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Changes in the total, free and bound activity of collagenase of the intramuscular connective tissue were studied during beef muscle maturation (2 degrees C). Lysosomes were isolated by differential centrifugation. The studies showed a relatively rapid release of collagenase from lysosomes and a comparatively high stability of the enzyme during slow autolysis of muscles.
The activity of acid phosphatase, a marker enzyme of lysosomal enzymes, in aqueous humor of wet-chamber stored eyeball and corneal storage medium was determined. The results showed a significant rise in the enzyme activity as the storage time increased, i.e. the degree of autolysis of the cornea progressed.
Effects of long-term immobilization stress and adaptation to short-term stressory actions on Ca2(+)-transport system were studied in sarcoplasmic reticulum (SR) of rat myocardium. The stress inhibited the rate of Ca2+ transport in SR, while adaptation not only optimized the Ca2(+)-pump functions but prevented the stress-induced impairments of its functions. After stress resistance of Ca2(+)-pump to endogenous impairing factors (in autolysis) was decreased 2-3-fold, whereas adaptation increased the SR membrane stability so distinctly that after simultaneous stress the membrane stability was maintained at the level which-was 2.5-times higher as compared with controls. Long-term stress caused also the higher output of Ca+2 from intracellular stores as compared with controls, while during adaptation and simultaneous stress and adaptation the loss of Ca2+ was practically absent. Protective effects of adaptation to short-term stressory actions on the Ca2(+)-transport system of cardiomyocytes are discussed.
Structure and enzymatic properties of myosin were studied in rat skeletal muscles during various periods of postmortal autolysis. Ca2+-ATPase activity of the protein was decreased within 3 hrs after death. At the same time, the structure of myosin, as shown by gel filtration in 8 M urea and disc electrophoresis in SDS polyacrylamide gel, was maintained. The phenomenon of "substrate inhibition" exhibited both native myosin and the protein isolated from the autolyzed muscle tissue. Enzymatic activity of myosin was markedly decreased within 12 hrs and 24 hrs after death. Degradation of the protein heavy chains was also observed.
In the course of organoheterotrophous growth, Pseudomonas carboxydoflava Z-1107 and Bacillus cereus 504 were found to synthesize the autoregulatory factor d2, a membranotropic lipid metabolite, and to accumulate it in the growth medium. At a low concentration, the factor activated respiration of the cells: at a high concentration, it inhibited respiration and induced autolysis of the cells. The physiological action of d2 was shown to be due to the effect of free fatty acids, in particular, palmitic, stearic and oleic acids. Oleic acid displayed the highest physiological activity, P. carboxydoflava Z-1107 became more susceptible to high concentrations of d2 and oleic acid, and respiration of the cells was activated by low concentration of these compounds when the culture passed from the exponential growth phase to the linear one. These results as well as data reported in literature about the action of fatty acids on biological membrane suggest that low concentrations of the factor d2 uncouple respiration and oxidative phosphorylation whereas high concentrations of the factor disorganize the structure of the cytoplasmic membrane by increasing its fluidity.
The method of electron microscopy was used to study the morpho-functional state of pancreatic islands of human fetuses at different terms (10-54 h) after clinical death. Within the periods of time in question the island secretory cells preserve the structure characteristic of each type (B, A, D). The development of symptoms of postmortem autolysis of the pancreatic island is not a chaotic fleeting physico-chemical reaction and is characterized by a definite sequence of cytological changes of endocrinocytes. A conclusion is made that cadaveric material of the pancreas is good for preparing the pancreatic island culture for using it in the following tissue substituting therapy of patients with diabetes mellitus.
From continuous passive-electrical measurements on autolyzing liver tissue are derived statements about the structural changes in time. In the early post mortem phase the autolysis is a cell-controled process, which suddenly goes over in an uncontroled degradation process. The changes of the uncontroled process are fitted. The results are statistically and thermodynamically interpreted by a condensation modell. The high complexity of the living resp. cell-controled system correlates to a relatively high structural entropy.
To understand the mechanism in postmortem autolysis better, processes in the postmortem degradation of myofibril proteins in the presence of protease inhibitors were studied. Male Wistar rats were given injections of the carboxyl-, thiol-, and serine-protease inhibitors, pepstatin, Ep-475[L-transepoxysuccinyl-leucylamide(3-methyl) butane; E-64-C], and chymostatin, via the femoral vein. Control rats were similarly treated with saline. Then, myofibril proteins were isolated from their cardiac and femoral muscles and from those of control animals at various times after death, and degradation of these myofibril proteins with time was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In cardiac muscle, alpha-actinin was degraded rapidly, followed by the heavy chain of myosin and light chain of myosin (L2). Actin and the light chain of myosin (L1) were degraded slowly. the degradations of the heavy chain of myosin, alpha-actinin, tropomyosin and L2 after 14 days were not inhibited by pepstatin, but were inhibited by Ep-475 and chymostatin. In skeletal muscle, L1 and L2 were degraded rapidly, followed by the heavy chain of myosin and alpha-actinin. Actin was degraded slowly and was still unchanged 2 weeks after death. The degradations of protein components were inhibited by pepstatin, Ep-475 and chymostatin. These results indicated that after death the components of myofibrils are degraded by various proteases at various rates depending on their properties or structures. This degradation is fundamentally the same in cardiac and skeletal muscles, but inhibitors have somewhat different effects on the postmortem degradation processes after death in the two types of muscle.
A considerable activity of digestive hydrolases (carbohydrases in particular) was found in fish during feeding. The enzyme activity increases 2-6-fold at low values of the pH due, probably, to autolysis. This mechanism is important for the digestion in fish and other wild animals.
In this work we have thrown light on the basis of our experiments on 530 rabbits', dogs' and human eyes preserved under various conditions on the influence of antioxidation substances on corneal preservation. The development of autolysis is highly dependent on the absence of antioxident - also important for the survival of the cornea. From our results we have drawn conclusions regarding the participation of antioxidant (-tocopherol, chondroitin sulphate) on the self-regulating oxidation of structural lipids in the cell membranes of the cornea, and its effects on preservation.
Protease Q, a recently discovered subtilisin-like protease, exhibited unusual stability in 10% sodium dodecyl sulfate (SDS) and 8 M urea solutions. In 2% SDS it exhibited higher activity than the control, and in 10% SDS it retained 50% of its original activity when azocoll was used as substrate. Kinetic studies showed that the decrease in the activity of protease Q in SDS solutions followed a first order kinetics with a half-life of 446, 278, 78, and 24 h in 1%, 2%, 5%, and 10% SDS, respectively, at 25 degrees C. Protease Q was also stable against autolysis. In 20 mM Tris-HCl buffer (pH 8.3) containing 1.0 mM CaCl2, protease Q had a half life of 2822 h and 725 h at room temperature and at 37 degrees C, respectively. The enzyme was able to completely hydrolyze beta-lactoglobulin, beta-casein, and keratin in 8-10 M urea.
In order to improve the stability of trypsin, an approach to knock out the autolytic site has been carried out in this investigation. Compared with trypsins from other species, the autolytic site Arg117-Val118 of rat trypsin is the most interesting candidate to work on. The Arg117 residue was designed to be deleted or replaced by other amino acid residues to destroy the autolytic site. With DNA site-directed mutagenesis method, one deletion mutant and several replacement mutants were selected. After expression and purification, the kinetic and anti-autolytic properties of mutant trypsins were studied. No net charge difference of the trypsin molecules was observed by native PAGE analysis. Kinetic studies show that the activities of mutants vary from one another. R117L gives 32 times the activity of wild type trypsin while R117C has no detective activity. Among 8 selected mutants with characteristic properties, 7 of them give prolonged half life during anti-autolytic assay with the exception of R117M which is more sensitive to autolysis.
Pep 5 and nisin are cationic bactericidal peptides which were shown to induce autolysis in Staphylococcus cohnii 22. In contrast to nisin, Pep 5 induced lysis could be stimulated in the presence of glucose. Addition of lipoteichoic acids (LTA) (D-alanine:phosphorus = 0.475:1) inhibited all effects of Pep 5 on susceptible cells in a molar ratio LTA:Pep 5 of 10:1. Treatment of S. cohnii 22 with Pep 5 or nisin for 20 min and subsequent washing with 2.5 M NaCl released autolysin activity. Crude preparations of the hydrolyzing enzymes produced free amino groups as well as polysaccharide fragments from the murein backbone, suggesting the presence of a muramidase or glucosamidase, and endopeptidase or amidase. Both enzyme activities were inhibited by lipoteichoic acid; they could be fully reactivated by addition of Pep 5 in sufficient concentrations. The velocity of hydrolysis was not influenced by nisin, whereas it was doubled in presence of Pep 5. The results are discussed in view of a possible mechanism of induction of lysis by Pep 5 and nisin.
Acid phosphatase activity is demonstrated employing p-nitrophenyl phosphate as substrate and lead acetate as coupler. The fine structural localization of the enzyme in starved planarian tissues is described. The method is used to pin-point starvation - induced acid phosphatase activity in relation to autophagy and crinophagy in the gland cells; autophagy, autolysis and cell death in parenchymal and gastrodermal cells and basement membrane lysis. Attention is also payed to the demonstration of muscle lysis. The histochemical implications of the method are discussed.
Disintegration substantially accelerates autolysis of yeast cells. Three proteases (A, B, and C) take part in the autolytic process, protease A being the activator of the other two enzymes. The role of proteases B and C in the process depends on temperature. At 40 degrees C both proteases are active while at 50 degrees C the major role is played by protease C. At 40 degrees C NaCl acts as inhibitor while at 50 degrees C it activates the process.
Isoelectric focusing was used to study the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase in lysosomes isolated from rat kidney. The isoelectric points of the main protein and hydrolase peaks were 1-1.5 units lower when electrofocusing was done in a pH 3-10 gradient than in a pH 10-3 gradient, apparently because the lysosomal constituents aggregated strongly at their isoelectric points and tended to settle somewhat in the gradient due to gravity. In the extended pH gradient the acidic form of each hydrolase occurred as asingle, relatively discrete peak. However, when pooled acidic fractions were refocused in a restricted pH gradient (pH 6-3 or 3-5) multiple acidic enzyme and protein components were resolved with isoelectric points between 2.7 and 5.1. When autolysis was minimized by extracting lysosomal fractions at alkaline pH (0.2% Triton X-100, 0.1%p-nitrophenyloxamic acid, 0.1 M glycine buffer, pH9) and including 0.1%p-NITROPHENYLOXAMIC ACID, AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND CATHEPSIN D, in the pH gradient, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in two forms, an acidic form with an isoelectric point of about 4.4, and a basic form with an isoelectric point close to 6.2, 6.7 and 8.0, respectively. Acid phosphatase occurred in three forms with isoelectric points of 4.1, 5.6 and 7.4. When some autolytic digestion was permitted by extracting lysosomal fractions in an acidic medium (0.2% Triton X-100, 0.1 M sodium acetate buffer, pH 5.2) AT 0-4DEGREES C and omitting p-nitrophenyloxamic acid from the gradient, the acidic form of beta-glucuronidase and the intermediate form of acid phosphatase were lost, the isoelectric points of the acidic forms of acid phosphatase, arylsulfatase and beta-N-acetylhexosaminidase were increased 0.6-1.2 units, and the isoelectric point of the basic forms of acid phosphatase, arylsulfatase and beta-glucuronidase was increased 0.5 unit. When lysosomal extracts were incubated with bacterial neuraminidase before electrofocusing, the acidic forms of acid phosphatase, arylsulfatase and beta-glucuronidase were largely lost, the isoelectric point of the acidic form of beta-N-acetylhexosaminidase was increased from 4.5 to 6.4, and the isoelectric points of the basic forms of all four hydrolases were increased 0.5-1.5 units. Autoincubation of lysosomal extracts in vitro at pH 5.2 PRODUCED SIMILAR, THOUGH LESS MARKED, effects. cont'd
Extracts of bovine pancreatic tissue are shown by HPLC to contain two distinct ternary complexes of procarboxypeptidase A (subunit I), chymotrypsinogen C (subunit II) and either proproteinase E or subunit III. It is shown that proproteinase E in the complex generates subunit III by removal of 13 N-terminal residues when the former is allowed to autolyze in solution or when catalytic amounts of isolated active proteinase E are added to it. Autolysis of proproteinase E was accompanied by the loss of potential activity towards specific synthetic substrates and occurred at a higher rate in pancreatic juice than in pancreatic tissue extracts, even when both were processed in the presence of serine protease inhibitors. We conclude that subunit III (also called truncated protease E) is an autolytic product of proproteinase E and not an ab initio component of the native ternary complex.
An ultrastructural investigation showed that there was a neurohaemal organ in the wall of the ampulla of the antennal pulsatile organ. The neurosecretory axon terminals occurred singly or in small groups, rather than closely packed together as in other neurohaemal organs. All axons contained the same type of neurosecretory granule. The granules had varying electron density and a diameter in the range 1000-2500 A. Some terminals contained small, elliptical electron-transparent vesicles and the axolemma was apposed to the stroma. Other terminals were large and enveloped by glial tissue and the contents of the terminals exhibited varying degrees of autolytic degeneration. Autolysis was characterized by the occurrence of dense bodies and multilaminate bodies which enclosed mitochondria and neurosecretory granules. It was suggested that the neurosecretory material affects antennal function.