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On the nature of the molecular heterogeneity of rat parotid amylase
Archs oral Bid. Vol. 17, pp. 595-600,
1972. Pergamon
SHORT
Press. Printed
in Great
Britain.
COMMUNICATION
ON THE NATURE OF THE MOLECULAR HETEROGENEITY OF RAT PAROTID AMYLASE M. R. ROBINOVITCH and L. M. SREEBNY Department of Oral Biology, School of Dentistry and Centre for Research in Oral Biology, University of Washington, Seattle, Washington 98105, U.S.A. Summary-Amylase was isolated from rat parotid tissue and its multiple forms were studied utilizing analytical, SDS-, and isoelectric focusing polyacrylamide gel electrophoresis. Investigations on total amylase, and on fractions prepared by preparative polyacrylamide electrophoresis revealed a major amylase isozyme which appears able to convert into two other isoenzyme forms, and a fourth minor isozyme apparently unrelated to the others.
AMYLASEis among the growing list of mammalian enzymes existing in more than one molecular form within the same species. Non-identity of amylases of different organs, as well as within the same organ, has been established. Thus, pancreatic amylase has been distinguished from salivary amylase in several species (SICK and NIELSEN, 1964; NORBY, 1964; OCER and BISCHOPS, 1966; BERK et al., 1966; NIELSEN, 1969; MALACINSKI and RUTTER, 1969; ALLAN, ZAGER and KELLER, 1970), and multiple forms have been shown to exist within both pancreas and salivary glands or their secretions (Muus and VNENCHAK, 1964; OGER and BISCHOPS, 1966; LAMBERTSand MEYER, 1967; WOLF and TAYLOR, 1967; Aw and HOBBS, 1968; STEINER and KELLER, 1968; NIELSEN, 1969 ; MALACINSKI and RUTTER, 1969 ; BOETTCHERand DE LA LANDE, 1969 ; KAUFFMAN et al., 1970). The molecular basis for the differences between various amylase isozymes has not been elucidated. In order to investigate the various possibilities for such differences, we have been studying the series of four amylase isozymes found in the rat parotid gland and saliva (ROBINOVITCH and SREEBNY, 1969). Our results, using preparative and several analytical polyacrylamide electrophoretic procedures, have led us to propose that there is more than one basis for molecular differences between the rat parotid amylase isozymes. Three of the four forms appear to have nearly the same, if not identical, molecular weight, and the main species in this group appears to convert to the other two forms under certain conditions of pH and temperature. The fourth isozyme seems not to be related to this family. The amylase used in the following experiments was isolated from the homogenates of parotid glands of adult, male, Sprague-Dawley rats as reported previously (ROBINOVITCH and SREEBNY, 1970), utilizing the technique of precipitation with glycogen in the presence of 40 per cent ethanol (SCHRAMM and LOYTER, 1966). The removal of glycogen dextrins from the amylase molecules, after washing, either by the charcoalCelite column step described by SCHRAMMand LOYTER (1966), or by the digestion of 595
596
M. R. ROBINOVI~~CH AND L. M. SREEBNY
the dextrins by amyloglucosidase as described by MALACINSU and RUTTER (1969), was unnecessary since the dextrins were removed during electrophoresis in polyacrylamide gel. The neutral sugar content of whole amylase, before and after passage through the preparative acrylamide gel apparatus, was assayed using the phenol-sulphuric acid procedure of DUBOIS et al. (1956). A standard curve for glucose was used. Amylase, before electrophoresis, contained the equivalent of 0.960 i 0.258 mg glucose/mg protein, and amylase, reconstituted and concentrated following electrophoresis, contained the equivalent of 0.0101 f 0.0007 mg glucose/mg protein. Amylase, isolated in this fashion and dissolved, finally, in 0.02 M NaH,PO, and 0.01 M NaCl at pH 6.9, gave an anionic analytical polyacrylamide gel pattern as shown in Fig. la. At the concentration of protein usually used for electrophoresis, three of the four isozyme bands are apparent, the appearance of the fourth and most anionic band requiring further loading of the gels with amylase due to its low concentration (ROBINOVIXH and SREEBNY, 1969). As a first step in defining the molecular differences between these amylase isozymes, preparative polyacrylamide gel electrophoresis was employed in an effort to separate and isolate the various species. The conditions for this separation have already been reported and the elution of a single prominent peak of protein with shoulders in both sides has been described (ROBINOVITCH and SREEBNY, 1970). This elution pattern is consistent with the analytical polyacrylamide gel results (Fig. la) suggesting that the prominent peak in the elution diagram corresponds to the heaviest disc on the analytical gel, referred to as band A,. When either the individual unconcentrated peak fractions or the recombined and concentrated fractions constituting the main peak area were analyzed on polyacrylamide gel, two heavy bands and a lighter band (bands A,, A, and A4 respectively) were seen rather than a single heavy band as would be expected (Fig. lb). The band with the least mobility in whole amylase (band A,) was not seen in the peak samples. When all the fractions of a preparative run were combined, concentrated and analysed in the same way, a pattern as shown in Fig. lc was obtained. It appeared that a shift had occurred between bands A,, A3 and A, in that A, diminished and A, and A, increased in proportion. Band A, appeared unchanged. If the whole amylase was suspended at pH 8.1, the pH at which the preparative electrophoresis and concentration was performed, and stored in the cold for a week or so, this shift also occurred, whereas it did not if the amylase was stored at pH 6.9. This shift occurred much more rapidly if the amylase was allowed to remain at room temperature at pH S. 1. A similar conversion amongst human salivary amylase isozymes has recently been reported (KAUFFMAN et al., 1970). As the separation in analytical polyacrylamide gels is dependent on both net charge and molecular size, the role of molecular weight alone was investigated by utilizing the dodecyl sulphate-polyacrylamide gel electrophoresis technique as described by WEBER and OSBORN (1969). Whole amylase samples, regardless of whether the shift described above had occurred, demonstrated one faint and one heavy band with molecular weights of 62,400 and 56,700 respectively according to their relative mobilities in the SDS-polyacrylamide gels (Figs. 2a and c). The peak samples from preparative electrophoretic runs showed only the heavy band (Fig. 2b). It therefore
MOLECULAR
HETEROCiENEtTY
OF RAT PAROTID
AMYLAsE
597
appeared that the amylase isozymes referred to in the analytical gels as A,, A3 and A, have molecular weights indistinguishable at the level of resolution of this system, Isozyme Al, not seen in the patterns of the peak fractions and apparently not involved in the shift, appears to have a distinctly different and larger molecular weight. The implication of the above findings is that isozymes A,, A, and A, are separated by ordinary analytical electrophoresis on the basis of differences in net charge. In order to test this supposition, isoelectric focusing in polyacrylamide gels was applied. Samples were analyzed using the L.K.B. “Ampholine” carrier ampholytes either of the pH 5-8 range or the pH 3-10 range according to the method described by WRIGLEY (1968). Gels were stained with bromophenol blue (AWDEH, 1969). Whole amylase at pH 6.9 demonstrated one major band (Fig. 3a), while both the peak fractions and the reconstituted whole amylase at pH 8.1 demonstrated two major bands (Figs. 3b and 3~). When the isoelectric focusing gels were tested for amylase activity on starch substrate slides before staining (ROBINOVITCH and SREEBNY, 1969), the relative amounts of protein, as revealed by the degree of staining in the gels, compared favorably with the relative degrees of starch hydrolysis in all the bands. Therefore, these bands do appear to represent intact amylase. If it can be assumed that these major bands correspond to those seen in the respective analytical gels, then band A, in Fig. la appears to be represented by the major band in Fig. 3a, and bands A2 and A, in Figs. lb and c correspond to the two major bands seen in Figs. 3b and 3c. The location, in the isoelectric focusing gel, of the less prominent band, A3 of Fig. la, is uncertain but may simply be the poorly resolved anodal edge of band A, in Fig. 3a. (Technical difficulties made it impossible to determine which minor bands in the isoelectric focusing gels might correspond to isozymes A, and A4). Therefore, the results of isoelectric focusing are consistent with the interpretation of the results of the analytical and SDS-polyacrylamide gel electrophoresis, in that isozymes A2 and A, (and perhaps A4) appear to be separated on the basis of a net charge difference due to differences in isoelectric points rather than on the basis of differences in molecular weight. The phenomenon of shift of isozyme A,, at least in part, to isozymes A, and A,, makes it unlikely that these proteins possess inherently different primary structures that would account for the differences in migration in the analytical electrophoretic system. It is more likely that this shift is brought about by some type of chemical modification of the isozyme A, molecule. Proteolysis also seems an unlikely possibility since there appears to be no measurable proteolytic activity in gland homogenates or in secretory granule fractions (SCHRAMMand DANON, 1961), and the molecular weights of isozymes AZ, A3 and A4 appear very similar, if not identical. Other chemical modifications could cause such a shift, however. For example, de-amidation has been reported to produce multiple forms of rat cytochrome c (FLATMARK and SLETTEN, 1968) and human carbonic anhydrase (FUNAKOSHI and DEUTSCH, 1969) under conditions similar to those reported here for amylase. The nature of the difference between isozyme A, and the other amylase isozymes remains unknown. Since the molecular weight appears to be significantly greater for A, than the AZ-A4 family, the difference may be in the primary protein structure or some other macromolecular component such as a polysaccharide, as suggested in the
M. R. ROBINOVITCHAND L. M. SREEBNY
598 case of
human parotid amylase isozymes by KAUFFMAN et al. (1970). Future experiments are planned in order to investigate these possibilities. The phenomenon of conversion of one isozyme form to another under near-physiological conditions underlines the need for very cautious interpretations of the role of genetic expression as a basis for the presence of multiple molecular forms. Certainly, it seems that, in the case of the rat parotid gland, and the human parotid gland (KAUFFMAN et al., 1970), some of the amylase isozymes may be the result of chemical modification of an enzyme molecule after translation, and these chemical modifications may be non-enzymic in nature. Acknowledgement-This study was supported by Research Career Grant 5-K3DE-33 195 from the National Institutes of Health. We thank Miss JEANNE IVERSEN and Dr. P. J. KELLER for their help in the preparation
of this manuscript.
R&urn&-L’amylase a ttt isolee du tissu de la parotide du rat et ses formes multiples ont CtCCtudiCes en utilisant analytiquement le SDS et en mettant du point isoClectriquement 1’Clectrophoriise de gel de la polyacrylamide. Des investigations sur l’amylase totale et sur des fractions preparies par electrophorese prkparatoire polyacrylamidique ont rev&? un isozyme majeur d’amylase qui parait capable de se convertir dans deux autres formes d’isoenzyme et un quatrieme isozyme apparement non-apparent6 aux autres. Zusammenfassung-Die Amylase wurde vom Parotidgewebe der Ratte getrennt und ihre vielfaltigen Formen studiert, mit Verwendung von analytischen SDS- und isoelektrischer fokussierter Polyakrylamid Gel-Elektrophorese. Untersuchungen von vollst%ndiger Amylase und auf prgparierten Teilchen durch vorbereitende Polyacrylamid Elektrophorese zeigten einen griisseren Amylase Isozym, welcher imstande zu sein scheint, sich in zwei andere Iso-Enzymformen zu verwandeln, und einen vierten kleineren Isozym, der nicht mit den anderen zu verbunden sein scheint.
REFERENCES ALLAN, B. J., ZAGER, N. I. and KELLER, P. J. 1970. Human pancreatic elastase and trypsinogen. Arch. biochem. Biophys. 136, 529-540.
proteins:
amylase, pro-
Aw, S. E. and HOBBS,J. R. 1968. Immunokinetic differences among human pancreatic, salivary and milk a-isoamylases. Immunochemistry 5, 135-137. AWDEH, Z. L. 1969. Staining method for proteins after isoelectric focusing in polyacrylamide gel. Science Tools 16, 42-43. BERK, J. E., HAYASHI,S., SEARCH,R. L. and HIGHTOWER,N. C., JR. 1966. Differentiation of parotid and pancreatic amylases in human serum. Am. J. Digest Dis. 11, 695-701. BOETTCHER,B. and DE LA LANDE, F. A. 1969. Electrophoresis of human saliva and identification of inherited variants of amylase isozymes. Aust. J. exp. Biol. Med. Sci. 47,97-103. D~OIS, M., GILLES,K. A., HAMILTON,J. K., REBERS,P. A. and SMITH,F. 1956. Calorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350-356. FLATMARK,T. and SLETTEN,K. 1968. Multiple forms of cytochrome c in the rat. J. biol. Chem. 243, 1623-1629. FUNAKOSHI,S. and DEUTSCH,H. F. 1969. Human carbonic anhydrases. J. biol. Chem. 244,3438-3446. KAUFFMAN,D. L., ZAGER, N. I., COHEN, E. and KELLER, P. J. 1970. The isoenzymes of human parotid amylase. Arch. Biochem. Biophys. 137, 325-339. LAMBERTS,B. L. and MEYER, T. S. 1967. In: Secretory Mechanisms of Salivary Glands (edited by SCHNEYER,L. H. and SCHNEYER,C. A.), pp. 313-325. Academic Press, New York. MALACINSKI,G. M. and RUTTER,W. J. 1969. Multiple molecular forms of a-amylase from the rabbit. Biochemistry
8, 4382-4390.
Muus, J. and VNENCHAK,J. M. 1964. Isozymes of salivary amylase. Nature, Land. 204,283-285.
MOLECULARHETEROGENEITY OF RAT PAROTIDAMYLASE
599
NIELSEN, J. T. 1969. Genetic studies of the amylase isoenzymes of the bank vole, Clethreonomys gIareola. Hereditas 61, 40&412. NORBY, S. 1964. Electrophoretic non-identity of human salivary and pancreatic amylases. Exp. Cell Res 36,663-636. OGER, A. and BISCHOPS,L. 1966. Les iso-enzymes de l’amylase. Clin. Chim. Acta 13,670-676. ROBINOVITCH,M. R. and SREEBNY,L. M. 1969. Separation and identification of some of the protein components of rat parotid saliva. Archs oral Biol. 14,935-949. ROBINOVITCH,M. R. and SREEBNY,L. M. 1970. Separation of rat parotid isoamylases by preparative polyacrylamide gel electrophoresis. Archs oral Biol. 15, 1381-1384. SCHRAMM, M. and DANON, D. 1961. The mechanism of enzyme secretion by the cell. I. Storage of amylase in the zymogen granules of the rat parotid gland. Biochim. biophys. Acta. 50, 102-112. SCHRAMM, M. and LOYTER, A. 1966. In: Methods in Enzymology (edited by COLOWICK, S. P. and KAPLAN, N. O.), Vol. VIII, pp. 533-537. Academic Press, New York. SICK, K. and NIELSEN, J. T. 1964. Genetics of amylase isozymes in the mouse. Heredifus 51,291-296. STEINER,J. C. and KELLER, P. J. 1968. An electrophoretic analysis of the protein components of human saliva. Archs oral BioL 13, 1213-1222. WEBER. K. and OSBORN. M. 1969. The reliabilitv of molecular weight determinations by dodecyl sulfate-polyacryl&ide gel electrophoresis. j. biol. Chem. 244, &OM12. WOLF, R. 0. and TAYLOR, L. L. 1967. Isoamylase of human parotid saliva. Nature, Lond. 213,11281129. WRIGLEY, C. 1968. Gel electrofocusing. Science Tools, 15, 17.
PLATE 1 OVERLEAF
600
M. R. ROBINOVITCH AND L. M. SREEBNY
PLATE 1 FIG. 1. Anionic analytical polyacrylamide gel electrophoresis of (a) 140 units of whole amylase stored in buffer at pH 6.9; (b) 140 units of amylase concentrated from the pooled peak fractions of a preparative polyacrylamide electrophoresis in buffer at pH 8.1; (c) 140 units of whole amylase concentrated from the total fractions of a preparative polyacrylamide electrophoresis in buffer at pH 8 * 1. The amylase isozymes are labelled A1 through A4 beginning with the band closest to the origin. The anode is toward the bottom of the figure. When tested on starch slides before staining, these bands demonstrated amylase activity proportional to the protein staining (ROBINOVITCHand SREEBNY,1969). FIG.
2. SDS-polyacrylamide gel electrophoresis of (a) 20 units of whole amylase stored in buffer at pH 6.9; (b) 20 units of amylase concentrated from the pooled peak fractions of a preparative polyacrylamide electrophoresis in buffer at pH 8.1; (c) 20 units of whole amylase concentrated from the total fractions of a preparative polyacrylamide electrophoresis in buffer at pH 8.1. In order to compute molecular weights from the relative mobilities of the bands, the following standards were run: bovine serum albumin purchased from Armour Laboratories, Chicago, Illinois (M.W. 68,000), crystalline catalase (beef liver) purchased from Worthington Biochemical Corp., Freehold, New Jersey (M.W. 6O,OOO),and crystallized and lyophilized ovalbumin (grade V) purchased from Sigma Chemical Co., St. Louis, Missouri (M.W. 43,000). FIG. 3. Isoelectric focusing electrophoresis of (a) 800 units of whole amylase stored in buffer at pH 6.9; (b) 800 units of amylase concentrated from the pooled peak fractions of a preparative polyacrylamide electrophoresis in buffer at pH 8.1; (c) 800 units of whole amylase concentrated from the total fractions of a preparative polyacrylamide electrophoresis in buffer at 8.1. The cathode is toward the bottom of the figure.
PLATE A.O.B.
1
f.p. 600
1972. Pergamon
SHORT
Press. Printed
in Great
Britain.
COMMUNICATION
ON THE NATURE OF THE MOLECULAR HETEROGENEITY OF RAT PAROTID AMYLASE M. R. ROBINOVITCH and L. M. SREEBNY Department of Oral Biology, School of Dentistry and Centre for Research in Oral Biology, University of Washington, Seattle, Washington 98105, U.S.A. Summary-Amylase was isolated from rat parotid tissue and its multiple forms were studied utilizing analytical, SDS-, and isoelectric focusing polyacrylamide gel electrophoresis. Investigations on total amylase, and on fractions prepared by preparative polyacrylamide electrophoresis revealed a major amylase isozyme which appears able to convert into two other isoenzyme forms, and a fourth minor isozyme apparently unrelated to the others.
AMYLASEis among the growing list of mammalian enzymes existing in more than one molecular form within the same species. Non-identity of amylases of different organs, as well as within the same organ, has been established. Thus, pancreatic amylase has been distinguished from salivary amylase in several species (SICK and NIELSEN, 1964; NORBY, 1964; OCER and BISCHOPS, 1966; BERK et al., 1966; NIELSEN, 1969; MALACINSKI and RUTTER, 1969; ALLAN, ZAGER and KELLER, 1970), and multiple forms have been shown to exist within both pancreas and salivary glands or their secretions (Muus and VNENCHAK, 1964; OGER and BISCHOPS, 1966; LAMBERTSand MEYER, 1967; WOLF and TAYLOR, 1967; Aw and HOBBS, 1968; STEINER and KELLER, 1968; NIELSEN, 1969 ; MALACINSKI and RUTTER, 1969 ; BOETTCHERand DE LA LANDE, 1969 ; KAUFFMAN et al., 1970). The molecular basis for the differences between various amylase isozymes has not been elucidated. In order to investigate the various possibilities for such differences, we have been studying the series of four amylase isozymes found in the rat parotid gland and saliva (ROBINOVITCH and SREEBNY, 1969). Our results, using preparative and several analytical polyacrylamide electrophoretic procedures, have led us to propose that there is more than one basis for molecular differences between the rat parotid amylase isozymes. Three of the four forms appear to have nearly the same, if not identical, molecular weight, and the main species in this group appears to convert to the other two forms under certain conditions of pH and temperature. The fourth isozyme seems not to be related to this family. The amylase used in the following experiments was isolated from the homogenates of parotid glands of adult, male, Sprague-Dawley rats as reported previously (ROBINOVITCH and SREEBNY, 1970), utilizing the technique of precipitation with glycogen in the presence of 40 per cent ethanol (SCHRAMM and LOYTER, 1966). The removal of glycogen dextrins from the amylase molecules, after washing, either by the charcoalCelite column step described by SCHRAMMand LOYTER (1966), or by the digestion of 595
596
M. R. ROBINOVI~~CH AND L. M. SREEBNY
the dextrins by amyloglucosidase as described by MALACINSU and RUTTER (1969), was unnecessary since the dextrins were removed during electrophoresis in polyacrylamide gel. The neutral sugar content of whole amylase, before and after passage through the preparative acrylamide gel apparatus, was assayed using the phenol-sulphuric acid procedure of DUBOIS et al. (1956). A standard curve for glucose was used. Amylase, before electrophoresis, contained the equivalent of 0.960 i 0.258 mg glucose/mg protein, and amylase, reconstituted and concentrated following electrophoresis, contained the equivalent of 0.0101 f 0.0007 mg glucose/mg protein. Amylase, isolated in this fashion and dissolved, finally, in 0.02 M NaH,PO, and 0.01 M NaCl at pH 6.9, gave an anionic analytical polyacrylamide gel pattern as shown in Fig. la. At the concentration of protein usually used for electrophoresis, three of the four isozyme bands are apparent, the appearance of the fourth and most anionic band requiring further loading of the gels with amylase due to its low concentration (ROBINOVIXH and SREEBNY, 1969). As a first step in defining the molecular differences between these amylase isozymes, preparative polyacrylamide gel electrophoresis was employed in an effort to separate and isolate the various species. The conditions for this separation have already been reported and the elution of a single prominent peak of protein with shoulders in both sides has been described (ROBINOVITCH and SREEBNY, 1970). This elution pattern is consistent with the analytical polyacrylamide gel results (Fig. la) suggesting that the prominent peak in the elution diagram corresponds to the heaviest disc on the analytical gel, referred to as band A,. When either the individual unconcentrated peak fractions or the recombined and concentrated fractions constituting the main peak area were analyzed on polyacrylamide gel, two heavy bands and a lighter band (bands A,, A, and A4 respectively) were seen rather than a single heavy band as would be expected (Fig. lb). The band with the least mobility in whole amylase (band A,) was not seen in the peak samples. When all the fractions of a preparative run were combined, concentrated and analysed in the same way, a pattern as shown in Fig. lc was obtained. It appeared that a shift had occurred between bands A,, A3 and A, in that A, diminished and A, and A, increased in proportion. Band A, appeared unchanged. If the whole amylase was suspended at pH 8.1, the pH at which the preparative electrophoresis and concentration was performed, and stored in the cold for a week or so, this shift also occurred, whereas it did not if the amylase was stored at pH 6.9. This shift occurred much more rapidly if the amylase was allowed to remain at room temperature at pH S. 1. A similar conversion amongst human salivary amylase isozymes has recently been reported (KAUFFMAN et al., 1970). As the separation in analytical polyacrylamide gels is dependent on both net charge and molecular size, the role of molecular weight alone was investigated by utilizing the dodecyl sulphate-polyacrylamide gel electrophoresis technique as described by WEBER and OSBORN (1969). Whole amylase samples, regardless of whether the shift described above had occurred, demonstrated one faint and one heavy band with molecular weights of 62,400 and 56,700 respectively according to their relative mobilities in the SDS-polyacrylamide gels (Figs. 2a and c). The peak samples from preparative electrophoretic runs showed only the heavy band (Fig. 2b). It therefore
MOLECULAR
HETEROCiENEtTY
OF RAT PAROTID
AMYLAsE
597
appeared that the amylase isozymes referred to in the analytical gels as A,, A3 and A, have molecular weights indistinguishable at the level of resolution of this system, Isozyme Al, not seen in the patterns of the peak fractions and apparently not involved in the shift, appears to have a distinctly different and larger molecular weight. The implication of the above findings is that isozymes A,, A, and A, are separated by ordinary analytical electrophoresis on the basis of differences in net charge. In order to test this supposition, isoelectric focusing in polyacrylamide gels was applied. Samples were analyzed using the L.K.B. “Ampholine” carrier ampholytes either of the pH 5-8 range or the pH 3-10 range according to the method described by WRIGLEY (1968). Gels were stained with bromophenol blue (AWDEH, 1969). Whole amylase at pH 6.9 demonstrated one major band (Fig. 3a), while both the peak fractions and the reconstituted whole amylase at pH 8.1 demonstrated two major bands (Figs. 3b and 3~). When the isoelectric focusing gels were tested for amylase activity on starch substrate slides before staining (ROBINOVITCH and SREEBNY, 1969), the relative amounts of protein, as revealed by the degree of staining in the gels, compared favorably with the relative degrees of starch hydrolysis in all the bands. Therefore, these bands do appear to represent intact amylase. If it can be assumed that these major bands correspond to those seen in the respective analytical gels, then band A, in Fig. la appears to be represented by the major band in Fig. 3a, and bands A2 and A, in Figs. lb and c correspond to the two major bands seen in Figs. 3b and 3c. The location, in the isoelectric focusing gel, of the less prominent band, A3 of Fig. la, is uncertain but may simply be the poorly resolved anodal edge of band A, in Fig. 3a. (Technical difficulties made it impossible to determine which minor bands in the isoelectric focusing gels might correspond to isozymes A, and A4). Therefore, the results of isoelectric focusing are consistent with the interpretation of the results of the analytical and SDS-polyacrylamide gel electrophoresis, in that isozymes A2 and A, (and perhaps A4) appear to be separated on the basis of a net charge difference due to differences in isoelectric points rather than on the basis of differences in molecular weight. The phenomenon of shift of isozyme A,, at least in part, to isozymes A, and A,, makes it unlikely that these proteins possess inherently different primary structures that would account for the differences in migration in the analytical electrophoretic system. It is more likely that this shift is brought about by some type of chemical modification of the isozyme A, molecule. Proteolysis also seems an unlikely possibility since there appears to be no measurable proteolytic activity in gland homogenates or in secretory granule fractions (SCHRAMMand DANON, 1961), and the molecular weights of isozymes AZ, A3 and A4 appear very similar, if not identical. Other chemical modifications could cause such a shift, however. For example, de-amidation has been reported to produce multiple forms of rat cytochrome c (FLATMARK and SLETTEN, 1968) and human carbonic anhydrase (FUNAKOSHI and DEUTSCH, 1969) under conditions similar to those reported here for amylase. The nature of the difference between isozyme A, and the other amylase isozymes remains unknown. Since the molecular weight appears to be significantly greater for A, than the AZ-A4 family, the difference may be in the primary protein structure or some other macromolecular component such as a polysaccharide, as suggested in the
M. R. ROBINOVITCHAND L. M. SREEBNY
598 case of
human parotid amylase isozymes by KAUFFMAN et al. (1970). Future experiments are planned in order to investigate these possibilities. The phenomenon of conversion of one isozyme form to another under near-physiological conditions underlines the need for very cautious interpretations of the role of genetic expression as a basis for the presence of multiple molecular forms. Certainly, it seems that, in the case of the rat parotid gland, and the human parotid gland (KAUFFMAN et al., 1970), some of the amylase isozymes may be the result of chemical modification of an enzyme molecule after translation, and these chemical modifications may be non-enzymic in nature. Acknowledgement-This study was supported by Research Career Grant 5-K3DE-33 195 from the National Institutes of Health. We thank Miss JEANNE IVERSEN and Dr. P. J. KELLER for their help in the preparation
of this manuscript.
R&urn&-L’amylase a ttt isolee du tissu de la parotide du rat et ses formes multiples ont CtCCtudiCes en utilisant analytiquement le SDS et en mettant du point isoClectriquement 1’Clectrophoriise de gel de la polyacrylamide. Des investigations sur l’amylase totale et sur des fractions preparies par electrophorese prkparatoire polyacrylamidique ont rev&? un isozyme majeur d’amylase qui parait capable de se convertir dans deux autres formes d’isoenzyme et un quatrieme isozyme apparement non-apparent6 aux autres. Zusammenfassung-Die Amylase wurde vom Parotidgewebe der Ratte getrennt und ihre vielfaltigen Formen studiert, mit Verwendung von analytischen SDS- und isoelektrischer fokussierter Polyakrylamid Gel-Elektrophorese. Untersuchungen von vollst%ndiger Amylase und auf prgparierten Teilchen durch vorbereitende Polyacrylamid Elektrophorese zeigten einen griisseren Amylase Isozym, welcher imstande zu sein scheint, sich in zwei andere Iso-Enzymformen zu verwandeln, und einen vierten kleineren Isozym, der nicht mit den anderen zu verbunden sein scheint.
REFERENCES ALLAN, B. J., ZAGER, N. I. and KELLER, P. J. 1970. Human pancreatic elastase and trypsinogen. Arch. biochem. Biophys. 136, 529-540.
proteins:
amylase, pro-
Aw, S. E. and HOBBS,J. R. 1968. Immunokinetic differences among human pancreatic, salivary and milk a-isoamylases. Immunochemistry 5, 135-137. AWDEH, Z. L. 1969. Staining method for proteins after isoelectric focusing in polyacrylamide gel. Science Tools 16, 42-43. BERK, J. E., HAYASHI,S., SEARCH,R. L. and HIGHTOWER,N. C., JR. 1966. Differentiation of parotid and pancreatic amylases in human serum. Am. J. Digest Dis. 11, 695-701. BOETTCHER,B. and DE LA LANDE, F. A. 1969. Electrophoresis of human saliva and identification of inherited variants of amylase isozymes. Aust. J. exp. Biol. Med. Sci. 47,97-103. D~OIS, M., GILLES,K. A., HAMILTON,J. K., REBERS,P. A. and SMITH,F. 1956. Calorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350-356. FLATMARK,T. and SLETTEN,K. 1968. Multiple forms of cytochrome c in the rat. J. biol. Chem. 243, 1623-1629. FUNAKOSHI,S. and DEUTSCH,H. F. 1969. Human carbonic anhydrases. J. biol. Chem. 244,3438-3446. KAUFFMAN,D. L., ZAGER, N. I., COHEN, E. and KELLER, P. J. 1970. The isoenzymes of human parotid amylase. Arch. Biochem. Biophys. 137, 325-339. LAMBERTS,B. L. and MEYER, T. S. 1967. In: Secretory Mechanisms of Salivary Glands (edited by SCHNEYER,L. H. and SCHNEYER,C. A.), pp. 313-325. Academic Press, New York. MALACINSKI,G. M. and RUTTER,W. J. 1969. Multiple molecular forms of a-amylase from the rabbit. Biochemistry
8, 4382-4390.
Muus, J. and VNENCHAK,J. M. 1964. Isozymes of salivary amylase. Nature, Land. 204,283-285.
MOLECULARHETEROGENEITY OF RAT PAROTIDAMYLASE
599
NIELSEN, J. T. 1969. Genetic studies of the amylase isoenzymes of the bank vole, Clethreonomys gIareola. Hereditas 61, 40&412. NORBY, S. 1964. Electrophoretic non-identity of human salivary and pancreatic amylases. Exp. Cell Res 36,663-636. OGER, A. and BISCHOPS,L. 1966. Les iso-enzymes de l’amylase. Clin. Chim. Acta 13,670-676. ROBINOVITCH,M. R. and SREEBNY,L. M. 1969. Separation and identification of some of the protein components of rat parotid saliva. Archs oral Biol. 14,935-949. ROBINOVITCH,M. R. and SREEBNY,L. M. 1970. Separation of rat parotid isoamylases by preparative polyacrylamide gel electrophoresis. Archs oral Biol. 15, 1381-1384. SCHRAMM, M. and DANON, D. 1961. The mechanism of enzyme secretion by the cell. I. Storage of amylase in the zymogen granules of the rat parotid gland. Biochim. biophys. Acta. 50, 102-112. SCHRAMM, M. and LOYTER, A. 1966. In: Methods in Enzymology (edited by COLOWICK, S. P. and KAPLAN, N. O.), Vol. VIII, pp. 533-537. Academic Press, New York. SICK, K. and NIELSEN, J. T. 1964. Genetics of amylase isozymes in the mouse. Heredifus 51,291-296. STEINER,J. C. and KELLER, P. J. 1968. An electrophoretic analysis of the protein components of human saliva. Archs oral BioL 13, 1213-1222. WEBER. K. and OSBORN. M. 1969. The reliabilitv of molecular weight determinations by dodecyl sulfate-polyacryl&ide gel electrophoresis. j. biol. Chem. 244, &OM12. WOLF, R. 0. and TAYLOR, L. L. 1967. Isoamylase of human parotid saliva. Nature, Lond. 213,11281129. WRIGLEY, C. 1968. Gel electrofocusing. Science Tools, 15, 17.
PLATE 1 OVERLEAF
600
M. R. ROBINOVITCH AND L. M. SREEBNY
PLATE 1 FIG. 1. Anionic analytical polyacrylamide gel electrophoresis of (a) 140 units of whole amylase stored in buffer at pH 6.9; (b) 140 units of amylase concentrated from the pooled peak fractions of a preparative polyacrylamide electrophoresis in buffer at pH 8.1; (c) 140 units of whole amylase concentrated from the total fractions of a preparative polyacrylamide electrophoresis in buffer at pH 8 * 1. The amylase isozymes are labelled A1 through A4 beginning with the band closest to the origin. The anode is toward the bottom of the figure. When tested on starch slides before staining, these bands demonstrated amylase activity proportional to the protein staining (ROBINOVITCHand SREEBNY,1969). FIG.
2. SDS-polyacrylamide gel electrophoresis of (a) 20 units of whole amylase stored in buffer at pH 6.9; (b) 20 units of amylase concentrated from the pooled peak fractions of a preparative polyacrylamide electrophoresis in buffer at pH 8.1; (c) 20 units of whole amylase concentrated from the total fractions of a preparative polyacrylamide electrophoresis in buffer at pH 8.1. In order to compute molecular weights from the relative mobilities of the bands, the following standards were run: bovine serum albumin purchased from Armour Laboratories, Chicago, Illinois (M.W. 68,000), crystalline catalase (beef liver) purchased from Worthington Biochemical Corp., Freehold, New Jersey (M.W. 6O,OOO),and crystallized and lyophilized ovalbumin (grade V) purchased from Sigma Chemical Co., St. Louis, Missouri (M.W. 43,000). FIG. 3. Isoelectric focusing electrophoresis of (a) 800 units of whole amylase stored in buffer at pH 6.9; (b) 800 units of amylase concentrated from the pooled peak fractions of a preparative polyacrylamide electrophoresis in buffer at pH 8.1; (c) 800 units of whole amylase concentrated from the total fractions of a preparative polyacrylamide electrophoresis in buffer at 8.1. The cathode is toward the bottom of the figure.
PLATE A.O.B.
1
f.p. 600