and their Inhibitors: Structure, Function and Applied Aspects (Turk. V. & Vitale .... Exopeptidase .... (2) an endogenous inhibitor is capable of completely inhibiting.
BIOCHEMICAL SOCIETY TRANSACTIONS
Matsubara, H. (1970)Methods Enzymol. 19.642-650 Murphy, G. & Sellars, A. (1980) in Collagenase in Normal and Pathological Connective Tissues (Woolley, D. E. & Evanson. J. M., eds.), pp. 65-82 Wiley, Chichester Otsuka, Y. & Goll, D. E. (1980) Fed. Proc. Fed. Am. Soc. Exp. Biol. 39,2044 Puizdar, V. & Turk, V. (198 1) FEES Lett. 132,299-304 Rifkin, D. B., Gross, J. L., Moscatelli, D. & Gabrielides, C. (1981) Acta Biol. Med. Germ. 40. 1259-1263 Riordan, J. F. (1973) in Food-Related Enzymes (Whitaker, J. R.. ed.). pp. 220-240, American Chemical Society, Washington Sasaki, M., Taniguchi, K. & Minakata, K. (198 1) J. Biochem. (Tokyo) 89. 169-177 Steinbuch, M. (1979) in Biological Functions of Proteinases (Mosbach Colloq. Ser.) (Holzer, H. & Tschesche, H.. eds.), pp. 207-222, Springer-Verlag. Berlin Subramanian, E. (1978) Trends Biochem. Sci. 3, 1-3 Sumi, H. & Toki. N. (1981) Proc. SOC.Exp. Biol. Med. 167. 530535 Suzuki, K., Tsuji, S., Kubota, S., Kimura, Y. & Imahori, K. (1981) J. Biochem. (Tokyo) 90,275-278 Swamy, K. H. S. S. & Goldberg, A. L. (1981) Nature (London) 292, 652-654
Szpacenko, A., Kay, J., Goll, D. E. & Otsuka, Y . (1981) in Proteinases and their Inhibitors: Structure, Function and Applied Aspects (Turk. V. & Vitale, Lj., eds.), pp. 151- 161, Pergamon Press, Oxford Tamai, M., Hanada, K., Adachi, T., Oguma, K., Kashiwagi, K., Omura, S. & Ohzeki, M. (1981)J. Biochem. (Tokyo) 90,255-257 Taniguchi, K., Ito, J. & Sasaki, M. (1981) J. Biochem. (Tokyo) 89, 179-1 84 Truglia, J. A., & Stracher, A. (198 1) Biochem. Eiophys. Res. Commun. 100,8 14-822 Tsuji, S. & Imahori, K. (198 1) J. Biochem. (Tokyo) 90,233-240 Umezawa, H. & Aoyagi, T. (1977) in Proteinases in Mammalian Cells and Tissues (Barrett, A. J., ed.), pp. 637-662, North-Holland, Amsterdam Vallee, B. L. (1979) in Regulatory Proteolytic Enzymes and their Inhibitors (Proc. FEES Meet. 1 Ith. Copenhagen) (Magnusson, S., Ottesen, M., Foltmann, B., Dano, K. & Neurath, H., eds.), pp. 57-67, Pergamon Press, Oxford Waxman, L. & Krebs, E. G. (1978)J. Biol. Chem. 253,5888-5891 Waxman, L.. Chung, C. H. & Goldberg, A. L. (1982) Fed. Proc. Fed. Am. SOC.Exp. Biol41,865
Wilkinson, K. D., Urban, M. K. & Haas, A. L. (1980) J. Biol. Chem. 255,7529-7532 Woolley, D. E. (1980) Ciba Found. Symp. 75,69-86
Muscle proteinases and their possible roles in muscle growth and meat texture DARREL E. GOLL, YUZURU OTSUKA, PETER D. NAGAINIS, SHRIDHAR K. SATHE, JOHN D. SHANNON and MICHIO MUGURUMA Muscle Biology Group, Department of Nutrition and Food Science, University of Arizona, Tucson, AZ 85721, U S A . Interest in identification and characterization of proteinases and other peptidases from muscle tissue has increased markedly during the last several years. It has long been suspected that muscle proteinases are responsible, at least in part, for the tenderization that occurs during post-mortem storage of meat, and recent findings have provided some intriguing insights on the nature of the proteolytic changes that may occur during post-mortem aging. Other studies have revealed that rate of muscle protein turnover may be one of the crucial factors limiting rate and efficiency of muscle growth in domestic animals. Theoretically, decreasing rate of muscle protein turnover could lead to startlingly large increases, up to 50%, in feed efficiency. Muscle tissue from vertebrates contains 18-22% of its wet weight (40-90% of its dry weight, depending on fat content) as protein and muscle proteins, like proteins in other tissues, are constantly undergoing metabolic turnover. All the available evidence indicates that intracellular proteinases are responsible for mediating metabolic turnover of proteins. It is not surprising, therefore, that interest has increased in the physiological roles and metabolic regulation of muscle proteinases.
Cellular origin of muscle proteinases Although an early report (Smith, 1948) indicated that, over a pH range of 4.0-8.0, aqueous extracts of muscle tissue had little or no proteolytic activity against endogenous proteins in a muscle extract, a large number of proteolytic activities were found in minced muscle tissue during the following several decades (Iodice et al., 1972; Koszalka & Miller, 1960; Tappel et al., 1962; Zalkin et al., 1962). The cellular origins of these proteolytic activities, however, were uncertain, and many investigators believed that they originated from macrophages or other non-muscle cells present in muscle tissue (Tappel, 1966). It was not until 1970 that Canonico & Bird (1970) demonstrated, by using density-gradient centrifugation, that striated-muscle tissue contains two classes of lysosomal-like particles. One of these two classes contained large amounts of cathepsin D and could be identified as originating from striated-muscle cells themselves, whereas the second class originated from macrophages and other connective-tissue cells. Since this early finding,
cathepsins B and D have been localized within striated-muscle cells by immunohistochemical procedures (Bird et al., 1978, 1980; Wildenthal et al., 1977), and cathepsins B, D, H, and L activities have been detected in homogeneous cultures of L, myoblasts (Bird et al., 1981). Table 1 shows a summary of lysosomal peptide hydrolases and indicates those that evidently occur in striated-muscle cells. The immunohistochemical localization of cathepsins B and D and of acid phosphatase activity (Seiden, 1973; Stauber & Bird, 1974) showed that lysosomal enzymes in striated-muscle cells are associated with the sarcotubular system in these cells. This finding accounted for failure of earlier morphological studies to observe structural entities resembling lysosomes in striated-muscle cells. Although it seems established that striated-muscle cells contain lysosomes and cathepsins B, D, H, and L, cellular origins of the neutral and alkaline proteinases that have been detected in minced muscle tissue are less clear. Only five or six of these proteolytic activities have been purified to an extent that would allow determination of their molecular parameters and careful comparison of their properties and catalytic activities. It has been demonstrated by immunohistochemical procedures that one of these five or six purified proteinases (Sanada et al., 1978; Yasogawa et al., 1978) originates from mast cells in striated-muscle tissue and not from muscle cells (Woodbury ef al., 1978). It seems likely that the proteinase activities described by Holmes et al. (1971), Mayer et al. (1974), Noguchi & Kandatsu (1976) and Murakami & Uchida (1978) are similar to, if not identical with, the alkaline serine proteinase of Katunuma et al. (1975; see Table 2), and therefore also originate from mast cells rather than from striated-muscle cells. On the basis of the loss of proteolytic activity after injection wtih '48/80', a compound that disrupts mast cells, it has been suggested that most, if not all, proteolytic activity having an alkaline pH optimum in minced muscle tissue originates from mast cells in the original tissue and not from striated-muscle cells (Drabikowski et al., 1977; McKee et al., 1979). In view of this evidence, the origin of the 14000-dalton proteinase that has been purified from the myofibrillar fraction of hamster cardiac muscle and that cleaves the LC, myosin light chain (Bhan et al., 1978; Malhotra et al., 1979) should be investigated (Table 2).
Neutral proteinases in muscle Only two neutral proteinases have been purified from muscle tissue (Table 2). The cellular origin of the neutral serine proteinase (Beynon & Kay, 1978) has not been established by 1982
598th MEETING, DUBLIN
Table 1. Properties of some lysosomal peptide hydrolases and their effects on muscle proteins Lysosomal peptide hydrolase Lysosomal carboxypeptidaseA (cathepsin A) Cathepsin B (cathepsin B,)
Type of hydrolase Exopeptidase (carboxypeptidase) Endopeptidase
Lysosomal carboxypeptidaseB (cathepsin B2) Dipeptidyl aminopeptidaseI (cathepsin C) Cathepsin D Cathepsin E Cathepsin G Cathepsin H
Side chain at active site -OH
PH optimum 5.0-5.5
Detected in muscle Muscle proteins cleaved Yes Myosin, myoglobin
Actin. myosin, intact myofibrils, collagen
Endopeptidase Endopeptidase Endopeptidase
3.0-5.0 2.5-3.5 6.5-7.5
Actin. myosin, intact myofibrils
Collagen, elastin. fibronectin
Endopeptidase (some aminopeptidase) Endopeptidase
Cathepsin N Cathepsin S Cathepsin T
Endopeptidase Endopeptidase Endopeptidase
-SH -SH -SH
3.0-4.5 3.0-4.5 6.0-7.5
(mast cells) Yes Actin. myosin Actin, a-actinin,myosin, troponin-I,troponin-T, collagen
Table 2. Some neutral and alkaline proteinases that have been isolated from minced muscle tissue Proteinase CAF Neutral serine proteinase Alkaline serine proteinase Alkaline myofibrillar proteinase Rat myofibrillar proteinase Muscle alkaline proteinase Myosin-cleaving proteinase Myosin light-chainproteinase Alkaline, cytoplasmic proteinase
Cation requirement Ca2+
Side chain at active site
-SH -OH -OH
?a2+or Mg2+ -
Unknown -OH -OH -SH -OH
immunohistochemical procedures, but this proteinase does not seem to originate from mast cells. It is extracted at low ionic strength, whereas high ionic strengths are generally required to extract the mast-cell proteinases, and its levels in minced intestinal smooth muscle are unaffected by treatment with compound 48/80 (Kay, 1980). The neutral serine proteinase degrades native proteins 100 to 300 times more rapidly than trypsin (Kay, 1980) and has been shown to degrade G-actin, myosin, a-actinin, troponin-T, and troponin-I rapidly and tropomyosin slowly (Kay et al., 1982). The Caz+-activated proteinase (hereafter termed ‘CAF’) has been purified from porcine (Dayton et al., 1976), chicken (Ishiura et al., 1978), and rabbit (Mellgren et al., 1982) skeletal muscle and from bovine cardiac muscle (Dayton & Schollmeyer, 1980a). C A F has been shown by immunohistochemical assays to be located inside striated-muscle cells at the Z-discs of myofibrils and at the cytoplasmic surface of the cell outer membrane (Dayton & Schollmeyer, 1980b). C A F was discovered because of its ability to remove Z-discs from striated-muscle fibrils (Busch et al., 1972). It subsequently was shown that C A F would degrade C-protein, tropomyosin, troponin-T and troponin-I, but not actin, a-actinin, or myosin (Dayton et al., 1975). Recently C A F has been shown to also degrade desmin and filamin. In contrast with the neutral serine proteinase, C A F has a very restricted activity, and even those proteins degraded by C A F are cleaved only to large peptides and not to small peptides or amino acids. On the basis of its ability to degrade those proteins or structures that seem to maintain myofibrillar proteins in their assembled states, it has been suggested that C A F initiates myofibrillar protein turnover VOl.
PH optimum 6.5-8.0 6.5-8.0 7.5-8.5 8-12 8.0-9.5 9.0-10.5 7.0-10.0 7.5-9.5 7.0-10.5
Reference Beynon & Kay (1978) Katunuma et al. (1975) Holmeseral. (1971) Mayer el al. (1974) Noguchi & Kandatsu (1976) Murakami & Uchida (1978) Bhan et al. (1978) Koszalka & Miller (1960)
(Dayton et al., 1975). This remains an attractive, although still unproven, hypothesis, because the extensive disruption of myofibrils caused by the neutral serine proteinase (Kay et al., 1982) is not usually observed in healthy skeletal muscle undergoing normal metabolic turnover. Because they evidently are not confined to membrane-bound particles like lysosomes, activity of the neutral serine proteinase and C A F must be under close regulation to prevent continuous and indiscriminate degradation of cellular proteins. Inhibitors of both proteinases have been purified (Carney et al., 1980; Otsuka & Goll, 1980), and a second form of C A F that is active at micromolar levels of Ca2+ has been purified (Szpacenko et al.. 1981). The present evidence suggests that C A F is regulated in at least three ways: ( I ) its limited specificity restricts its action; (2) an endogenous inhibitor is capable of completely inhibiting it; and (3) it exists, most of the time, in a state that requires millimolar Ca2+for activity, and levels of free intracellular Caz+ rarely, if ever, get this high in healthy living cells. The nature of the factors that interfere with the CAF-CAF-inhibitor interaction or that mediate the transformation between the high- and low-Ca2+-requiring forms is still unknown. Beynon, R. J. & Kay, J. (1978) Biochem. J. 173.29 1-298 Bhan, A., Malhotra, A. & Hatcher, V. B. (1978) in Protein Turnover and Lysosome Function (Segal, H. L. & Doyle, D. J., eds.), pp. 607-618, Academic Press, New York Bird, J. W. C., Spanier, A. M. & Schwartz, W. N. (1978) in Protein Turnover and Lysosome Function (Segal, H. L. & Doyle, D. J.. eds.). pp. 589-604, Academic Press, New York Bird, J. W. C., Carter, J. H.. Triemer, R. E., Brooks, R. M. & Spanier. A. M. (1980) Fed. Proc. Fed. Am. Soc. Exp. Biol. 39.20-25
BIOCHEMICAL SOCIETY TRANSACTTONS
Bird, J. W. C., Roisen, F. J., Yorke, G., Lee, J. A., McElliott, M. A., Triemer, D. F. & St. John, A. (1981) J. Histochem. Cytochem. 29, 43 1-439 Busch, W. A., Stromer, M. H., Goll, D. E. & Suzuki, A. (1972)J. Cell Biol. 52,367-381 Canonico, P. G. & Bird, J. W. C. (1970) J. Cell Biol.45,321-333 Carney, I. T., Curtis, C. G., Kay, J. & Bicket, N. (1980) Biochem. J. 185,423-433 Dayton, W. R. & Schollmeyer, J. V. (1980a) 25. Mol. Cell. Cardiol. 12, 538-550 Dayton, W. R. & Schollmeyer, J. V. (1980b)J.Cell Biol. 8 7 , 2 6 7 ~ Dayton, W. R., Goll, D. E., Stromer, M. H.. Reville, W. J., Zeece, M. G. & Robson, R. M. (1975) Cold Spring Harbor Conf. Cell Proliferation; Vol. 2: Proteases and Biological Control (Reich, E., Rifkin, D. B. & Shaw, E., eds.), pp. 551-577, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Dayton, W. R., Goll, D. E., Zeece, M. G., Robson, R. M. & Reville, W. J. (1976)Biochemisrry 15, 215C2158 Drabikowski, W., Gorecka, A. & Jakubiec-Puka, A. (1977) I n / . J. Biochem. 4 6 1-7 1 Holmes, D., Parsons, M. E., Park, D. C. & Pennington, R. J. (1971) Biochem. J . 1 2 5 , 9 8 ~ Iodice, A. A,, Chin, J., Perker, S. & Weinstock, I. M. (1972) Arch. Biochem. Biophys. 152, 166-174 Ishiura, S., Murofushi, H., Suzuki, K. & Imahori, K. (1978) J. Biochem. 84,225-230 Katunuma, N., Kominami, E., Kobayashi, K., Banno, Y., Suzuki, K., Chichibu, K., Hamaguchi, Y. & Katsunuma, T. (1975) Eur. J. Biochem. 5237-50 Kay, J. (1980) CIBA Found. Symp. 75,219-225 Kay, J., Siemankowski, L. M., Siemankowski, R. F., Greweling, J. A. & Goll, D. E. (1982) Biochem. J. 201,279-285 Koszalka, T. R. & Miller, L. L. (1960)J. Biol. Chem. 235,665-668
Malhotra, A., Huang, S. & Bhan, A. (1979)Biochemistry 18,46 1-467 Mayer, M., Amin, R. & Shafir,E. (1974)Arch. Biochem. Biophys. 161. 20-25 McKee, E. E., Clark, M. G., Beinlich, C. J., Lins, J. A. & Morgan, H. E. (1979)J. Mol. Cell. Cardiol. 11. 1033-1051 Mellgren, R. L., Repetti, A., Muck, T. L. & Easly, J. (1982) J. Biol. Chem. in the press Murakami, U . & Uchida, K. (1978) Biochim. Biophvs. Acta 525, 2 19-229 Noguchi, T. & Kandatsu, M. ( I 976) Agric. Biol. Chem. 40,927-933 Otsuka, Y. & Goll, D. E. (1980) Fed. Proc. Fed. Am. SOC.Exp. Biol. 39,2044 Sanada, Y., Yasogawa, N. & Katunuma, N. (1978) 5. Biochem. (Tokyo) 83,27-33 Seiden, D. (1973) Z . Zelyorsch 144,467-473 Stauber, W. T. & Bird, J. W. C. (1974) Biochim. Biophys. Acta 338, 234-245 Smith, E. L. (1948)J. Biol.Chem. 173. 553-569 Szpacenko, A., Kay, J., Goll, D. E. & Otsuka, Y. (1981) in Proteinases and Their Inhibitors: Structure, Function. and Applied Aspects (Turk, V., & Vitale, Lj., eds.), pp. 15 1-161, Pergamon Press. Oxford Tappel, A. L. (1966) in The Physiology and Biochemistty oJMuscle as a Food (Briskey, E. J., Cassens, R. G. & Marsh, B. B., eds.). pp. 237-249, The University of Wisconsin Press. Madison Tappel, A. L., Zalkin, H., Caldwell, K. A.. Desai. 1. D. & Shibko. S . (1962) Arch. Biochem. Biophys. 96,340-346 Wildenthal, K., Decker, R. S., Poole, A. R. & Dingle, J. T. (1977) J. Mol. Cell. Cardiol. 9, 859-866 Woodbury, R. G., Everitt, M., Sanada, Y.. Katunuma. N.. Lagunoff, D. & Neurath, H. (1978) Proc. Natl. Acad. Sci. U.S.A. 75.53 1 1-53 I3 Yasogawa, N., Sanada, Y. & Katunuma, N. (1978) J. Biochem. (Tokyo)83, 1355-1360 Zalkin, H., Tappel, A. L., Caldwell, K. A., Shibko. S., Desai. 1. D. & Holliday, T. A. (1962) J. Biol. Chem. 237.2678-2682
Proteolysis in milk and dairy products
Cows' milk contains approx. 3.5% (w/w) protein, about 80% of which is classified as casein, a mixture of four phosphoproteins, as,-, a,*-, /?- and K-CaSeinS, in the approximate proportions 4:1:4: 1. All the caseins are insoluble at pH4.6 and exhibit considerable microheterogenity. The a,,-, as2-and /3-caseins are insoluble at Ca2+ concentrations greater than 6 m ~ but , are stabilized by rc-casein in the form of coarse colloidal particles, micelles, with molecular weights of approx lo*. Proteolysis of K-casein results in destabilization of the caseinate system. The caseins have an open, largely random structure which, inter alia, makes them readily susceptible to proteolysis, and because the caseins, especially P-casein, are strongly hydrophobic, casein hydrolysates tend to be bitter. The proteins soluble at pH 4.6, i.e. the whey proteins, are very heterogeneous. The principal proteins, @-lactoglobulin and a-lactalbumin, are typical globular proteins with a high degree of secondary and tertiary structure. In undenatured form, they are remarkably resistant to proteolysis, even by very active, broad-specificity proteinases. Consequently, with a few minor applications of exogenous proteinases, proteolysis in milk and dairy products is concerned with the caseins. Major reviews and texts on the milk protein system include: McKenzie (1971), Lyster (1972), Swaisgood (1973, 1982), Farrell & Thompson (1974), Schmidt & Payens (1976), Slattery (1976), Whitney et al. (1976), Davies & Holt (l979), Schmidt (1980, 1982), Brunner (1981), Fox (1982) and Dalgleish (1982a,b).
that it may be of bacterial origin persisted for many years. It is now well-established that milk does contain at least one proteinase which has assumed considerable technological significance in recent years. The subject has been reviewed by Humbert & Alais (1979), Visser (1981), Fox (1981a), Suhren (1981) and Reimerdes (1981, 1982). The principal indigenous milk proteinase, which is associated with the casein micelles, has been highly purified by several methods, including affinity chromatography on Sepharoselysine. It is a serine proteinase, probably identical with plasmin, with optimum activity at pH7.5-8.0 and 37OC. and mol.wt. 48000; it is very heat-stable and survives even UHT (ultra high) processing (142OC for 3s). Like trypsin. it is highly specific for bonds adjacent to lysine residues and in milk is most active on @-casein,which it hydrolyses to y-caseins and proteose peptones. a,,-Casein is also very susceptible, but the products have not been identified. a,,-Casein is hydrolysed slowly. but rc-casein and whey proteins are very resistant (P-lactoglobulin is a weak inhibitor). The commercial significance of milk alkaline proteinase is still unclear. Since its activity in milk increases with advancing lactation, it may be responsible for deterioration in the processing characteristics of !ate-lactation milk. It may be responsible for age-gelation in UHT milks, but addition of trypsin inhibitors to aseptic milk does not prevent gelation, suggesting physico-chemical changes as causative agents (Harwalker, 1982). It contributes to a limited extent to proteolysis in cheese. Milk is also reported to contain an acid proteinase, thrombin and aminopeptidases, the action and significance of which have not been clarified.
Indigenous milk proteinases The presence of a low level of indigenous proteinase in milk was suggested by the work of Babcock about 1890, but doubts
Endogenous proteinases in dairy products Milk and dairy products contain a variety of micro-organisms
P. F. FOX Department of Food Chemistry, University College, Cork, Ireland