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Isolation and the Properties of Muscle Lysosomes

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Isolation and the Properties of Muscle Lysosomes ISOLATION AND PROPERTIES OF MUSC LS LYSOSOMES* JOHN W. C . BIRD DEPARTMENT OF PHYSIOLOGY RWERS UNIVERSITY In a classical series of papers beginning with Berthet and de hve (1951) and summarized by de Duve (1959) it was demonstrated that in osmotically protected homogenates of rat liver certain hydrolytic enzymes with acid pH optimum displayed the phenomenon of latency, The latency property was demonstrated by the fact that the enzymes were practically unreactive to their substrates when conditions to keep the particles intact were maintained. The enzymes became maximally active toward substrates only when the homogenates were subjected to physico-chemical treatments which would disrupt membranes, On the basis of differential centrifugation experiments, it was proposed that the hydrolases belonged to a distinct particle, and the name "lysosome" was proposed (de me, et al., 1935). de Duve (1959) suggested that the lysosomal enzymes were primarily catabolic in function, and that being segregated from surrounding cytoplasm by a bounding membrane they were prevented from digesting their host cell, It was also stated, unfortunately, that under certain circmstances the lysosomes acted as "suicide bags" by releasing their contents into the cell and thus resulting in cell death (de Dwe, 1963). I say "unfortunate, 'I because the "suicide bag concept" masked for several years the profound significance of the lysosome in noma1 cell physiology. Many disciplines have contributed to the development of the lysosome concept, especially in defining lysosomal functions with respect to the normal economy of the cell. It is now thought that the several types of lysosomes form a complex intracellular digestive system, whereby macromolecules brought into the cell, or "worn out" cellular components are organized into vacuoles, which in turn fuse with primary lysosomes formins: secondary lysosomes. Digestion occurs in the secondary lysosomes and the end products are transported into the cell's cytoplasm by passive diffusion or active transport (de Duve and Wattiaux, 1966). In some cells the end products of digestion may be secreted from the cell as useful by-products. Undigestible material 1s either excreted by exocytosis, or may remain in the cell in the form of a residual body such as lipofuscin granules in muscle and nerve tissue (Novikoff, 1962). * Presented st the 24th Annual Reciprocal Meat Conference of the Anerican Meat Science Association, 1971 . 68 Several workers have enphasieed, however, that the lysosomes and other component8 of the vacuolar apparatus are not developed to the same degree of complexity in all cell types (de Dwe, 19678 Novikoff, 1962; and Straus, 1967). The vacuolar apparatus is a morphological specialieation which is normally present in those cells nhose phyaiological function requires an efficient and economical catabolic machinery. A survey of the number of lysosomes found in different types of cells shows that lysosomes are more numerous in epithelial cells of organs having a phagocytic, absorptive, or secretory function (Straw, 1967) where a highly developed catabolic machinery is essential to the cell or organ. In skeletal muacle fibers, which do not have phagocytia or secretory functions, microscopy had failed to demonstrate the presence of lysosomes or lysosome-like granules. The failure to find lysosome-like morphological entities in normal nuecle cells sparked a controversy a few years ago as to the origin of aaid hydrolase aativity in muscle tissue. Tappel (1966) stated that the particles described by bioohemical studies me totally contributed by the non-muscle cella of nurscle tissue. Furthermore, in certain myopathies a dramatic increase in macrophages, leucocytes and lymphocytes occurs which is coincident with increases in total lyaosomal ensyme activities. Since the cells that comprise the connective tissue components of muscle are known to have among the highest concentrations of lysosomal enzymes, Kohn (1969) has calculated that under certain situations they could account for all the lysosomal enzyme activity found in muscle tissue. Van Flaet, et al. (1968) reported that lysosome-like structures were not found in nornal skeletal muscle fibers of rabbits. Pellegrino and F’ranzini (1963) emphatically stated that no lysosome has ever been detected in normal muscle fibers by electron microscopy. Maier and Zainan (1965) concluded that acid phosphatase, the lysosomal marker enzyme most frequently used in histochemical studies, could not be demonstrated in normal rat skeletal nusale fibers, Smith (1965) reported that the histochemical reactions for acid phosphatase in normal rabbit muscle is scanty and confined to bloed vessels (i.e., endothelial connective cells). The experimental cenclusions of the morphologists and biochemists have been difficult to reconcile. Thua, the experiments I will discuss are an attempt to resolve the existing controversy over the nature of the lysosomes in normal skeletal musale. I must also confess my prejudices. My basic working hypothesis Is that all normal musole protein catabolism is initiated and completed by lysosomal acid proteaaes, which axe indigenous to the muscle fibers. I an not aware of any carefully characterized or proven neutral proteases in muscle tissue. The alkaline protease of Kescalka and Miller (1960) and the neutral protease of Kohn (1965) have recently been shown, in an elegant study by Willemot, Ialanne and Berlinguet (1969), to be xanthine oxidase. I am certainly not opposed to neutral proteases, as the- occurence would simplify matters greatly, I look forward with great anticipation to a paper currently &press by Noguchi and Kandatsu, of the University of Tokyo, on the purifi- cation and properties of a new alkaline protease in rat skeletal muscle (e,e. Chem., personal communication from Noguchi), I should also report that Professor B, Sylven demonstrated a purified lysosomal cathepsin B from tumors that had a broad pH spectrum from to 8.0, with good activity at pH 7.0, using urea denatured Edestrin3.5 as substrate (International Research Conference of Lysosomes, Louvain, 1970) There have been occasional suggestions that acid pH is "unphysiological" and that acid hydrolases can, therefore, have little significance in living organisms (Barrett ' 1969). Although the pH optima of these enzymes are well removed from neutrality, several of them have been shown to have sufficient activity at the higher pH to be highly significant in the long time-scale of physiological processes. Moreover, the available evidence from the use of indicators (Rous, 1925) points to an extremely acid pH in the digestive wicuoles within phagocytic cells. For today's discussion, first let us review the essential hiochemical criteria for lysosomes in muscle tissue; Sedimentation properties (figure 1 ). Using differential centrifugation, and expressing the enzyme activity as mean relative specific activity of the fractions versus their mean relative protein content, the area of each block representing a fraction is thus proportional to the percentage of activity recovered in the corresponding fraction, and its height to the degree of purification achieved over the homogenate (de Dwe et al., The fractions are represented on the abscissa in the order in which1955). they were isolated, i.e., from left to right: N, nuclear, M, heavy mitochondrial$ L, light mitochondrial; P, microsomal; and S, final supernatant fraction. It is seen that the highest RSA is found in the light mitochondrial fraction, which has been demonstrated to be the lysosome-rich fraction in other tissues, Structure-linked latency (table 1 ), If the acid hydrolase is contained within a non-permeable membrane, chemical or physical forces which disrupt this membrane should allow accessibility of the enzyme to its substrate, In this slide we see that increasing the duration of homogenization increases the amount of free activity, i.e., increases the amount of enzyme available to react with substrate. The thermal labilization of acid hydrolases from liver, kidney and muscle lysosomes is shown in figure 2. Muscle lysosomes seem more resistant to thermal incubation than are liver and kidney lysosomesr When the homogenates are incubated in neutral or acid media, the rate which lysosomal enzymes are released into the non- sedimentable fraction is several times greater for liver and kidney than for muscle, 70 0 2 I Q 0 l2 II I 0r: s c JJO c 3 d 8 m z N a i Q)c A I 0 rc)- 0 C Y 71 TABLE 1. INFLUENCE OF PRELIMINARY BLENDING OF MUSCLE ON ENZYME LATENCY Enzyme activity" X VirTis l?ree Total Free (%a> Aryl Sulfatase 1 59.8 101 .o 59.2 2 75.6 117.0 64.6 4 106.8 106.7 100.1 Ribonuclease 1 33.1 58.6 56.4 2 39.1 60.0 65.2 4 65.3 62.3 104.8 Muscles were cut into small pieces with scissors, then blended in 0.25 M sucrose with a VirTis "45" homogenizer at top speed for 2 seconds, for the number of times indicated in the table. This was followed by three up-and-down passes in a Dual homo- genizer. Homogenates were assayed for free and total activities as described in text, except that incubation times were 15 minutes, *RNase = A O.D./mg protein per hr x 10-5; aryl sulfatase = mpoles nitrocathechol/ma protein per hr . 72 pK 5.0 8 p-NPPa 8 e f4 0 20 10 ,o 20 pGluc 6 ' 10 0 40 I 20 t 0 1 2 INCUBATION TZME (Hours)u Figure 2. Thermal labilization of acid hydrolases from liver, kidney and muscle lysosomal particles. e, kidney; a, liver; 0, muscle (from Canonico, 1969). The effect of freezing and thawing on lysosomes is presented in figure 3. A total mitochondrial preparation was frozen by immersing the test tube in a mixture of dry ice and acetone, and thawed by placing the tubes in a 38OC incubator. It is noted that one freeze- than treatment released 5% of the cathepsin activity, whereas subsequent freeze-thaw cycles released an additional 10% of enzyme.
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