Acid Protease Activity Research Articles

A comparative study of the biological activities of venoms from snakes of the genus Agkistrodon (moccasins and copperheads)

1. 1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, alkaline phosphomonoesterase, 5′-nucleotidase, hyaluronidase, arginine ester hydrolase, phospholipase A, l-amino acid oxidase and protease activities of 31 samples of venom from three species of Agkistrodon ( A. bilineatus, A. contortrix and A. piscivorus) and 10 venom samples from five other related species belonging to the same tribe of Agkistrodontini were examined. 2. 2. The results indicate that interspecific differences in certain biological activities of the Agkistrodon venoms are more marked than individual variations of the activities, and that these differences can be used for differentiation of the species. Particularly useful for this purpose are the phosphodiesterase, arginine ester hydrolase and anticoagulant activities of the venoms. 3. 3. Venoms of the subspecies of A. contortrix and A. piscivorus do not differ significantly in their biological activities.

Toxic impact of phosphamidon on protein degradation in phasic and tonic muscles of marine prawn,Penaeus indicus(H. Milne Edwards)

Levels of various protein fractions, (sarcoplasmic, myosin, actin, non-collagen and collagen) and the rate of their degradation by proteases were studied in phasic and tonic muscles of marine prawn, Penaeus indicus following acute (2 d) and chronic (15 d) exposure to sublethal concentration of phosphamidon. During exposure, greater decrease in sarcoplasmic protein fraction was observed in phasic muscle as compared to other myofibrillar proteins. But the sarcoplasmic protein content showed an elevation in tonic muscle. The changes in protein fractions were more pronounced during acute exposure than chronic exposure both in phasic and tonic muscles. These changes were correlated with the elevation of the acidic, neutral and basic protease activities during acute and chronic exposure. Free amino acids were increased during acute exposure, while they showed a significant decrease during chronic exposure in both the muscles. These results indicate that protein metabolism in both phasic and tonic muscles was significantly altered following phosphamidon exposure. These differential responses observed at acute and chronic exposure indicate the operation of compensatory mechanisms to mitigate the phosphamidon toxic stress.

A comparative study of the biological properties of venoms from snakes of the genus Vipera (true adders)

1. 1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, hyaluronidase, alkaline phosphomonoesterase, 5′-nucleotidase, arginine ester hydrolase, phospholipase A, l-amino acid oxidase and protease activities of 26 samples of venoms of 13 taxa of Vipera were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. 2. The results indicate the presence of certain common characteristics among the venoms, particularly if V. russelli is excluded from the comparison. The results also support the recently proposed reassignment of V. russelli to a separate genus. 3. 3. The data show that information on venom biological properties can be used for differentiation of venoms of many species of Vipera. Particularly useful for this purpose are the protease, phosphodiesterase, phospholipase A and the procoagulant activities and the Sephadex G-75 gel filtration patterns of the venoms.

Isolation and immunochemical characterization of fractions from membranes of Aspergillus fumigatus with protease activity

Two fractions exhibiting acid protease activity (AFPI and AFPII) were isolated by extraction of membrane vesicles of Aspergillus fumigatus with Triton X-100. These two fractions produced single bands in both polyacrylamide and sodium dodecyl sulfate polyacrylamide gel electrophoresis and showed apparent molecular weights of 73,000 and 43,000, respectively. Molecular weights determined by gel filtration in the absence and presence of Triton X-100 and sedimentation velocities in analytical ultracentrifugation indicated hydrophobic characteristics, since both fractions readily aggregated and complexed with Triton X-100; both exhibited elevated enzyme activities in the presence of Triton X-100. Carbohydrate content was 93% for AFPI and 85% for AFPII. The enzymatic fractions demonstrated different pH optima in the acid range as well as different temperature stabilities. Both protease fractions cross reacted in double immunodiffusion, while in crossed immunoelectrophoresis both demonstrated five precipitin peaks, each with similar patterns. AFPI demonstrated two additional precipitin peaks in crossed immunoelectrophoresis. As determined by crossed immunoaffinoelectrophoresis, the protease fractions demonstrated galactose and mannose residues. In biotin-avidin enzyme-linked immunosorbent assay both fractions reacted with allergic bronchopulmonary aspergillosis and aspergilloma sera. It can be concluded that two fractions with protease activity of A. fumigatus reported here may be of significance in Aspergillus-induced diseases.

Variation among lines selected for body size in the fractional rate of degradation of protein and acid protease activity in the muscle of quail (Coturnix coturnix japonica)

Variation among lines selected for body size in the fractional rate of degradation of protein and acid protease activity in the muscle of quail (Coturnix coturnix japonica)

Macrophages possess both neutral and acidic protease activities toward low density lipoproteins

Low density lipoproteins (LDL) have been strongly implicated in the pathogenesis of atherosclerosis. We have studied the proteolytic degradation of these lipoproteins by macrophages, which are a major cellular constituent of atherosclerotic lesions. Mouse peritoneal macrophages contained both an acidic and a less active but distinct neutral/alkaline protease activity toward human 125I-labelled LDL. The acidic activity had a pH optimum of 4.5 and the neutral/alkaline activity one of 8–8.5. The acidic activity started to plateau with increasing lipoprotein concentrations whereas the neutral activity was directly proportional to the lipoprotein concentration up to at least 150 μg of protein/ml. The acidic protease activity had a complex time course whereas the neutral activity was directly proportional to the time of incubation up to at least 48 h. Leupeptin (35 μM) and pepstatin (5 μM) inhibited the acidic activity by about 70% individually and almost entirely in combination, indicating that cathepsins B and D are important in the degradation of LDL by lysosomal cathepsins. In contrast, there was little, if any, inhibition of the neutral protease activity by leupeptin or pepstatin. The acidic protease activity was increased by both DL-dithiothreitol (5 mM) and disodium EDTA (1 mM) whereas the neutral protease activity was increased by dithiothreitol but inhibited partially by EDTA. The possible significance of macrophage neutral and acidic protease activities toward LDL in atherosclerosis needs to be assessed.

Surface acid proteinase (gp63) of Leishmania mexicana: A metalloenzyme capable of protecting liposome-encapsulated proteins from phagolysosomal degradation by macrophages

Acid proteinase activity is associated with the major surface glycoprotein (gp63) of both extracellular promastigotes and intracellular amastigotes of the parasitic protozoan, Leishmania mexicana. The enzyme purified by monoclonal affinity chromatography from promastigotes is strongly inhibited by metal ion chelators, which is reversible by the addition of Zn(II). This proteinase loses its activity after dialysis against 1,10-phenanthroline. The apoenzyme thus prepared is reactivated substantially by Zn(II) and partially by Cu(II), Cd(II), Co(II), or Ni(II). From the recently published structure of the gene encoding gp63, we identify hitherto unrecognized sequences, which can be aligned to the consensus zinc-binding sites of other known metalloproteinases. Anti-gp63 polyclonal antibodies, but not the monoclonals, precipitate similar molecules from amastigotes. These molecules differ slightly from gp63 in electrophoretic mobility but have similar endopeptidase activity. Phagolysosomal degradation by macrophages of proteins entrapped in liposomes is prevented by coating them with native gp63. This protection is lost with heat denaturation of gp63 to kill its enzymatic activity. The proteolytic activity of the metalloenzyme on the surface of these parasites may thus protect their membrane from cytolytic damages during their survival, differentiation, and multiplication in the phagolysosomes of macrophages.

Enhanced acid protease activity of lysosomes from papillary thyroid carcinoma.

In vitro lysosomal acid protease activity was studied in human papillary thyroid carcinoma (n = 13). As a control, morphologically normal thyroid tissue from the same patient was used in each individual case of carcinoma. Although a marked variation may be observed between individual cases, each examined papillary thyroid carcinoma showed significantly greater activity of acid proteases, both per unit weight of wet thyroid tissue and per unit of lysosomal proteins, in comparison to the corresponding control (range, 24%-248%). In conclusion, it is suggested that enhanced proteolytic activity of lysosomal acid proteases in papillary carcinoma is probably a result of disturbance in catabolic degradation of the thyroglobulin molecule in malignantly transformed thyroid tissue.

Early systemic effects on the hepatic mitochondria of tumour bearing mice

The activities of citrate synthase and cytochrome c oxidase, mitochondrial marker enzymes, were evaluated in the liver of Ehrlich ascites carcinoma-bearing mice during the life span of the inoculated animals. After 10 days of tumour transplantation, when cell proliferation has ceased, a 30 – 40% decrease of these activities was detected in both liver and kidney. Simultaneously, an increase in the total acidic proteinase activity (50 – 60%) was observed. The gel filtration profiles of liver proteinase activities from inoculated animal extracts displayed different patterns to those of the controls; low molecular weight proteinase activities appear to be enhanced in the livers of tumour-bearing animals.

Production of swine pepsinogen by protein-producing Bacillus brevis carrying swine pepsinogen cDNA

An expression-secretion vector, pNU100, was constructed, utilizing the promoter and coding sequences for the signal peptide and nine amino-terminal amino acids of the middle wall protein, to produce foreign proteins by protein-producing Bacillus brevis. Expression of swine pepsinogen cDNA in B. brevis was examined with pNU100 as a vector. The recombinant swine pepsinogen synthesized by B. brevis was found to accumulate extracellularly in the form of a soluble protein and to have acid protease activity. The acid protease activity was completely inhibited by pepstatin. Furthermore, the recombinant pepsinogen was converted autocatalytically to pepsin under acidic conditions. This indicates that B. brevis produces a pepsinogen with the same conformation as authentic pepsinogen. Efficient production of the enzyme (11 mg/l) was achieved by regulating the pH of the medium. The enzyme produced by B. brevis remained stable on cultivation for a long period, up to 40 h. This is suggested to be due to a unique property of protein-producing B. brevis, i. e. a deficiency in extracellular protease production.

Evidence for selection of virulent sub-populations ofEntamorba histolyticaby cholesterol

Quantitatively much higher Concanavalin A (Con. A) agglutinability, haemolytic potency, and activities of acid hydrolases, namely phosphatase (EC 3.1.3.2), ribonuclease (EC 2.7.7.16), deoxyribonuclease (EC 3.1.4.5) and proteinase--were observed in a virulent strain of Entamoeba histolytica (IP-106), as compared to attenuated and avirulent strains (200-NIH) and DKB respectively. In addition, significant differences in these parameters were observed among clonal cultures derived from the latter two cultures by cultivation of single amoebic cells picked out by micromanipulation. Repeated sub-culturing of parent cultures of both these strains in cholesterol-enriched medium resulted in marked enhancement of all the above activities, but no such change occurred in the derived clonal cultures following similar cholesterol treatment. The implication of these findings in relation to enhancement of the virulence of E. histolytica by cholesterol is discussed.

Cytoplasmic pH regulation and chloride/bicarbonate exchange in avian osteoclasts.

Osteoclasts resorb bone by first attaching to the bone surface and then secreting protons into an isolated extracellular compartment formed at the cell-bone attachment site. This secretion of protons (local acidification) is required to solubilize bone hydroxyapatite crystals and for activity of bone collagen-degrading acid proteases. However, the large quantity of protons required, 2 mol/mol of calcium, would result in an equal accumulation of cytosolic base equivalents. This alkaline load must be corrected to maintain cytosolic pH within physiologic limits. In this study, we have measured cytoplasmic pH with pH-sensitive fluorescent compounds, while varying the extracellular ionic composition of the medium, to determine the nature of the compensatory mechanism used by osteoclasts during bone resorption. Our data show that osteoclasts possess a chloride/bicarbonate exchanger that enables them to maintain normal intracellular pH in the face of a significant proton efflux. This conclusion follows from the demonstration of a dramatic cytoplasmic acidification when osteoclasts that have been incubated in bicarbonate-containing medium are transferred into bicarbonate-free medium. This acidification is absolutely dependent on and proportional to medium [Cl-]. Furthermore, acidification is inhibited by the classic inhibitor of red cell anion exchange, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, and by diphenylamine-2-carboxylate, an inhibitor of chloride specific channels. However, the acidification process is neither energy nor sodium dependent. The physiologic importance of chloride/bicarbonate exchange is demonstrated by the chloride dependence of recovery from an endogenous or exogenous alkaline load in osteoclasts. We conclude that chloride/bicarbonate exchange is in large part responsible for cytoplasmic pH homeostasis of active osteoclasts, showing that these cells are similar to renal tubular epithelial cells in their regulation of intracellular pH.

Mycelial distribution in rice Koji

Mycelial distribution of Aspergillus oryzae in rice koji was measured to elucidate the steric growth mode in the solid-state culture. Mycelial density showed a exponential distribution against the distance from the surface of rice koji. On the basis of this phenomenon, mycelial distribution was expressed by a mathematical model and total mycelia were shown by the following equation: Y t= ac∫e b( R− r) r 2d r+ Y s where Y t is the total mycelia in koji, the first term in right side is the mycelia in the inner part of rice grain and Y s is the surface mycelia. The variable r is the distance from the center of the koji, R is the radius of the koji, and a, b, and c are parameters. The values of each parameter were obtained by the least squares method using the experimental data and the distribution of mycelia both in the surface and in the matrix of rice grain was shown quantitatively. The steric growth mode of fungi in rice grain was shown using these techniques. The mycelial distribution and enzyme composition in rice koji made in sake factories were examined and the results were analyzed by a principal component analysis. The results suggested that when the degree of mycelial penetration is low the activities of acid protease and acid carboxypeptidase are high.

Physiological and biochemical characterization of a suspensionculture system for sustained exponential growth of Nicotiana silvestris

A strong approach to understanding the regulation of enzymes in metabolic pathways, such as those responsible for amino acid biosynthesis, is to follow enzyme levels throughout the growth curve of higher plant cells in suspension culture. The rise and fall of enzyme levels can be traced as a function of physiological stage of growth Subculturing, as typically carried out by low‐factor dilution of stationary phase cells, yields a system suitable for the study of changes in enzyme and metabolite levels that accompany the transition from stationary‐phase physiology to exponential‐phase physiology. However, the short duration of exponential growth in such subculture protocols is inadequate to avoid carryover effects from previous stationary‐phase physiology. Suspension cultures of Nicotiana silvestris Speg, et Comes (2N = 24) were used to demonstrate substantial carryover levels of acid phosphatase, alkaline phosphatase and protease activities. A subculture routine is described for maintaining cell populations in exponential phase indefinitely. About 10 generations of sustained exponential growth is required to approach a true balanced state of exponential growth. Such exponential phase populations consist of cells termed EE cells. EE‐cell populations were similar to cells that have been in exponential phase for only a few generations (E cells), with respect to doubling time (about 40 h) and to minimal density of diluted populations able to resume growth (about 500 cells ml−1). EE cells possess a high content of soluble protein; they are smaller and more aggregated than are E cells. Upon dilution into fresh medium, EE cells resume exponential growth without a lag. In contrast to E cells, EE cells exhibit properties of balanced growth, since proportionate increases in cell number, dry weight, wet weight and packed‐cell volume were observed. E cells, sampled at different elapsed times of growth, are likely to differ in metabolite, enzyme and cell properties, whereas EE cells exhibit near‐constant properties.

Subcellular Localization of Proteases in Developing Leaves of Oats (Avena sativa L.)

The distribution and subcellular localization of the two major proteases present in oat (Avena sativa L. cv Victory) leaves was investigated. Both the acidic protease, active at pH 4.5, and the neutral protease, active at pH 7.5, are soluble enzymes; a few percent of the enzyme activity was ionically bound or loosely associated with organellar structures sedimenting at 1000g. On the average, 16% of the acidic protease could be washed out of the intercellular space of the leaf. Since isolated protoplasts contained correspondingly lower activities as compared to crude leaf extracts, part of the acidic activity is associated with cell walls. No neutral protease activity was recovered in intercellular washing fluid. Of the activities present in protoplasts, the acidic protease was localized in the vacuole, whereas the neutral protease was not. The localization of the acidic protease in vacuoles did not change during leaf development up to an advanced stage of senescence, when more than 50% of the leaf protein had been degraded. These observations indicate that protein degradation during leaf senescence is not due to a redistribution of acidic protease activity from the vacuole to the cytoplasm.

The thoracic surgical spectrum of acquired immune deficiency syndrome

A rat model has been developed to study the local effects of burn injury on the underlying muscle tissue. Protein turnover was measured in soleus muscle incubated in vitro in which both tyrosine release and protein synthesis was measured. A scald injury (3 seconds) to a small area of one hindlimb produces an increase in muscle proteolysis and is without effect on the soleus muscle of the contralateral leg. A very high concentration of indomethacin (40 μmol/L) had no effect on proteolysis in the control muscle but specifically inhibited burn-induced protein breakdown. However, since other cyclooxygenase inhibitors (aspirin and ibuprofen), lipoxygenase inhibitors (ETYA, NDGA, and esculetin), and mepacrine (a phospholipase inhibitor) had no effect on protein breakdown, it is unlikely that a product of arachidonic acid metabolism maintains the increased proteolysis in vitro. In addition, endogenous production of prostaglandin E2 (PGE2) was not different in muscles from burned and control legs. Probes of the proteolytic pathway using inhibitors show that the burn-induced stimulation of proteolysis is consistent with the stimulation of lysosomal protease activity. These results are supported by the observation of increased acid protease activity in muscle homogenates from the burned leg. The best hypothesis that explains these data is that a lysosomal pathway of protein degradation may be enhanced by burn. Products of arachidonic acid metabolism do not appear to maintain burn-induced proteolysis in muscle, although their role in initiating the pathological changes in vivo cannot be excluded.

Characterization of enzymes that degrade crab exoskeleton: II. Two acid proteinase activities

AbstractSpecific proteins extracted from the membranous layer of the exoskeleton of the Bermuda land crab, Gecarcinus lateralis, are degraded at acid pH values by two proteinase activities (AP I and AP II) extracted from integumentary tissues. The pH optimum of AP I was about 5; it does not bind to cation exchange resin and was strongly inhibited by pepstatin A. The pH optimum of AP II was about 4; it binds to a cation exchange resin and was inhibited by cysteine proteinase inhibitors. AP II shared some characteristics with the human lysosomal proteinase cathepsin B. Unlike cathepsin B, which binds to the cysteine proteinase inhibitor E‐64 in a 1:1 ratio, AP II was not completely inhibited by E‐64, and only at the lower concentrations of inhibitor was there a linear relationship between enzyme activity and amount of inhibitor.

Nucleotide sequence and expression in Escherichia coli of cDNA of swine pepsinogen: involvement of the amino-terminal portion of the activation peptide segment in restoration of the functional protein

A clone, pSPcA2, which carries the full-length swine pepsinogen cDNA was isolated. The coding sequence comprised the signal peptide [15 amino acids (aa)], the activation peptide segment (44 aa) and mature pepsin (327 aa). The deduced amino acid sequence agrees with the published sequence with two exceptions. Asparagine instead of aspartate is present at aa positions 19 and 308. Two types of plasmids, pAS and pUC tacSPc series, were constructed for expressing swine pepsinogen cDNA. These plasmids directed the synthesis of polypeptides which were detected by employing an antibody to swine pepsinogen. However, all the polypeptides formed aggregates and showed no acid protease activity. Only the protein directed by pAS5 regained the acid protease activity after renaturation procedures. The activity was completely inhibited by pepstatin. Furthermore, the renatured pAS5 protein was spontaneously converted to pepsin under acidic conditions. The presence of Arg-8 in the activation peptide segment appears important for the stabilization of the pepsinogen molecule.

Soluble and bound acid protease activity of myelin from bovine cerebral white matter and spinal cord

Isolated myelin of bovine spinal cord was found to degrade exogenous myelin basic protein (MBP) at pH 4.4. Electrophoretic peptide patterns were consistent with limited proteolysis of MBP. Some of the proteolytic activity was soluble at increased ionic strength, some remained bound, withstanding extraction at 37 degrees C for up to 12 hr. While being measurable with exogenous MBP, bound protease degraded neither bound MBP nor any other major intrinsic myelin protein. Both soluble and bound protease activity was completely inhibited by pepstatin A. The patterns of limited proteolysis of MBP they produced were identical. Myelin of cerebral white matter also exhibited soluble and bound acid protease activity which was likewise inhibited by pepstatin A. Protease activity of spinal cord and cerebral myelin is therefore suggested to be due to a cathepsin D-like endopeptidase, present in a loosely and tightly bound form. Both forms increased by 50 to 80% in activity when myelin was isolated from mixtures of white and cortical gray matter. While increased soluble activity of myelin is consistent with binding of cathepsin D of lysosomal origin during the isolation of myelin the tightly bound form might point to a principal mechanism through which exogenous proteins may become attached to the myelin sheath in vivo.

Cathepsin D is membrane-associated in macrophage endosomes.

Previously we identified an acid protease activity which was located in the endosomes of rabbit alveolar macrophages (Diment, S., and Stahl, P.D. (1985) J. Biol. Chem. 260, 15311-15317). In this study, the endosomal protease is identified as cathepsin D by immunoprecipitation with polyclonal antibodies raised against rabbit cathepsin D and by NH2-terminal sequence. In order to elucidate the mechanism for targeting of cathepsin D to endosomes, we first examined the membrane association of cathepsin D with light (rho = 1.05 g/ml) and heavy density (rho = 1.1 g/ml) vesicles from Percoll density gradients. After sequential washes, 8.4 and 21.9% of cathepsin D activity remained associated with heavy and light density vesicles, respectively. This membrane-associated cathepsin D could not be solubilized in either buffer at pH 5.0 containing mannose 6-phosphate and EDTA or in buffer at pH 10.6. Solubilization required the detergent Triton X-100. To determine whether membrane-associated cathepsin D was found in endosomes, the enzyme was radioiodinated within endosomes and lysosomes with internalized lactoperoxidase. The membrane-associated form was detected in endosomes, but much less in lysosomes. Biosynthetic studies combined with the same extraction procedure revealed that macrophage cathepsin D is first synthesized as an inactive membrane-associated precursor. The precursor is processed to an active, membrane-associated form and then to the active soluble form found in lysosomes. Our studies provide evidence that 1) cathepsin D is in endosomes of macrophages; 2) cathepsin D is transported to endosomes as a membrane-associated form; and 3) the membrane-associated form is a biosynthetic precursor for the soluble form found in endosomes and lysosomes.