Acid Protease Activity Research Articles

Acid protease and formation of multiple forms of glucoamylase in batch and continuous cultures of Aspergillus niger

In order to identify factors responsible for production of multiple forms of glucoamylase (GA) by Aspergillus niger Bo-1, the fungus was cultured in both complex and defined media in pH-controlled batch fermenters and chemostats. At all culture conditions three forms of GA were produced with molecular weights of approx. 91 (GAI), 73 (GAII), and 59 kDa (GAIII). Data from batch fermentations with constant pH 3.0 and 5.0 showed a uniform distribution of extracellular GA forms throughout the fermentations and independent of culture growth phases. Furthermore, steady-state data from chemostat cultivations at constant pH 3.0 and 5.0 showed a similar distribution of extracellular GA forms and established that the nitrogen concentration of the medium (C/N ratio) did not affect the distribution of multiple forms of GA. The extracellular acid protease activity was only moderate when the fungus was cultivated in batch and continuous fermentations with a constant pH of 3.0 or 5.0, whereas acidification of both complex and defined batch culture media after 20 h of growth induced a significant secretion of acid protease(s). The protease(s) present in the complex medium catalysed modification of the extracellular profile of the multiple forms of GA by degradation of GAI to GAII, whereas no proteolytic processing of the GA profile was observed in the defined medium. In vitro experiments confirmed that the pH-induced modifications of the GA multiple-form profile were caused by proteolytic and not spontaneous degradation of the GA forms at low pH. It was concluded that the observed modifications of the extracellular profile of GA isoforms in A. niger Bo-1 are due to changes in pH and medium composition.

Effect of Glyphosate on Aromatic Amino Acid Metabolism in Purple Nutsedge (Cyperus rotundus)1

The effect of glyphosate on aromatic amino acid metabolism in purple nutsedge sprouted tubers and shoots was investigated. Glyphosate at 33.5 mM caused inhibition of bud elongation, increased total free amino acid concentration, and caused rapid accumulation of shikimic acid in sprouted tubers. However, only one aromatic amino acid, tryptophan, decreased quickly to 22% of control 3 d after treatment (DAT) and remained low afterwards. This suggests that the inhibition of bud elongation is due to the rapid accumulation of shikimic acid and the repression of tryptophan synthesis. Foliar application of glyphosate at 14.5 mM to purple nutsedge shoots resulted in the rapid accumulation of glyphosate which was rapidly converted to its metabolite, aminomethylphosphoric acid. Free amino acids in leaves were also increased by glyphosate 3 DAT. The reduction in soluble protein 5 DAT and increased acid protease activity 3 DAT suggests that the late accumulation of free amino acids partially resulted from protein hydr...

Increase of Candida cell virulence by anticancer drugs and irradiation.

The influence of anticancer drugs and irradiation on Candida cell proliferation, adherence to HeLa cells and susceptibility to antifungal drugs (amphotericin B and miconazole) and neutrophils were examined using two Candida albicans strains. After treatment with 5-fluorouracil (25 microg/ml to 250 microg/ml), cis-diammine-dichloroplatinum (10 microg/ml to 100 microg/ml), peplomycin (0.5 microg/ml to 5 microg/ml) or 137Cs (20 Gy to 40 Gy) for 3 days or more, surviving Candida cells proliferated more rapidly than did untreated control cells. Anticancer agent-pretreated Candida cells revealed an increased adhesion to HeLa cells corresponding to an increase of binding to the lectins. The concentration of half limited colony formation (IC50) of amphotericin B and miconazole was increased to near two-fold that of the control by pretreatment of Candida cells with the anticancer agents, except peplomycin, which only weakly increased IC50. In addition, the enolase and Candida acid proteinase activities in the culture supernatants were increased by pretreatment with the drugs and irradiation. Correspondingly, surviving Candida cells after these treatments were resistant to neutrophils, with a reduction to half of the killing. These results indicate that anti-cancer drugs and irradiation potentiate the virulence of Candida cells, or they eliminate Candida cells with low virulence, thereby enhancing the risk of oral and systemic candidiasis.

清酒麹の酵素活性に及ぼす原料米タンパク質組成の影響

It was examined whether the protein components of raw material rice influence enzyme production by the Koji fungus. The ratio of the protein components varied greatly among the varieties of raw material rice. A negative high correlation was recognized between prolamin and glutelin, and a positive high correlation was recognized between prolamin and globulin. Furthermore, a negative high correlation was recognized between glutelin and globulin. High levels of activity for α-amylase and glucoamylase were caused by raw material rice which contained low levels of prolamin and globulin and high levels of glutelin. Acid protease activity was intense in the raw material which had high levels of globulin and low levels of glutelin. Interestingly, acid carboxypeptidase was not at all influenced by the protein components of the raw material rice.

Functional defects of NK cells treated with chloroquine mimic the lytic defects observed in perforin-deficient mice.

NK cells are the primary effectors mediating acute rejection of incompatible bone marrow cell grafts. To reduce rejection, we evaluated the ability of chloroquine (CHQ) to prevent perforin-dependent NK cell activity. Perforin is a key cytotoxic component released from the lytic granules of activated NK cells. Generation of functional perforin requires an acidic protease activity that occurs in the secretory, lytic lysosomes. Our hypothesis was that CHQ, a lysosomotropic reagent, would raise the pH of the acidic compartment in which perforin is processed and thereby block perforin maturation and cytotoxicity. We have measured NK cytotoxicity in vivo by clearance of YAC-1 tumor cells from the lungs and by rejection of incompatible bone marrow transplants and in vitro by cytolysis of YAC-1 and Jurkat cells. The engraftment of bone marrow cells was monitored by recolonization of the spleen with hemopoietic cells from transplants of MHC class I-deficient bone marrow cells into lethally irradiated recipient mice. Transplant rejection was compared in two inbred strains of mice: 129, which apparently use perforin-dependent cytotoxicity, and C57BL/6, in which rejection can be perforin-independent. CHQ treatment reduced NK cell activity in 129 mice in which perforin is important for mediating rejection. CHQ affected the fraction of NK cell cytolysis that was Fas independent. In addition, we found that CHQ prevents perforin processing by LAK cells in vitro. These data indicate that CHQ may impair rejection of incompatible bone marrow transplants and other functions mediated by NK and cytotoxic T cells.

Analysis of Two Aspergillus nidulans Genes Encoding Extracellular Proteases

vanKuyk, P. A., Cheetham, B. F., and Katz, M. E. 2000. Analysis of two Aspergillus nidulans genes encoding extracellular proteases. Characterization of prtAΔ mutants, generated by gene disruption, showed that the prtA gene is responsible for the majority of extracellular protease activity secreted by Aspergillus nidulans at both neutral and acid pH. The prtAΔ mutation was used to map the prtA gene to chromosome V. Though aspartic protease activity has never been reported in A. nidulans and the prtAΔ mutants appear to lack detectable acid protease activity, a gene (prtB) encoding a putative aspartic protease was isolated from this species. Comparison of the deduced amino acid sequence of PrtB to the sequence of other aspergillopepsins suggests that the putative prtB gene product contains an eight-amino-acid deletion prior to the second active site Asp residue of the protease. RT-PCR experiments showed that the prtB gene is expressed, albeit at a low level.

Conception of a Bioelectromagnetic Signal System via the Collagen Fibril Network; Biochemical Conclusions and Underlying Coherent Mechanism. II. Energetic Aspects, Acid and Neutral Proteases, and the Phenomenon of Coherence

This paper presents a bioelectrical conception of connective tissue regulation in bone, cartilage, and tendon, as well as other mechanically stressed connective tissues, based on the biological hypothesis of a biosensor and nerve-like signal conducting function of the native collagen fibril in the extracellular matrix. The various levels of existing conceptions of bioelectrical connective tissue regulation as well as some questions of classical connective tissue research (e.g., neutral and acid protease activity) are discussed from this electrophysiological point of view. Part I presented the topic in the form of classical biophysics and physicochemistry. This paper, Part II, makes use of the concept for a discussion of the “living state” of the extracellular matrix, biochemical aspects of acid and neutral protease activity, and nanoelectronic, relativistic, and coherent aspects of connective tissue regulation.

Conception of a Bioelectromagnetic Signal System via the Collagen Fibril Network; Biochemical Conclusions and Underlying Coherent Mechanism. I. Solid State Effects and Hierarchical Bioelectrical Regulation

This paper presents a bioelectrical conception of connective tissue regulation in bone, cartilage, and tendon as well as other mechanically stressed connective tissues, based on the biological hypothesis of a biosensor and nerve-like signal conducting function of the native collagen fibril in the extracellular matrix. The proposed bioelectrical collagen function and its interdisciplinary test offer a new approach to various clinically important questions. This first paper (Part I) presents the topic in the form of classical biophysics and physicochemistry. Part II attempts to make good use of the concept in discussing the “living state” of the extracellular matrix and the biochemical aspects of acid and neutral protease activity, as well as nanoelectronic, relativistic, and coherent aspects of connective tissue regulation.

Effect of Organic Nitrogen Sources on Raw Starch-Digesting Glucoamylase Production of Rhizopus sp. MKU 40

In a liquid cultivation of Rhizopus sp. MKU 40, supplementation of the medium with 1.5% (w/v) organic nitrogen sources (neopeptone, casein from milk, and meat extract) had a slightly positive effect on glucoamylase (GA) (EC 3.2.1.3) activity compared with the medium lacking organic nitrogen sources. The addition of organic nitrogen sources induced production of protease. Supplementation of the medium with 1.5% (w/v) organic nitrogen sources resulted in an acid and neutral protease activity of 11-25 U/mL and 12-20 U/mL, respectively. The co-existence of GA-I {a highly raw starch-digesting glucoamylase (RSDG)} and protease in the same medium leads to the production of Ga-II (an extremely weak RSDG) from GA-I. As a result the RSDG activity in the medium decreases. Raw starch adsorption rates of a medium without organic nitrogen sources were 100%, because the medium contained only GA-I. In contrast, the media supplemented with organic nitrogen sources had low starch adsorption rates because the media contained both GA-I and GA-II. The results presented in this paper indicate that supplementation of the culture medium of Rhizopus strains with organic nitrogen sources negatively affects GA-I production.

Physiological and Oxidative Response to NaCl of the Salt Tolerant Grevillea ilicifolia and the Salt Sensitive Grevillea arenaria

Summary A comparative study of shoot cultures of two Grevillea species exposed to various levels of NaCl (for 6 weeks) indicated that Grevillea ilicifolia was significantly more tolerant to salt than Grevillea arenaria . Growth (explant height and numbers of shoots and leaves) in G. ilicifolia significantly declined at salt concentrations greater than 100 mmol · L -1 , whilst growth of G. arenaria was significantly impaired at 40 mmol. L -1 NaCl. Protochlorophyll, chlorophylls and carotenoids were significantly reduced as the salt concentration was increased, with both of the Grevillea species being equally sensitive to NaCl. The rate of decline of protochlorophyll and chlorophyll b with NaCl exposure was greater than that of chlorophyll a and carotenoids for both species. Anthocyanin pigments on the other hand significantly increased with salt stress in both of the Grevillea species. Levels of Na and Cl were significantly elevated during salt exposure, while K, Ca and Mg levels were markedly reduced. The levels of malondialdehyde significantly declined in both species as a result of salt exposure, and this was accompanied by significant increases in both acid protease and neutral protease activities. Four other key enzymes involved in oxidative stress and tissue degradation (catalase, polyphenol oxidase, superoxide dismutase and lypoxygenase) were also significantly increased in both Grevillea species as a result of NaCl treatment. The main difference between the two Grevillea species appeared to be that the more salt tolerant G. ilicifolia had significantly lower levels of the oxidative/degrading enzymes, compared with the more salt sensitive G. arenaria .

Macrophage responses to Toxoplasma antigens in vitro: a possible role in inflammatory lesions in toxoplasmosis.

Toxoplasma antigen and Toxoplasma immune complex were shown to induce increased production and release of acid hydrolases from macrophage cell line P388D in a concentration-dependent manner. Antigen concentrations of 10-50 microg/ml gave a 2-4-fold increase in the activities of acid proteinase, acid phosphatase, and phospholipase A2 compared with control cells without antigen. Results were similar for immune complex concentrations of 30-80 microg/ml compared with controls. No significant lactate dehydrogenase activity was detected in the culture medium, indicating that enzyme release was selective and not due to cell death. These results suggest that increased release of acid hydrolases may play a role in the inflammatory lesions observed in Toxoplasma encephalitis.

Phagocytosis inhibition of clam and mussel haemocytes by Perkinsus atlanticus secretion products.

The in vitro effects of the secretion products (SP) released by cultured Perkinsus atlanticus on the phagocytic activity of two shellfish species, Ruditapes decussatus and Mytilus galloprovincialis were determined. Haemocytes from clam and mussel were incubated with Perkinsus SP, and the number of phagocytosed particles were counted. The particles assayed were zymosan, Escherichia coli and Vibrio tapetis. Phagocytosis of zymosan was measured at 15 and 21°C, and different incubation times in SP were tested. In all cases, phagocytosis was inhibited in SP-treated haemocytes. Phagocytosis of zymosan was inhibited to a greater extent in haemocytes from mussel than from clam. No differences were found between phagocytosis at 15 and 21°C, and the decrease in the phagocytic activity was evident within 30min of haemocyte incubation in SP. Clam haemocyte phagocytosis of V. tapetis was inhibited to a greater extent than for zymosan and E. coli, while phagocytosis by the mussel haemocytes was inhibited to the same extent for the two bacterial species tested. Otherwise, the phagocytic index was higher for clam haemocytes than for mussel haemocytes in all the experiments. SP contained high protein concentration and acid phosphatase and protease activity. These results suggest that the products secreted by Perkinsus atlanticus may have a negative effect on the cellular defence response of these bivalve molluscs.

Immunological control of scab mites: digestive enzymes as candidate compounds

Phosphatases, C4 and C8 esterases, leucine and valine aminopeptidases, n-acetyl-β-glucosaminidase, β-glucosidase, β-galactosidase and β-glucuronidase were active in extracts of scab mites ( Psoroptes spp.) raised on sheep or rabbits. Trypsin and chymotrypsin activities were not detected. Haemoglobin was hydrolysed by a detergent-soluble fraction of the mite extracts in a pH-dependent fashion with an optimum of pH 3–5. Acid proteinase activity was greater in mites raised on rabbits than in those raised on sheep. Inhibitors of cysteine, serine and metallo-proteinases failed to inhibit the hydrolysis of H–Pro–Thr–Glu–Phe–Phe(NO 2)Arg–Leu–OH while pepstatin A, a specific inhibitor of aspartic proteinases, totally inhibited its hydrolysis at a concentration of 1 nM.

Serotype distribution and secretory acid proteinase activity of Candida albicans isolated from the oral mucosa of patients with denture stomatitis.

Denture stomatitis is the most common form of oral Candida infection in humans. In the current study, the distribution of Candida albicans serotype A and B as well as the activity of the secreted acid proteinase were determined in clinical isolates from patients with denture stomatitis. It was found that 70% of individuals with clinical signs of denture stomatitis exhibited fungal growth, with C. albicans representing the most frequently isolated species (75%). Of the C. albicans isolates, 75% were serotype A and 25% were serotype B, representing a significant increase of serotype B compared to a control group of non-denture-wearing HIV-seronegative individuals with oral candidiasis, but no significant difference compared with isolates from HIV-seropositive patients, who also exhibited a high percentage of serotype B. The mean secretory acid proteinase activity of C. albicans isolates from denture stomatitis patients (2796 +/- 819 U/l) was statistically not different from the mean secretory acid proteinase activity in non-denture-wearing HIV-seronegative individuals (2324 +/- 1487 U/l). Both values were significantly lower than the mean secretory acid proteinase activity of C. albicans from HIV-seropositive individuals (4256 +/- 2372 U/l). No correlation exists between the C. albicans serotype and the amount of secreted acid proteinase, indicating that serotype and secretory acid proteinase expression are two independent pathogenetic factors in oral candidiasis. These results indicate that there seems to be strain selection for C. albicans serotype B in denture stomatitis. These results further indicate that increased secretion of the acid proteinase seems to be of pathogenetic significance in the candidiasis of HIV-seropositive patients but not in denture stomatitis. Nevertheless, the secretory acid proteinase is likely to be an important pathogenetic factor also in denture stomatitis, where an increased secretion of the acid proteinase may not be required because of decreased salivary flow and a low pH under the denture, which will result in a high enzymatic activity.

Stratum corneum thiol protease (SCTP): a novel cysteine protease of late epidermal differentiation.

Proteolytic enzymes play crucial roles in the formation of the stratum corneum barrier tissue and in its subsequent maturation. Despite this, the proteases involved in stratum corneum physiology are not well characterized. Hence, studies were performed to identify these proteolytic enzymes present in the peripheral layers of this tissue using a combination of tape stripping and zymography. Using this approach, a novel human cysteine protease was identified and characterized, and named stratum corneum thiol protease (SCTP). Gelatin zymography revealed that SCTP is composed of two variants with apparent molecular weights of 34 and 35 kDa which do not correspond to any previously described stratum corneum protease. Mechanistically SCTP belongs to the cysteine proteinase class as shown by: (1) acid protease activity, (2) a requirement for mild reducing conditions, and (3) the specific inhibition of activity by E64 and Z-phe-ala-diazomethylketone. Further analysis using concanavalin A affinity chromatography demonstrated that the two 34 and 35 kDa variants are both glycoproteins, which, after removal of the oligosaccharide sidechains with the specific enzyme N-glycopeptidase F, reveal a single active core protease of 32 kDa. SCTP did not crossreact with antibodies raised against the lysosomal cysteine proteases cathepsins B, H or L, thereby distinguishing it from the classical cysteine cathepsins. Localization studies revealed that SCTP is present at all depths in the stratum corneum, thereby precluding microbial contamination as the enzyme source. Moreover, it was also present at all body sites investigated, except for the hyperkeratotic palmoplantar stratum corneum. SCTP was found to be a product of late differentiation in cultured human keratinocytes; the enzyme was synthesized by differentiated calcium-switched cells and secreted into the medium, whereas nondifferentiated basal keratinocytes did not produce this protease. Moreover, human fibroblast cultures did not produce the enzyme, suggesting that SCTP is not produced by the dermis and hence is epidermal specific. The function of SCTP is unknown, but the observed gelatinolytic activity coupled with its secretion into the medium by cultured keratinocytes indicates that physiologically it is responsible for the degradation of extracellular structural proteins. Furthermore, the optimal activity at acid pH suggests that it can function in the acidic environment of the stratum corneum. It remains to be elucidated whether this enzyme has a role in desquamation.

Hydrolytic enzymes of Psoroptes cuniculi (Delafond)

Phosphatases, C4 and C8 esterases, leucine and valine aminopeptidases, N-acetyl-β-glucosaminidase, β-glucosidase, β-galactosidase and β-glucuronidase were detected in extracts of the parasitic mite Psoroptes cuniculi. Lipase, trypsin-like and chymotrypsin-like activities were not present. Haemoglobin was hydrolysed by a detergent-soluble fraction of the mite extracts with a maximum hydrolysis between pH 3 and 5. Acid proteinase activity was greater against haemoglobin than bovine serum albumin. Inhibitors of cysteine, serine and metallo-proteinases failed to inhibit the hydrolysis of H–Pro–Thr–Glu–Phe–Phe(NO 2)–Arg–Leu–OH while pepstatin A inhibited its hydrolysis in a dose-dependent manner (IC 50 8.02×10 −11M (±0.30×10 −11). Thermal inactivation of the proteolytic activity followed an exponential decay pattern. Typical K m and V max values were 7.2×10 −5 (±0.7×10 −5) M −1 and 1.13×10 −3 (±0.05×10 −3) OD unit −1 min −1 respectively. Acid proteinase activity eluted from a size exclusion column in a single, major peak representing a molecular weight range of 21–24.5 kDa. The major endoproteinase of P. cuniculi therefore appears to be a cathepsin D-like aspartic proteinase.

Application of an artificial neural network and genetic algorithm for determination of process orbits in the koji making process

In order to estimate α-amylase, glucoamylase, acid proteinase, and acid carboxypeptidase activities in koji from the process variables and initial conditions of the koji making process, artificial neural network (ANN) models (ANN-10, -11, -15, and -21) were constructed with 10, 11, 15, and 21 input variables, respectively. These models could estimate the enzyme activities with high accuracy. Temperature and humidity orbits were then acquired by a genetic algorithm searching in the reverse direction using ANN-10, -11, -15, and -21 (GA-10, -11, -15, and -21). The orbits acquired by GA-15 and -11 were almost identical to the actual orbits, but those acquired by GA-21 and -10 were different. Enzyme activities acquired by GA-15 had 1.3% errors compared with the target values, while those aquired by GA-11 had 9.7% errors. GA-15 was, therefore, selected as the most suitable algorithm and was used to determine temperature and humidity orbits for target enzyme activities. Test koji making was then carried out according to the orbits acquired. As a result, the enzyme activities of the koji produced were almost the same as the target values.

Digestive enzyme activity during larval development of the Senegal sole (Solea senegalensis)

The activities of the main digestive enzymes (proteases, amylase, lipase) as well as those of acid and alkaline phosphatases were assessed during the larval development of the Senegal sole, Solea senegalensis. Important variations in specific activities of all the enzymes were observed during the period of study and were mostly related to the beginning or the end of metamorphosis. Both acid and alkaline protease activities were identified at early stages of development. Acid proteases accounted for only 10% of total protease activity in a 9 days-old larva, but exceeded 75% in a 33 days-old individual. Maximum lipase activity was related to the development of exocrine pancreas (days 6 to 10) and to metamorphosis. It is suggested this can be related to the metabolism of lipid reserves taking place during this morphological change. Activity of alkaline phosphatase decreased from day 5 to day 20 but therafter revealed a sharp increase, possibly linked to the development of enterocytes. The pattern of development of the main digestive enzymes found in S. senegalensis is similar to that described in other flatfish species.

Do digestive enzymes set a physiological limit on growth rate and food conversion efficiency in the Atlantic cod (Gadus morhua)

The objective of this study was to determine the potential sites of maximal growth limitation in Atlantic cod (Gadus morhua). Forty cod were reared in ten sea water tanks. Fish were randomly divided into 5 groups, a control group (injected with saline solution) and 4 experimental groups that received different levels of recombinant bovine somatotropin (rbST: 1.0, 2.0, 4.0 and 10.0 μg g fish−1 2 weeks−1) by injection in the abdominal cavity. Fish were fed ad libitum 3 times a week during four weeks. We measured individual body mass, growth rate, food intake and food conversion efficiency. At the end of the experiment, we measured the activity of acid proteases in the stomach; trypsin and chymotrypsin in the pyloric caeca; alkaline phosphatase (ALP) and glutamyltransferase (GGT) in the intestine. No significant differences were observed in growth rate, food intake or food conversion efficiency among the five groups. Trypsin, chymotrypsin, ALP and GGT activities when expressed in U g fish−1 were correlated with growth rate. These enzymes were also correlated with food ingestion except for trypsin when expressed in U mg protein−1. Trypsin was the only enzyme that showed a significant correlation with food conversion efficiency. Our conclusion is that, at the level of digestion, trypsin is the only enzyme measured that could be suspected to potentially limit growth rate in cod.

Elevation of Acid Protease Activity in the Mantle Muscle of Female Japanese Common Squid <i>Todarodes pacificus</i> during Maturation

Elevation of Acid Protease Activity in the Mantle Muscle of Female Japanese Common Squid <i>Todarodes pacificus</i> during Maturation