Browsing by Author "Gevers, Wieland"

Now showing 1 - 9 of 9
  • Thumbnail Image
    Item
    Open Access
    Apolipoprotein biosynthesis and turnover in mammalian small intestine
    (1994) Combrinck, Marc Irwin; Gevers, Wieland
    The mammalian small intestine is a major site (second in total activity only to the liver) for the synthesis and secretion of plasma apolipoproteins, and contributes significantly to overall whole-body lipid dynamics. A prominent feature of the small intestine is its exposure to periodic loads of meals often containing dramatically varying amounts or types of food components, including lipids such as tri-acylglycerols, cholesterol and cholesteryl esters. Since the trans-epithelial transport of most of these latter materials requires the elaboration of particles partially covered by apolipoproteins, the regulation of the biosynthesis or, more correctly, the availability of these proteins is an important and as yet little-understood problem. Previous studies have been conducted on systems which, for one or the other reason, have not permitted the following questions to be satisfactorily or coherently answered: Does the ingestion of fat-containing meals, either acutely or chronically, increase the rate of biosynthesis of intestinal apolipoproteins such as apo B-48, and is this the principal method of matching the "demand" with the supply of this "packaging material" needed for fat transport across the intestinal epithelial cells? Alternatively, does the maintenance of a large steady-state intracellular pool in the face of variations in intracellular apolipoprotein degradation, controlled by acute or chronic lipid ingestion, produce the required "match" between supply and demand for these proteins (as has recently been suggested in studies on liver cells)? An in vitro system was therefore devised whereby sheets of intestinal epithelial cells (enterocytes) were freshly isolated from the jejuna of adult male Syrian golden hamsters and incubated for several hours in a medium supporting steady-state protein synthesis, in a manner which was assumed to be similar to the activity just before the killing of the donor animals. (Hamsters appear on various grounds to be a better small-animal model of human lipoprotein metabolism than the more commonly studied rats). The isolated epithelial cell sheets produced primary apolipoprotein products that could be extracted from the cells or detected in the incubation media, free from the subsequent modifications that they are known to undergo in vivo. Hamsters maintained on a low-fat chow were either studied as such or subjected to a variety of dietary treatments designed to maximize (over short or long time periods) intracellular apolipoprotein requirements for the "packaging" of tri-acylglycerol-rich lipoproteins, especially chylomicrons: acute bolus administration of lipid into the gut; overnight feeding of fat-enriched food; and chronic (six week) fat feeding. Using specific antisera and immuno-precipitation techniques, apo B-48 and two other principal intestinal apolipoproteins were shown to be synthesized in the steady state by intestinal cell sheets derived from control animals and from those subjected to acute or chronic fat-containing diets. Secretion took place, however, only when prior fat exposure of the donor intestines had occurred. Pulse-chase labelling was used to compare the rates of apolipoprotein synthesis, degradation and secretion in the same cell sheet preparations. The rates of apolipoprotein B-48 synthesis did not vary significantly under conditions of low or high trans-epithelial lipid flux, supporting findings derived from in vivo experimental systems. In contrast with data from other systems, however, the biosynthesis of apolipoprotein A-IV was not reproducibly increased on fat challenge. The rates of apo B-48 degradation varied significantly and were markedly reduced under conditions of fat feeding. The experiments permit a choice between the two alternatives mentioned above: Ingestion of fatty foods, either acutely or over long periods of time, does not increase the rates of biosynthesis of apolipoproteins such as apo B-48; but variations in the rate of intracellular degradation of this and probably other apolipoproteins allows the intestinal cells to match their requirements for lipid-transporting molecules to the demands of any given situation, relying in each case on a large steady-state intracellular pool maintained by "constitutive" biosynthesis. Importantly, there seems also to be a specific, possibly related effect of fat feeding on the secretion of lipoproteins into the intestinal extracellular fluid. These conclusions coincide with those obtained by other workers from studies of apolipoprotein B dynamics in isolated hepatocytes and in the hepatoma-derived liver cell line, Hep G2. The mechanisms underlying these phenomena are as yet unresolved.
  • Thumbnail Image
    Item
    Open Access
    Apoliprotein B metabolism in hamster livers, studied in vitro
    (1990) Hayward, Nicola Margaret; Gevers, Wieland
    This study aimed to investigate lipoprotein metabolism in male hamsters fed diets considered to be atherogenic in humans. Livers from adult male hamsters were selected to study aspects of apolipoprotein B metabolism. Isolated hepatocytes in suspension were compared with those maintained under tissue culture conditions. Liver slices were also prepared and compared with isolated suspended hepatocytes. Freshly prepared hepatocytes from the animals were incubated with radiolabelled precursors in suspension, or they were maintained under tissue culture conditions; liver slices were also investigated. The rates of total protein synthesis were of the same order in each of these systems, but protein secretion was impaired in liver slices, probably as a result of diffusion problems associated with the altered architecture of the sliced tissue. Albumin constituted 40 - 50% of the secreted proteins in each system. The rates of VLDL synthesis were increased in cells and slices prepared from animals previously fed sucrose- or fat-rich diets, but the secretion of VLDL was inhibited when diets contained unsaturated fat. The overall synthesis of apolipoprotein B was enhanced by fat-feeding; in the case of suspended hepatocytes, secretion of this protein was decreased when the preceding diet contained fats that were unsaturated; while in the case of liver slices, secretion was paradoxically enhanced. Apolipoprotein B was not degraded at significant rates in hepatocytes prepared from either control or fat-fed hamsters.
  • Thumbnail Image
    Item
    Open Access
    ASSAf turns 20: Young enough to be dynamic and old enough to be trusted with its mission
    (2016) Gevers, Wieland
    The International Society for Burns Injuries (ISBI) has published guidelines for the management of multiple or mass burns casualties, and recommends that 'each country has or should have a disaster planning system that addresses its own particular needs.' The need for a national burns disaster plan integrated with national and provincial disaster planning was discussed at the South African Burns Society Congress in 2009, but there was no real involvement in the disaster planning prior to the 2010 World Cup; the country would have been poorly prepared had there been a burns disaster during the event. This article identifies some of the lessons learnt and strategies derived from major burns disasters and burns disaster planning from other regions. Members of the South African Burns Society are undertaking an audit of burns care in South Africa to investigate the feasibility of a national burns disaster plan. This audit (which is still under way) also aims to identify weaknesses of burns care in South Africa and implement improvements where necessary.
  • Thumbnail Image
    Item
    Open Access
    Isoflurane : interaction with hepatic microsomal enzymes
    (1992) Bradshaw, Jennifer Jean; Gevers, Wieland; Ivanetich, Kathryn M
    lsoflurane interacts with cytochrome P-450 in rat and human hepatic microsomes and the Δ6- and Δ5-desaturases in rat hepatic microsomes. The interaction of isoflurane with cytochrome P-450 results in its metabolism to fluoride ion and organofluorine metabolites. The cytochrome P-450 isozymes catalysing the defluorination of isoflurane were assessed in hepatic microsomes from phenobarbital-, β-naphthoflavone- and pregnenolone-16α-carbonitrilepretreated and untreated rats. One or more of the cytochrome P-450 isozymes induced by phenobarbital and pregnenolone-16α-carbonitrile appear to defluorinate isoflurane, but those induced by β-naphthoflavone do not. From a comparison of the extent of defluorination of isoflurane in hepatic microsomes from phenobarbital- and pregnenolone-16α-carbonitrile-pretreated rats, and their Kₘ and Vₘₐₓ values, it appears that isoflurane is defluorinated by one or more isozymes induced by both phenobarbital and pregnenolone-16α-carbonitrile. The major isozyme is probably cytochrome P-450PCN1. The metabolites of isoflurane were identified in human and phenobarbital-induced rat hepatic microsomes. In microsomes from phenobarbital-pretreated rats, isoflurane is metabolised to fluoride ion and trifluoroacetaldehyde; trifluoroacetic acid is not produced in measureable amounts. The trifluoroacetaldehyde produced binds to microsomal constituents. In human hepatic microsomes, the organofluorine metabolite is identified as trifluoroacetic acid. It is proposed that isoflurane is metabolised by different pathways in human and phenobarbital-induced rat hepatic microsomes. The interaction of isoflurane with the cyanide-sensitive factors was assessed by several criteria. Firstly, using the reoxidation of cytochrome b₅ as an index of fatty acid desaturase activity, isoflurane appears to interact with the Δ6- and/or Δ5-desaturases, but not the Δ9-desaturase. Secondly, these results were confirmed and clarified by the use of direct assays to measure the fatty acid desaturase activity. Using the direct assay, we confirmed that isoflurane did not inhibit the Δ9-desaturase and inhibited Δ6-desaturation of linoleic acid, but not the Δ6-desaturation of α-linolenic acid. The inhibition of the Δ6-desaturation of linoleic acid occurred at low millimolar concentrations of isoflurane. lsoflurane inhibits the Δ5-desaturation of eicosa-8, 11, 14-trienoic acid to a small extent which is only apparent at much higher concentrations of isoflurane than that which inhibits the Δ6-desaturase. Further studies focussed on measurement of the activity of Δ6-desaturase in order to attempt to study the kinetics of the inhibition of the Δ6-desaturase by isoflurane: Δ6-desaturase activity was assessed using hepatic microsomes as the source of the enzyme and linoleic acid as substrate precursor. In the course of these studies, we identified a number of factors that affected the apparent activity of the Δ6-desaturase in hepatic microsomes. These included significant levels of endogenous substrate and competing reactions in the hepatic microsomes. Endogenous substrate levels were quantified and corrected for. We then resorted to computer modelling to extract the kinetics of the Δ6-desaturase free of contributions from acyl-CoA synthetase and lysophospholipid acyltransferase, as well as enzyme decay. The kinetics of isoflurane inhibition of the Δ6-desaturase were then superimposed and studied by computer modelling.
  • Thumbnail Image
    Item
    Open Access
    Ligandin in the steroidogenic tissues of the rat : characterisation, distribution and development
    (1982) Eidne, Karin Ann; Gevers, Wieland
    One of the main problems in the field of multifunctional proteins such as ligandin is the possibility that multiple forms and isoproteins may exist. Two forms of liver ligandin [ GSH (reduced glutathione) S-transferase B] have been described, a heterodimeric form consisting of equal amounts of Ya (22000 daltons) and Yc (25000 daltons) subunits, and a homodimeric form containing only Ya. Because rat testis ligandin, prepared by the standard technique of anion-exchange and molecular exclusion chromatography, contains more Yc subunit than Ya, it has been claimed that testis and liver ligandin are different entities (Bhargava, Ohmi, Listowsky and Arias (1980) J. Biol. Chem. 255, 724-727). This thesis investigated the nature and character of ligandin in the steroid-producing tissues of the rat. A comparative study was undertaken to establish whether testis ligandin differed from liver ligandin. Different methods of purification were used to investigate testis ligandin and its relationship to other GSH S-transferases in steroidogenic tissues. Testis ligandin purified by immunoaffinity chromatography using anti-liver YaYa ligandin antiserum yielded a product identical with liver preparations (Yc=Ya). This suggests that the differences previously described may be due to contamination of testis ligandin by a closely related species. Testis ligandin prepared by the standard technique was similar to that previously reported, containing more Yc than Ya. Cross-linking studies of standard testis ligandin preparations with dimethylsuberimidate showed more than one band in the 50000 dalton region, further strengthening the view that these testis ligandin preparations may be contaminated. Since this contaminant was likely to be another GSH S-transferase, sodium dodecyl sulphate/ polyacrylamide-gel-electrophoretic analysis was performed on testis GSH S-transferases separated by CM-cellulose chromatography. GSH S-transferase AA which was present in large amounts, was shown to migrate in the same region as Yc subunit. CM-cellulose chromatography of a 'pure' standard testis ligandin preparation revealed significant amounts of GSH S-transferase AA migrating as Yc subunit, in addition to ligandin consisting of equal amounts of Ya and Yc subunits, indicating that testis ligandin is identical with liver ligandin and that previously described differences are due to a contaminant identified as GSH S-transferase AA. Studies on ligandin in other steroid-synthesising tissues showed that ovary and adrenal ligandin prepared by standard techniques also contained more Yc than Ya. Separation of ovary GSH S-transferases on CM-cellulose showed that GSH S-transferase B, the peak reacting with anti-liver YaYa ligandin antisera contained equal amounts of Ya and Y c subunits, suggesting a situation similar to that in the testis exists. Glutathione peroxidase II activity of testis and ovary GSH S-transferases was investigated. Fractions corresponding to GSH S-transferase AA, A and B exhibited activity with cumene hydroperoxide. The considerable glutathione peroxidase activity of GSH S-transferases in testis and ovary suggest a protective function for the cells of gonadal tissue against oxidative damage to essential intracellular components. Further attempts to clarify the function of ligandin in the steroid-synthesising tissues were made. The pattern of gonadal ligandin development during early life, puberty and pregnancy determined by radioimmunoassay was found to parallel serum steroid hormone concentrations. This correlation was not observed in liver or kidney. Ligandin was localised to specific cells of the steroid synthesising tissues using immunocytochemical techniques. These findings suggest that there may be a functional link between steroidogenic cells, or products of their activity and certain GSH S-transferases. Phenobarbital pre-treatment did not have any effect on developing testis, ovary or adrenal ligand in concentrations. Immunocytochemical localisation of ligandin in rat steroid-producing tissues using a peroxidase anti-peroxidase (PAP) technique with anti-liver YaYa ligandin antiserum as the first antibody, showed staining in the testis to be limited to the interstitial (Leydig) cells. Stromal cells of the ovary and the fascicular, glomerular and reticular zones of the adrenal cortex also contained immunoreactive material. PAP staining with anti-testis ligandin antisera (testis ligandin prepared using the standard technique) showed far greater intensity of staining in these tissues, presumably due to reaction with both ligand in and GSH S-transferase AA. This study has clarified the structural aspects of testis ligandin and demonstrated identity with liver ligandin. Ontogeny of ligandin in the steroidogenic tissues and localisation to specific regions in these tissues suggests a functional link between ligandin, GSH S-transferases, GSH peroxidases and activity of steroidogenic tissue.
  • Thumbnail Image
    Item
    Open Access
    Non-lysosomal protein degrading systems in chicken skeletal muscle
    (1990) Arnold, Jane Elizabeth; Gevers, Wieland
    In an attempt to understand the roles played by the ubiquitin-dependent and calpain pathways in protein degradation in chicken skeletal muscles, biochemical studies were conducted on components of these two systems as well as their potential endogenous and exogenous substrates. ATP- and ubiquitin-dependent breakdown of endogenous proteins (measured by tyrosine release) or exogenous proteins (measured by the appearance of trichloroacetic acid-soluble radiolabel after incubation with 125I-lysozyme) took place in muscle extracts; the specific activities of these processes were significantly lower than those detected in rabbit reticulocytes. Conjugation of ubiquitin to a subset of endogenous proteins was detected by incubating muscle extracts (fraction II: depleted of ubiquitin by DEAE-cellulose chromatography) with 125I-ubiquitin and Mg2+-ATP, followed by analysis of the radiolabelled conjugates by one-dimensional polyacrylamide gel electrophoresis in the presence of SOS, and autoradiography. Discrete conjugates were formed with apparent molecular weights between 30 -100 000, as well as a large number of undifferentiated entities of higher molecular weights. Conjugation of ubiquitin to the exogenous protein lysozyme was detected only when fresh, as opposed to previously frozen fraction II preparations were assayed: three bands were obtained, as opposed to the six ubiquitin conjugates formed by reticulocyte extracts. The muscle system catalyzed the ubiquitination of partially purified myofibrillar proteins, principally myosin and possibly actin. Fractionation of the ubiquitin-activating enzymes into El and E2 on the one hand, and E3 on the other, permitted mixing experiments to be conducted by means of conjugation assays, and confirmed the low content of E3 in muscle as opposed to reticulocytes. Fraction II from muscle displayed ubiquitin conjugate-degrading activity but again this was less active than in reticulocytes. A number of other proteolytic activities, independent of ubiquitin, were also present. Isopeptidases, active on 125I-ubiquitin conjugates were strongly inhibited by sulphydryl alkylating agents such as N-ethylmaleimide. The overall picture of the ubiquitin pathway in muscle is one where many proteins may be converted into long-lived conjugates but not in all cases requiring the action of E3: some E3-dependent protein degradation undoubtably does occur in this physiologically basal system. Formation of a ubiquitin conjugate of the ubiquitinactivating enzyme (E1) and some of the ubiquitin carrier proteins (E2 's) was detected during incubations of 125Iubiquitin and ATP lasting 2 hr or longer. Because treatment of such systems with NaOH, even at early times during the incubations, greatly enhanced the appearance of the same entities, the phenomenon appeared to be one of auto-, rather than E3-mediated ubiquitination. The bonds involved had properties compatible with their being peptidic in nature, and their formation occurred from ubiquitin thiolesters bound to E1 and E2. The protease inhibitor and alkylating agent, TLCK, when pre-incubated with fraction II for 2 hr before the addition of 125I-ubiquitin and ATP, greatly enhanced the subsequent auto-ubiquitination of E1 in the absence of NaOH treatment, and caused the inhibition of its adenylate-forming and thiolester-transferring activities: thus ubiquitin transfer to E2's and further to other acceptors was markedly impaired. such an inactivation of El by TLCK may, in a manner analogous to that described in the thermolabile ts85 mutants (Finley et al., 1984), be the basis of the action of this agent to block the cell cycle in late G2 or early M phase (Schnebli & Haemmerli, 1974). TLCK-induced inactivating auto-ubiquitination of El may be an important tool for the study of ubiquit-independent processes which (apart from possible intrinsic protease activity), all appear to require the activity of this enzyme. The number of calpain species existing in chicken skeletal muscle is controversial with only one (Ishiura et al., 1978) or three (Wolfe et al., 1985) species having been reported. When extracts of chicken skeletal muscle were applied to a DEAE-cellulose column and the bound protein eluted in a linear salt gradient, two calpain activities, separated from their endogenous inhibitors (calpastatins), were detected. The first eluting activity, "calpain I", was active at low ca2+ concentrations, was heat-labile and had a lower apparent molecular weight on gel filtration when compared with the later eluting activity which appeared to be a typical calpain II species. "Calpain I". was not an autolytic product of calpain II but appeared to be derived from a more heat-stable calpain I species. A proportion (up to 14%) of the calpains in crude muscle extracts was bound to membrane fractions in the presence of ca2+; this could be removed by EGTA treatment. In addition, membrane-bound fractions examined by 9el filtration contained calpain· forms of an apparent molecular · weight lower than that of calpain which had not been membrane-associated. Membrane binding of the calpains (especially of calpain II), may be important in physiological activation.
  • Thumbnail Image
    Item
    Open Access
    Non-lysosomal protein degrading systems in chicken skeleton muscle
    (1990) Arnold, Jane Elizabeth; Gevers, Wieland
    In an attempt to understand the roles played by the ubiquitin-dependent and calpain pathways in protein degradation in chicken skeletal muscles, biochemical studies were conducted on components of these two systems as well as their potential endogenous and exogenous substrates. ATP- and ubiquity tin-dependent breakdown of endogenous proteins (measured by tyrosine release) or exogenous proteins (measured by the appearance of trichloroacetic acid-soluble radiolabel after incubation with 125rlysozyme) took place in muscle extracts; the specific activities of these processes were significantly lower than those detected in rabbit reticulocytes. Conjugation of ubiquitin to a subset of endogenous proteins was detected by incubating muscle extracts (fraction II: depleted of ubiquitin by DEAE-cellulose chromatography) with 125rubiquitin and Mg2+-ATP, followed by analysis of the radiolabelled conjugates by one-dimensional polyacrylamide gel electrophoresis in the presence of sos, and autoradiography. Discrete conjugates were formed with apparent molecular weights between 30 -100 000, as well as a large number of undifferentiated entities of higher molecular weights. Conjugation of ubiquitin to the exogenous protein lysozyme was detected only when fresh, as opposed to previously frozen fraction II preparations were assayed: three bands were obtained, as opposed to the six ubiqui tin conjugates formed by reticulocyte extracts. The muscle system catalyzed the ubiquitination of partially purified myofibrillar proteins, principally myosin and possibly actin. Fractionation of the ubiquitin-activating enzymes into El and E2 on the one hand, and E3 on the other, permitted mixing experiments to be conducted by means of conjugation assays, and confirmed the low content of E3 in muscle as opposed to reticulocytes. Fraction II from muscle displayed ubiquitin conjugatedegrading activity but again this was less active than in reticulocytes. A number of other proteolytic activities, independent of ubiquitin, w~re also present. Isopeptidases, active on 125I-ubiquitin conjugates were strongly inhibited by sulphydryl alkylating agents such as N-ethylmaleimide. The overall picture of the ubiquitin pathway in muscle is one where many proteins may be converted into long-lived conjugates but not in all cases requiring the action of E3: some E3-dependent protein degradation undoubtably does occur in this physiologically basal system. Formation of a ubiquitin conjugate of the ubiquitinactivating enzyme (El) and some of the ubiquitin carrier proteins (E2 's) was detected during incubations of 125rubiqui tin and ATP lasting 2 hr or longer. Because treatment of such systems with NaOH, even at early times during the incubations, greatly enhanced the appearance of the same entities, the phenomenon appeared to be one of auto-, rather than E3-mediated ubiquitination. The bonds involved had properties compatible with their being peptidic in nature, and their formation occurred from ubiguitin thiolesters bound to El and E2. The protease inhibitor and alkylating agent, TLCK, when pre-incubated with fraction II for 2 hr before the addition of 125I-ubiquitin and ATP, greatly enhanced the subsequent auto-ubiqui tination of El in the absence of NaOH treatment, and caused the inhibition of it adenylate-forming and thiolester-transferring activities: thus, ubiquitin transfer to E2's and further to other acceptors was markedly impaired. Such an inactivation of El by TLCK may, in a manner analogous to that described in the thermolabile ts85 mutants (Finley et al., 1984), be the basis of the action of this agent to block the cell cycle in late G2 or early M phase (Schnebli & Haemmerli, 1974). TLCK-induced inactivating auto-ubiquitination of El may be an important tool for the study of ubiquitindependent processes which (apart from possible intrinsic protease activity), all appear to require the activity of this enzyme. The number of calpain species existing in chicken skeletal muscle is controversial with only one ( Ishiura et al. , I 1978) or three (Wolfe et al., 1985) species having been reported. When extracts of chicken skeletal muscle were applied to a DEAE-cellulose column and the bound protein eluted in a linear salt gradient, two calpain activities, separated from their endogenous inhibitors (calpastatins), were detected. The first eluting activity, "calpain I", was active at low ca2+ concentrations, was heat-labile and had a lower apparent molecular weight on gel filtration when compared with the later eluting activity which appeared to be a typical calpain II species. "Calpain I”. was not an autolytic product of calpain II but appeared to be derivea from a more heat-stable calpain I species. A proportion (up to 14%) of the calpains in crude muscle extracts was bound to membrane fractions in the presence of ca2+; this could be removed by EGTA treatment. In addition, membrane-bound fractions examined by 9el filtration contained calpain• forms of an apparent molecular · weight lower than that of calpain which had not been membraneassociated. Membrane binding of the calpains (especially of calpain II), may be important in physiological activation.
  • Thumbnail Image
    Item
    Open Access
    Protein degradation in rat skeletal muscle : intracellular, non-lysosomal enzyme systems and their endocrine control
    (1982) Ismail, Firhaad; Gevers, Wieland
    Two manuscripts based on the work reported in this thesis were submitted and accepted for publication, the abstracts of which follow: SOLUBLE AND PARTICULATE FORMS OF MUSCLE ALKALINE PROTEINASE SHOW DIFFERENTIAL SENSITIVITY TO ENDOGENOUS INHIBITOR(S): Firhaad Ismail and Wieland Gevers, (Biochemistry International, In Press). Membrane-free washed myofibrils derived from rat skeletal muscle homogenates contained a chymostatin-sensitive protease(s) which acted on associated myofibrillar proteins, at an optimum pH of 8.5, much less rapidly at low ionic strength (insoluble myofilaments) than at high salt concentrations (solubilized proteins). When the myofibrillar fraction was added to the particle-free cytosol prepared from the muscle extracts, proteins of the cytosol were also degraded, but the activity in this case was much more pronounced at low ionic strength. This was because inhibitor(s) of the proteinase present in the cytosol fraction were only effective at high ionic strength when all the myofibrillar (and associated) proteins were in solution. The protease was separated from the bulk of the myofibrillar proteins by gel chromatography at high ionic strength. On dialysis against a low-salt buffer, part of the enzyme was precipitated. The putative cytosolic inhibitor(s) were again only effective on the soluble enzyme at high ionic strength. A HIGH MOLECULAR WEIGHT CYSTEINE ENDOPEPTIDASE FROM RAT SKELETAL MUSCLE: Firhaad Ismail and Wieland Gevers, (Biochim. Biophys. Acta, In Press). A cytosolic enzyme of high molecular weight (about 500000), which attacks native or denatured proteins (inter alia casein, globin and hexokinase) was purified about 1000-fold from mixed rat skeletal muscles, including muscles freed of mast cells by prior treatment of the animals with the degranulator, compound 48/80. Peptides of varying size were generated from radio-actively labelled globin, but no free amino acids were formed; free tyrosine was also not released from azocasein. The pH optimum was 7.5 and the presence of an essential cysteine group was suggested because dithiothreitol (1 mM) stimulated the activity and N-ethylmaleimide (5 mM) and p-chloromercuriphenylsulphonic acid (1 mM) were inhibitors. The activity was markedly inhibited by Zn²⁺ but not by leupeptin, chymostatin or pepstatin. The enzyme was stabilized by ATP, at concentrations as low as 0.1 mM, against inactivation at 42°C. The endopeptidase was clearly separated on gel chromatography from another large protease, also sensitive to Zn²⁺, but with marked aminopeptidase activity and the properties of Hydrolase H. The activity levels of the protease, assayed after chromatography on Sepharose 68 of high-speed supernatant fractions, did not vary significantly in skeletal muscle samples which were derived from denervated, starved, diabetic or hyperthyroid animals, in all of which the abnormal physiological states expressed themselves as enhanced rates of tyrosine release by incubated soleus and extensor digitorum longus muscles. Nevertheless, the enzyme described here may be part of an ATP-dependent, multi-component proteolytic system similar to that already known to be present in reticulocytes.
  • Thumbnail Image
    Item
    Open Access
    Purification and some properties of an alkaline protease from rat skeletal muscle
    (1981) Bosch, Benjamin; Gevers, Wieland
    Various alkaline proteases derived from skeletal muscle have been described by a number of researchers and have been purified to varying degrees. Such alkaline proteases may play an important role in the metabolism of myofibrillar and other muscle proteins and as such deserve to be fully characterised. In this study, a major myofibrillar alkaline protease was purified from rat skeletal muscle. The enzyme degraded both denatured casein and azocasein and had a pH optimum of 9,0. The molecular mass was 32 250 ± 650. The presence of a second, minor alkaline protease was demonstrated using three different separation techniques as well as by inhibitor studies. The major protease was insensitive to inhibition by pepstatin and leupeptin, whilst 90 % of the activity was expressed in the presence of 2 mM EGTA. A moderate degree of inhibition was observed in the presence of soybean trypsin inhibitor and the protease was markedly sensitive to chymostatin. A similar alkaline protease was partially purified from rat cardiac muscle using the same purification procedure. Incubation of washed myofibrils in the presence of sodium pyrophosphate released a factor into the supernatant, the removal of which facilitated the separation of myofibrillar alkaline protease from the myofibrils. The factor appeared to be necessary for binding of the alkaline protease to the myofibrillar proteins but its removal did not disrupt the binding of proteolytic activity already attached to the myofibrillar proteins. An inhibitor of myofibrillar alkaline protease was demonstrated which is, in principle, capable of playing an important regulatory role in controlling the activity of these enzymes and thereby of myofibrillar protein catabolism.