Acid Protease Activity Research Articles

Partial characterisation of human and porcine adipose acidic protease activity

1. 1. An aspartic protease was isolated from human and porcine white adipose tissue and from isolated human adipocytes. The three preparations appeared to represent the same enzyme. 2. 2. Electrofocussing of all three preparations revealed two bands corresponding to a pI of 3.6 and 4.4 respectively. On PAGE a single band in the same position was obtained in all three cases. 3. 3. Both the porcine and human fractions were optimally active at pH 3.4, using acid denatured haemoglobin as substrate, and both activities were strongly inhibited by pepstatin and iodoacetate. 4. 4. The K m values for haemoglobin for the porcine and human proteases were 0.16 and 0.14 mM respectively, whereas V max values of 30 and 33 units · nmol −1, respectively, were obtained.

Physiological properties of liver, gonads and muscle during maturation of female atlantic salmon, Salmo salar. Comparison between a control and xenobiotics containing fish feed

1. Atlantic salmon, Salmo salar, in their 4th year were maintained in a sea-based farm in the Baltic Sea, salinity 0.2–0.4%. The fish were fed a control diet or a diet containing, among the lipid-soluble xenobiotics, 60 parts of dioxin per 10 12 parts of fish oil. 2. In the fish that attained sexual maturity the liver and gonadal wet weight increased, but decreased after stripping of the roe. Vitellogenesis was also apparent as an elevated level of plasma vitellogenin which was higher after than 2 months prior to removal of the roe. 3. In muscle protein content was highest prior to the removal of the roe. RNA content decreased with time. Following the taking of the roe glycogen content and acid proteinase activity were elevated. 4. Comparison between the feeding groups showed, in the fish fed the experimental diet, a higher gonadal wet weight and plasma vitellogenin content, and in muscle, after stripping of the roe, a lower glycogen content and an elevated level of acid proteinase activity.

Physiological changes in skeletal muscle by maturation-spawning of non-migrating female atlantic salmon, Salmo salar

1. 1. Physiological properties of epaxial muscle of non-migrating female Atlantic salmon ( Salmo salar) were compared between non-spawning and spawning fish. 2. 2. The rise in blood plasma vitellogenin, and liver and gonadal weights confirmed the maturation-spawning condition. 3. 3. In the spawning females DNA, glycogen and acid proteinase activity were elevated per g wet weight of muscle, while protein and RNA content of the sarcoplasmic fraction and RNA of the myofibrillar fraction were diminished. 4. 4. The data suggest that characteristic changes occur in skeletal muscle by the process of maturation and spawning, which are independent of the physical load of upstream migration.

Aspartic proteinase from barley grains is related to mammalian lysosomal cathepsin D

Resting barley (Hordeum vulgare L.) grains contain acid-proteinase activity. The corresponding enzyme was purified from grain extracts by affinity chromatography on a pepstatin-Sepharose column. The pH optimum of the affinity-purified enzyme was between 3.5 and 3.9 as measured by hemoglobin hydrolysis and the enzymatic activity was completely inhibited by pepstatin a specific inhibitor of aspartic proteinases (EC 3.4.23). Further purification on a Mono S column followed by activity measurements and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the affinity-purified enzyme preparation contained two active heterodimeric aspartic proteinases: a larger 48k Da enzyme, consisting of 32-kDa and 16-kDa subunits and a smaller one of 40 kDa, consisting of 29-kDa and 11-kDa subunits. Separation and partial amino acid sequence analysis of each subunit indicate that the 40-kDa enzyme is formed by proteolytic processing of the 48k Da form. Amino-acid sequence alignment and inhibition studies showed that the barley aspartic proteinase resembles mammalian lysosomal cathepsin D (EC 3.4.23.5).

A comparative study of the biological properties of venoms of some old world vipers (Subfamily viperinae)

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 30 samples of venoms from nine species (12 taxa) of the old world vipers (Subfamily Viperinae) including snakes from the genera Bitis, Causus, Cerastes, Echis, Eristicophis and Pseudocerastes, were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. 2. Examination of the biological properties of the venoms of the Viperinae tested indicates the presence of common venom biological characteristics at the various phylogenic levels. 3. 3. Venoms of most species of the Viperinae examined exhibited characteristic biological properties at the species level, and this allows the differentiation of the Viperinae species by differences in their biological properties. 4. 4. Particularly useful for this purpose, are the effects of venom on kaolin-cephalin clotting time of platelet poor rabbit plasma and the Sephadex G-75 gel filtration pattern and arginine ester hydrolase activity of the venom.

Response of epaxial muscle and liver to 17-β estradiol in fed and starved Atlantic salmon (Salmo salar)

Presmolt salmon, 16 months old and weighing 54.5 ± 1.2 g, were continuously exposed to 17-β estradiol by implanting a capsule containing the hormone. The fish and their non-hormone-treated controls were fed or starved. At the end of the third week the fish were killed. After hormone treatment the vitellogenin content of the fed fish was 37.9 ± 4.7 mg and of the starved fish 44.9 ± 3.3 mg/ml of serum. Hormone treatment decreased liver glycogen of the fed fish to 26 mg from 58 mg and of the starved fish to 11 mg from 28 mg/g wet weight of tissue. In muscle a significant decrease in glycogen was noted as the result of starvation. Acid proteinase activity per g wet weight of liver was unchanged. As compared with the starved controls, acid proteinase activity in liver per 100 g body weight was significantly elevated in all estradiol-treated fish. Myosin heavy chain content per g wet weight of muscle and per mg of DNA decreased as a result of hormone treatment. A diminished protein content in muscle after hormone treatment of both the fed and starved fish was paralleled by an increase in acid proteinase activity. In the fed fish, muscle aspartate amino transferase (EC 2.6.1.1) increased significantly after hormone treatment, whereas alanine amino transferase (EC 2.6.1.2), glutamate dehydrogenase (EC 1.4.1.3) and malic enzyme (EC 1.1.1.40) were not changed significantly. Estradiol treatment of the starved fish had no effect on the activities of the metabolic enzymes. Alanine amino transferase decreased significantly as a result of starvation.In conclusion, ingestion of food combined with hormone treatment decreased muscle protein content and increased acid proteinase and aspartate amino transferase activity. Food deprivation combined with hormone treatment followed a similar pattern but was without effect on the amino acid metabolizing enzymes. Muscle glycogen content decreased as a result of starvation. Hepatic glycogen content declined as a result of hormone treatment and starvation alone. The carbohydrates in liver appeared to be utilized for energy production during starvation and/or estradiol treatment. The amino acids derived from muscle proteins were made available by the stimulation of proteinases in hormone-treated fed and starved fish and they were utilized for protein synthesis or as energy source.

Change in the acid protease activity in plasma of patients with disseminated intravascular coagulation

Lysosomal protease was determined in the serum of patients with disseminated intravascular coagulation (DIC) to clarify whether the platelet count is an appropriate diagnostic index which allows the early initiation of treatment. The platelet count and the serum level of cathepsin D, a lysosomal protease, were determined in 60 patients diagnosed to have DIC. The cathepsin D activity could not be detected in the sera of healthy individuals with a platelet count of 100,000 or above, but was detectable in the serum of DIC patients with a platelet count of 100,000, and this activity increased as the platelet count decreased to 70,000 and 50,000, and was about 5 times higher at a platelet count of 30,000 than at 70,000. In DIC patients, the decrease in the platelet count was correlated with the increase in the serum lysosomal protease activity. The appearance of cathepsin D activity in the serum of DIC patients is considered to reflect the release of lysosomal enzyme activities from damaged organs, and the treatment for DIC must be initiated before the platelet count decrease below 100,000, and cathepsin D activity then appears in the serum. At a platelet count of 30,000 or less, DIC becomes established, and no therapeutic effects can be expected because of the associated multiple organ failure.

INFLAMMATORY RESPONSE TO PARTICLES IN THE RAT LUNG: SECRETION OF ACID AND NEUTRAL PROTEINASES BY BRONCHOALVEOLAR LEUCOCYTES

Leucocyte proteinases are considered to be central to the tissue damage that is associated with chronic inflammatory lung disease. Both acid and neutral proteinases have the ability to degrade connective tissue molecules and both may therefore play a part in tissue proteolysis in inflamed lungs. In this study we have used a rat model of lung inflammation to investigate levels of acid and neutral proteinase activity in the bronchoalveolar region of control and inflamed lungs. We assessed the ability of proteinases, secreted by resident and inflammatory bronchoalveolar leucocytes, to damage the connective tissue molecule fibronectin. Inflammatory bronchoalveolar leucocytes had greater ability to mediate connective tissue damage than had resident alveolar macrophages and the tissue proteolysis was mediated, in the main, by proteinases active at neutral rather than at acid pH. The increased secretion of proteinase by inflammatory leucocytes was dependent on in vivo stimulation: exposing resident or inflammatory bronchoalveolar leucocytes in vitro to particulate or soluble stimuli had no effect in increasing their ability to degrade fibronectin or secrete the inflammogenic proteinase, plasminogen activator. Thus, neutrophils in the bronchoalveolar region of the rat lung have proteolytic activity which is mediated largely by neutral proteinases.

Enzymatic and Microbial Interactions in Response to Pitch Pine Root Growth

AbstractRoot growth stimulates many soil metabolic and enzymatic activities through, in part, loss of photosynthate as root exudates. Because of the abundance and structural simplicity of the carbonaceous compounds comprising root exudates, compared with native soil organic matter, it can be hypothesized that not only the magnitude but also the relationship between various soil microbial and enzymological processes vary with proximity to the growing root. To test this hypothesis, metabolism of amino acids and glucose, microbial growth, microbial biomass C, and potential acid phosphatase, protease, and invertase activities were examined in pitch pine (Pinus rigida Miller) rhizosphere and near rhizosphere soils. The trees were grown in microcosms (total volume ≈145 cm3) in the greenhouse in O or A horizon soils (Lakehurst sand, a mesic, coated Haplaquodic Quartzipsamment) from the New Jersey Pinelands. The microbial and enzymological activities in the organic‐matter‐rich O horizon soil were 2‐ to 25‐fold greater than those detected in the organic‐matter‐poor A horizon soils throughout the study. Phosphatase activity and glucose and amino acid metabolic rates did not correlate with tree root mass, whereas protease activity, microbial growth rate, and microbial biomass C varied with root mass. No effect of proximity to the root on these relationships was detected. In the A horizon soil after 22 wk of growth, amino acids and glucose metabolic rates declined with increasing root mass, whereas the microbial growth rate increased. Phosphatase activity was not detected in the A horizon soil. The interrelationship of these activities was evaluated in O horizon soil. Significant (P <0.05) correlations were detected between microbial biomass C and the various indices of microbial activity, but glucose metabolism and phosphatase and proteolytic activities varied independently of microbial biomass C. In conclusion, significant effects of tree root growth on enzymatic and metabolic activities were detected, but, with a few minor exceptions, relationships between these enzymological and metabolic activities were not altered by proximity to the growing tree root.

Effect of blood leucocyte depletion on the inflammatory response of the lung to quartz

This study determined the effect of blood leucocyte depletion on the early inflammatory response of the lung to α-quartz. F344 N rats were instilled intratracheally with either physiological saline or 2 or 5 mg of α-quartz suspended in saline. One day prior to the instillation, half of the rats received an ip injection of rabbit antiserum that had been raised against rat neutrophils. The other half of the rats received an ip injection of normal rabbit serum. One day after the instillation of saline or quartz, the animals were euthanized and observed for changes in blood cell numbers, lung histopathology, and bronchoalveolar lavage fluid (BALF) content of indicators of an inflammatory response and cytotoxicity. The rabbit antiserum depleted the blood of most white blood cells of all types. BALF fluid from saline-instilled animals did not differ between the white blood cell-depleted and the nondepleted animals except for a 20% reduction in numbers of alveolar macrophages in the depleted animals. BALF fluid from the nondepleted, quartz-instilled animals had a dose-dependent increase in content of neutrophils and protein (indicator of an increase in the permeability of the alveolar/capillary barrier) as well as an increase in lactate dehydrogenase and glutathione reductase (cytoplasmic enzymes whose presence extracellularly indicates cytotoxicity), alkaline phosphatase (indicator of type II cell secretory activity), β-glucuronidase, and acid proteinase (lysosomal enzymes) activities. The higher dose of quartz also elicited an increase in LTB 4 and PGE 2 content of BALF. GSH content of BALF was decreased by the quartz exposure. The depletion of blood white blood cells prevented the influx of neutrophils into the alveoli of the quartz-exposed rats and decreased the BALF markers of capillary permeability and cytotoxicity (protein content and extracellular cytoplasmic enzymes). The absence of neutrophils in the alveoli had no effect on the lysosomal content of BALF, indicating that the neutrophils were not the source of these enzymes in nondepleted rats exposed to α-quartz. The quartz-induced elevation of LTB 4 in BALF was not observed in depleted rats, suggesting that neutrophils may be the source of the increase in this leukotriene in the BALF. Both the GSH content and the alkaline phosphatase activity in BALF were enhanced in the absence of alveolar neutrophils. The enhancement of GSH in BALF is consistent with the neutrophils being the source of reactive oxygen species that deplete GSH. The increased alkaline phosphatase activity in the BALF of both the depleted and nondepleted animals is consistent with the type II cell hypertrophy that was induced by quartz instillation and was neutrophil independent. The study provides information on the probable sources of some of the inflammatory mediators in BALF and suggests that neutrophils contribute to the acute toxicity accompanying the inflammatory response of the lung to quartz. The neutrophil is not required. however, for the production of type II cell hypertrophy.

Metabolic changes related to the acceleration of jack pine germination by osmotic priming

Jack pine (Pinus hanksinna Lamb.) seeds were primed in 10% (w/w) polyethylene glycol8000 for 2-6 days and their germination, as well as soluble protein, amino acid, and ATP contents compared with unprimed germinating seeds. Primed seed germinated about 2 days earlier than unprimed seed and had a high ATP content early in the priming period. Pepstatin-sensitive acid proteinase activity, which was high even in dry seeds, correlated poorly with germination in primed seed.

Biochemical changes and in‐vitro protein digestibility of the endosperm of germinating dolichos lablab

AbstractA gradual decrease was noted in total protein, soluble protein and protein fractions such as albumin, globulin, prolamine andglutelin in the endosperm of Dolichos lablab L germinated for 8 days. Prolamine showed a maximum decrease of 85% whereas only a 25% decrease was noted in the globulin fraction. Both acid and alkaline protease activities increased during germination, and the acid protease activity was consistently higher than the alkaline protease activity throughout. Electrophoretic studies showed that high molecular weight polypeptide bands disappeared with the appearance of new low molecular weight polypeptide bands in the endosperm proteins of the germinating seeds. Total phenolics and tannin contents and trypsin inhibitory activity also increased during the progressive germination. Germination increased the in‐vitro protein digestibility of the endosperm of Dolichos lablab in the presence of proteolytic enzymes like pepsin, trypsin and pancreatin. It showed a negative but non‐significant correlation with trypsin inhibitory activity, phenolics and tannin contents. Amylase activity also increased gradually up to the sixth day of germination which coincided with the increase in the reducing sugars.

Batchwise ethanol fermentation with shochu distillery waste

A novel vacuum distillation procedure at low temperature (35–40°C) was applied to shochu distillation to produce a shochu with a mild aroma. The resulting kome (rice) shochu distillery waste contained a large amount of viable yeast (7 × 10 6 cells/ml), glucoamylase activity (70 U/ml), acid protease activity (720 U/ml), and neutral protease activity (420 U/ml). Secondary ethanol fermentation of the mash composed only of glucose as a carbon source and kome or mugi (barley) shochu distillery waste was properly effected. About 10% of ethanol (v/v) was produced in the fermented mash at 30°C within 3 d. In contrast, distillery waste discharged by conventional distillation at high temperature (55–60°C) could not be used for secondary ethanol fermentation at all. The filtrate of secondarily-fermented distillery waste, containing ethanol and possessing a fine aroma fortified with higher alcohols and volatile esters during ethanol fermentation, could be useful for the production of a mirin-like liquor for cooking, Akazake, a characteristic red-colored, sweet alcoholic beverage produced in Kumamoto prefecture only, and a bath additive containing ethanol, a fine aroma, and enzymes. The solid cake of the fermented mash could be used in the same way as conventional sake-kasu, sake-cake. Here, a novel system of utilizing shochu distillery waste, conventionally treated as wastewater, was established economically using a simple vacuum distillation procedure without a need of any complicated process.

A comparative study of the biological properties of some venoms of snakes of the genus Bothrops (American lance-headed viper)

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 26 samples of venoms from 13 species of Bothrops were determined, and the Sephadex G-75 gel filtration patterns for some of the venoms also examined. 2. 2. The results show that while there are considerable individual variations in the biological activities of many of the Bothrops venoms tested, there are some common characteristics at the genus and species levels. 3. 3. The differences in the biological properties of the Bothrops venoms tested can be used for the differentiation of most Bothrops species examined.

Intracellular activation of digestive zymogens in rat pancreatic acini. Stimulation by high doses of cholecystokinin.

The mechanism by which digestive zymogens become activated during acute pancreatitis remains poorly understood. Given the ability for cholecystokinin (CCK) to induce pancreatitis in vivo, the effects of high dose CCK on preparations of isolated pancreatic acini were examined. Using an immunologic technique for the detection of zymogen activation, CCK was found to stimulate the conversion of procarboxypeptidase A1 to a 35-kD form having the same net charge and electrophoretic mobility as purified recombinant carboxypeptidase A1. This enhanced conversion was proportional to the dose of CCK (maximal at 100 nM), and time dependent. CCK also produced changes in the electrophoretic mobility of procarboxypeptidase B and chymotrypsinogen 2 immunoreactivity, consistent with activation of these zymogens. These events were detectable only within acinar cell pellets and not in the incubation medium, suggesting an intracellular site of conversion. The conversion of procarboxypeptidase A1 to its active form was inhibited by pretreatment with the weak base chloroquine (40 microM) and the protonophore monensin (10 microM). This conversion was also inhibited by pretreatment with the serine protease inhibitor benzamidine (10 mM) but not the cysteine protease inhibitor E64 (100 microM). The results suggest that high dose CCK stimulates the intracellular activation of digestive zymogens within isolated pancreatic acini. This event appears to require an acidic subcellular compartment and serine protease activity.

Production, Purification and Properties of Acid Proteinase from Genus Monascus.

The screening was carried out to find strains in Monascus fungus that would produce a high activity of acid proteinase. Monascus sp. No. 3404 was found to have the highest activity of the enzyme, which was formed by addition of defatted soybean meal to the medium.The enzyme was purified to homogeneity from the culture broth of this organism grown on defatted soybean meal. The molecular weight of the purified enzyme was determined to be 43000 by gel filtration method and 46000 by SDS-polyacryl amide gel electrophoresis method. The optimum pH was 3.2 and the optimum temperature was 50°C. The enzyme was active on human hemoglobin, milk casein and bovine serum albumin, and Km values for them were calculated to be 0.80, 1.45 and 1.80mg/ml, respectively. The enzyme was markedly inhibited by pepstatin A and chymostatin, and Ki values for them were calculated to be 28nM and 0.13mM, respectively.

Proteolysis of peripheral nerve myelin in acute experimental allergic neuritis

Proteolysis of peripheral nerve myelin was studied in rats with experimental allergic neuritis (EAN). In vitro measurements using rat sciatic nerve homogenate and denatured bovine myelin as a substrate showed two myelin specific enzyme activities at pH 3.8 (inhibited by pepstatin) and pH 5.8 (inhibited by PMSF) in the normal rat and newly appearing activities at pH 2.8 (inhibited by pepstatin) and pH 5.0 (not characterized) in the EAN rat. In EAN the proteolytic activity was not restricted to myelin substrate but degraded total sciatic nerve protein as well. Endogenous sciatic nerve protease at pH 5.8 did not significantly change in activity during the course of disease. On the contrary, activity of acid protease at pH 2.8 corresponded well to the disease. Myelin degradation in EAN, therefore, appears to be mainly due to exogenous non-tissue protease.

Identification of Cystatin C, a Cysteine Proteinase Inhibitor, as a Major Secretory Product of Human Alveolar MacrophagesIn Vitro

The major inhibitor of the cysteine class of proteinases found in human body fluids, such as spinal fluid, milk, and seminal plasma, is cystatin C. In this study we show that human bronchoalveolar fluid also contains cystatin C and examine cystatin C expression by alveolar macrophages in vitro. Immunoprecipitation of extracts of metabolically labeled cells and immunoblotting of cellular extracts and culture media show that cystatin C is synthesized as a 14 (+/- 0.5) kilodalton (kD) protein and that greater than 90% of the protein is released as the 14 kD product into the culture supernatant (26.5 +/- 6.8 ng per 10(6) cells per 24 h). Cystatin C is not one of the most abundant proteins secreted during the first 24 h in vitro, representing approximately 10 to 12% of the total protein released by normal nonsmoker macrophages. Alveolar macrophages obtained from cigarette smokers or nonsmoker macrophages exposed to zymosan in vitro released 10 to 55% less cystatin C than nonsmoker macrophages. We also assayed culture supernatants from macrophages of smokers and nonsmokers for functional cystatin C. Supernatants of nonsmoker macrophages inhibited cathepsin B-like amidolytic activity in a fluorometric assay at pH 5.5. The inhibition was blocked by adsorption with Sepharose-coupled cystatin C antibodies and the inhibitor subsequently recovered from the Sepharose beads. In contrast, supernatants from smoker macrophages had obvious cathepsin B-like activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in cathepsin B + L and calcium-dependent protease activities among breed type and their relationship to beef tenderness.

Activities of acidic proteases (cathepsin B + L) and neutral, calcium-dependent proteases (CDP) were quantified to determine whether differences in proteolytic activity could explain differences in meat tenderness among breed types. Steers (n = 32) of known percentage Angus (A) and Brahman (B) breeding were used to establish differences in meat tenderness (A; 3/4A-1/4B; 1/2A-1/2B; 1/4A-3/4B). Samples were removed from the longissimus muscle within 1 h postmortem and within 2 h were frozen for subsequent determination of cathepsin B + L, CDP-I, CDP-II and CDP-inhibitor activities. Warner-Bratzler shear (WBS) was assessed after 1, 5 and 10 d of postmortem aging. Taste panel evaluations, conducted on steaks that were subjected to 5 d of aging, detected no differences. At d 1, WBS did not differ among breed types; however, by d 10 of aging, steaks from Angus steers were more tender (P less than .05) than steaks from 1/2B and 3/4B steers. The Angus and 1/4B steaks had significantly more (P less than .05) cathepsin B + L activity than the 3/4B. The CDP had no relationship with WBS; however, CDP-inhibitor was positively related to d-1 WBS (r = .41, P less than .05). Cathepsin B + L activity was negatively related to WBS at d 10 (r = -.44, P less than .05). These data suggest that differences in meat tenderness among breed types may be explained partially by differences in proteolytic enzyme activity.

Yolk degradation in tick eggs: I. Occurrence of a cathepsin L‐like acid proteinase in yolk spheres

In crude extracts of eggs of the soft tick Ornithodoros moubata, maximum degradation of vitellin is at pH 3-3.5, whereas no proteolysis is detected at neutral or weakly acidic pHs. Acidic proteolysis is maintained at high level throughout embryonic development, and rapidly decreases in the larva, during the high phase of yolk degradation. Proteinase, acid phosphatase, and N-acetylglucosaminidase are localized within the yolk spheres; these can be considered as lysosomal-like organelles containing both substrate (vitellin) and the degradative machinery. Proteolytic activity has been essentially attributed to a cathepsin L-like enzyme through substrate specificity and inhibitors. The molecular weight is 37,000 to 39,000 as shown using gelatin-containing SDS-PAGE activity gels. At neutral pH the enzyme binds to vitellin, as demonstrated by gel filtration and PAGE under nondenaturing conditions. Acid proteinase activity at pH 5-6 is undetectable both with proteins and synthetic substrates, but is strongly increased after preincubation at pH 3-4. Activation at low pH could be important in the regulation of yolk degradation.