule content. 1~G. R. Bartlett. J. Biol. Chem. 234, 446 (1959). ~2 M. A. K. Markwell, S. M. Hass, L. L. Bieber, and N. E. Tolbert, Anal. Biochem. 37, 206 (1978). ~3The estimated levels of contamination constitute upper limits because the most probable contaminants of the granule membrane fraction are outer mitochondrial membranes rather than intact mitochondria. The former will have much higher amine oxidase specific activities, hence will contribute less lipid and protein per unit of activity as contaminants.
84
SPECIALIZED METHODS
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FIG. 3. Electron micrograph of the secretion granule membrane fraction. Membranes appear as smooth-surfaced, mostly closed vesicles, in some cases multivesicular, apparently owing to resealing of larger membrane pieces around smaller membrane fragments. Bar = 1 /tm.
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RAT PAROTID SECRETORY MEMBRANES
85
Radioactive protein found in the final fraction (detected by scintillation counting and by fluorography 14 of 3H-labeled polypeptides resolved in SDS-gel electrophoretograms) is thought to compete and hence is used to approximate the extent of trapping and/or adsorption of residual content to the purified granule membranes. In both cases the membranes are freed of more than 99.99% of the contaminant markers. Although these levels of contaminant removal suggest that the granule membranes are highly purified, residual amylase in the membrane fraction is estimated to represent 5% of the total protein of this fraction (based on the specific activity of purified rat parotid amylase: 2780 units per milligram ofproteinlS). Consequently, we estimate that, taken together, mitochondrial protein and residual secretory protein constitute at most 10-15% of the protein of the granule membrane fraction. Enzyme Activities Associated with the Plasmalemma and Secretion Granule Membranes
Assays for several enzymes known to mark plasma membranes of other cell types indicate that our parotid plasmalemmal fraction is not significantly enriched in alkaline phosphatase and leucyl aminopeptidase (l A-fold and 1.2-fold higher specific activities than the homogenate, respectively) and is only modestly enriched in 5'-nucleotidase (3.8-fold over the homogenate). As shown in Table II, two enzymes, sodium-potassium adenosine triphosphatase (Na+,K+-ATPase) and 7-glutamyltransferase (7-glutamyltranspeptidase; GGTPase) exhibit large enrichments. Although distributions of these markers are discussed in detail elsewhere,16 several features are worthy of mention here. I. The low enrichments for alkaline phosphatase and 5'-nucleotidase favor an origin of the plasma membrane sheets in acinar cells, since histochemical studies have suggested that these activities are associated with other parotid cell types. 17,18 Consequently, these markers are of little use where the isolation of membranes involved in exocytosis in parotid tissue is intended; morphological observations provide the key means of analysis for apical secretory surface. 2. Na+,K+-ATPase is a classical marker of the basolateral domain in polarized epithelia. Although the specific activity increases substantially in discontinuous gradient centrifugation, ~ 70% of the activity of P5 pellets 14 R. A. Laskey and A. D. Mills, Eur. J. Biochem. 56, 335 (1975). 15 T. G. Sanders and W. J. Rutter, Biochemistry 11, 130 (1972). 16 p. Arvan and J. D. Castle, J. Cell Biol. 95, 8 (1982). ~7j. R. Garrett and P. A. Parsons, Histochem. J. 5, 463 (1973). ~8S. Yamashina and K. Kawai, Histochemistry 60, 255 (1979).
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SPECIALIZED M E T H O D S
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TABLE II ENZYMES MARKING PAROTID PLASMALEMMA AND SECRETION GRANULE MEMBRANESa
Relative Enzyme Na+,K+-ATPase b
Fraction Homogenate
Plasma membranes Homogenate
Secretion granules (23% o f total amylase) Granule membranes GGTPase a
Homogenate
Plasma membranes Homogenate
Secretion granules (23% o f total amylase) Granule membranes
Specific activity (per/tmol lipid
Recovery (%)
specific activity
100 4.6 100 0.4
1.0 20. l 1.0 0.07
-----
ND ~
--
--
100 5.9 100 4.4
1.0 25.6 1.0 0.3
-0.68 ---
2.8
30.6
0.21
phosphate)
a With the exception of data reported for Na+,K+-ATPase recovery in granules, which represent the average o f two experiments, all data reported constitute the averages for at least six different experiments. t, Na+,K+_ATPase has been shown to be equivalent in rat parotid tissue to potassium-stimulated p-nitrophenylphosphatase and is quantitated by measuring p-nitrophenol formation in an assay mixture containing 5 mMp-nitrophenylphosphate, 0.1 M imidazole, pH 7.5, 0.01 M MgCIz, 0.75 m M EGTA, 0.01 M NaCI, and 0.01 M either KCI or choline chloride. " N D , not detected. a y-Glutamyltransferase [S. S. Tate and A. Meister, J. Biol. Chem. 249, 7593 ( 1974)].
rather than floats to the interface band. This activity is likely to be associated with large plasma membrane-containing structures intimately contacting basement membranes. We are anticipating that for the purified fraction the Na+,K+-ATPase activity resides in the lateral membrane segments emanating from the junctional complexes in the plasmalemmal sheets. 3. 7-Glutamyltransferase has been shown to be highly enriched on the free surface of epithelial cells, notably those specialized for secretion and absorption.~9 We favor the notion of a similar localization in parotid acinar cells, especially since, in contrast to Na+,K+-ATPase, ~ 70% of the GGTPase activity of P5 is found in the interface band. Consequently, these two enzymes may constitute markers for distinct plasmalemmal domains in parotid acinar cells and in the isolated membrane sheets. 4. 7-Glutamyltransferase is present in secretion granule fractions at an average yield (six separate preparations) of 4.4% of the total homogenate ~9 A. Meister aod S. S. Tate, Annu. Rev. Biochem. 45 559 (1976).
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RAT PAROTID SECRETORY MEMBRANES
87
activity. We estimate that ~ 17% of the total tissue GGTPase is associated with secretion granules according to the assumption that amylase approximates a granule-specific marker and that we recover about one-quarter (23%) of the total tissue amylase activity in the purified granule fraction. Since granule-associated protein represents a large percentage of the total protein of parotid, GGTPase is actually depurified (threefold in Table II) in preparing a secretion granule fraction. The recovery of GGTPase activity in the steps following granule lysis is 98%. After taking into account the loss of purified granules during sucrose dilution and centrifugation prior to lysis, we find that 80% of the GGTPase activity of the granule fraction is recovered in the purified membranes. Most of the remaining activity is found in the load and pellet fractions of the discontinuous gradient. 5. The presence of GGTPase activity in granule membranes (and the apparent absence of Na+,K+-ATPase) suggests that there is a compositional overlap of membranes that serve as fusion partners during exocytosis. Further, the overlap is qualitative but apparently not quantitative, since the specific activity of GGTPase relative to lipid phosphorus (as a reliable normalization for this membrane-associated enzyme) for the granule membrane fraction is 30% that of the plasmalemmal fraction. Considering that the plasma membrane-associated activity may be concentrated in regions comprising the apical surface, the quantitative difference in surface concentration may be as much as 10-fold. 2° These studies provide the rationale for a detailed immunocytochemical study of GGTPase localization. Further, they form the basis for future detailed investigations of compositional similarities and differences between membranes that attain functional continuity and of the resulting implications to the mechanisms of both exocytosis and membrane recycling.
20 At this point 10-fold clearly represents an estimate for two reasons: ( 1) The value is based on data obtained with purified fractions, rough microsomes; and mitochondria (which are low-level contaminants of the plasmalemmal fraction) contain very low activities of GGTPase. Hence their contribution of lipid phosphorus exceeds their contribution of GGTPase to the plasmalemmal fraction. (2) At present we do not have quantitative estimates of the fraction of surface area of isolated plasma membrane that constitutes apical region.