Inactivation of uridine nucleosidase in yeast. Purification and properties of an inactivating protein.

It has been previously demonstrated in our laboratory that uridine nucleosidase (EC 3.2.2.3) is subjected in yeast to inactivation. An inactivating fraction has been isolated and purified to homogeneity with a procedure which includes gel filtration, adsorption chromatography, and electrofocusing techniques. The molecular weight of the enzyme, estimated either by sodium dodecyl sulfate disc gel electrophoresis or by gel filtration is approximately 44,000. No quaternary structure was evidenced. The inactivating activity possesses proteolytic activity against casein and hemoglobin with pH optima of 2.5 and 3.2, respectively. The optimal pH for uridine nucleosidase inactivation is around 4.7. The inactivating activity as well as the proteolytic activity of the preparation can be inhibited by IA but not by IB2 and IC, yeast macromolecular inhibitors for proteinase A (EC 3.4.23.8), B (EC 3.4.22.9), and C (EC 3.4.12.8), respectively. The apparent isoelectric point is pH 4.03. The carbohydrate content is 8.5%. A comparison of the properties of the inactivating protein with those of known yeast proteinases leads to the conclusion that it is identical with the enzyme previously designated as proteinase A, which for the first time has been obtained homogeneous and characterized. It has been shown that proteinase A could play a physiological role in the uridine nucleosidase inactivation process when it is associated, as a complex, with proteinase B.     

Esterase A is a proteinase from rat urine that can activate plasminogen

A proteinase which can activate human, dog and rat plasminogen to plasmin has been isolated from the urine of female rats, using affinity chromatography on benzamidine-coupled Sepharose. Inhibition by diisopropylfluorophosphate, tosyl-L-lysine chloromethylketone and benzamidine classified the enzyme as trypsin-like. The proteinase has weak activity on alpha-casein and hemoglobin, but will not lyse fibrin clots. It readily cleaves arginyl amides, including synthetic substrates specific for human glandular kallikrein and other serine proteinases. A chromogenic substrate for human urokinase (pyro Glu-Gly-Arg-pNA) is a poor substrate for the rat proteinase. Characteristics of the enzyme, such as its molecular weight (25 900), kinetic parameters and inhibition by aprotinin, indicate that this proteinase is esterase A, described by several investigators. Esterase A is shown not to be a true urinary plasminogen activator but rather is a unique arginine-specific proteinase. Urokinase-like and kallikrein-like activity are part of a broader proteolytic activity displayed by this enzyme.     

Rapid purification of secretory acid proteinase from Candida albicans and its characterization.

Secretory acid proteinase from C. albicans was purified from culture supernatant to apparent homogeneity by ion-exchange chromatography. Two isozymes of the proteinase were resolved using a novel chromatofocusing method. The enzyme, which appears to be a glycoprotein, consists of a single polypeptide chain with glutamine at the N-terminus. Its molecular weight is about 45,000 and isoelectric point is pH 4.6. At pH 5, the proteinase is stable at 45 degrees C for at least 15 min. It has a broad substrate specificity. With BSA as a substrate, Km was determined to be 1.6 x 10(-4) M. The enzyme is inhibited by pepstatin and thus is a carboxyl proteinase. It undergoes autocatalytic digestion at or below pH 5.0. The kinetics of induction of proteinase by various proteins are also reported.     

Mechanism of Excretion of a Bacterial Proteinase: Factors Controlling Accumulation of the Extracellular Proteinase of a Sarcina Strain (Coccus P)

It has been known that the extracellular proteinase of Coccus P is found only in cultures grown in the presence of Ca²⁺. It is now shown that this cation is required neither for synthesis, excretion, or activation of a zymogen nor as a prosthetic factor necessary for enzymatic activity. The only function of Ca²⁺ is to stabilize the active structure of the enzyme molecule, presumably by substituting for absence of S-S bridges. In the absence of Ca²⁺, the excreted proteinase undergoes rapid autodigestion and, instead of the active protein, its hydrolytic products are accumulated in the culture fluid. In minimal medium and under conditions of enzyme stability [presence of Ca²⁺ and Ficoll (Pharmacia)], Coccus P accumulates the proteinase at a gradually reduced speed although the rate of cultural growth remains constant. It is shown that this decline in rate of accumulation is caused by the excreted proteinase itself, possibly acting on its own precursor emerging from the cell in a form susceptible to proteolytic attack and not amenable to Ca²⁺ protection. A proteinase precursor is actually demonstrable in a calciumless culture at the onset of the enzyme accumulation which follows Ca²⁺ addition. It is suggested that excreted proteins require an unfolded (or incompletely folded) structure to cross the cell envelope.     

Purification of a cysteine proteinase from Carica candamarcensis L. and cloning of a genomic putative fragment coding for this enzyme.

We describe the purification of a cysteine proteinase from latex of Carica candamarcensis, hereby designated CC23. The enzyme has been purified by ion-exchange chromatography and behaves electrophoretically as a monomer of M(r) 23,000 and optimal pH of 8.0. It displays a basic isoelectric point, has one cysteine residue in the active site by titration with E-64, confirmed by DNA sequencing, and responds to proteinase inhibitors as a classic cysteine proteinase. The K(m) and k(cat)/K(m) for CC23 using BAPNA were respectively 14.7 +/- 1.8 x 10(-4) M and 1.3 x 10(3) M(-1) s(-1). Therefore, the catalytic efficiency of CC23 is sixfold higher than that of CC-I, another proteinase from the same plant. DNA primers were designed to amplify by PCR a genomic sequence related to this enzyme. An 895-bp DNA fragment was cloned and sequenced. It shows strong homology with chymopapain isoform IV from C. papaya. The translated sequence is similar to that of chymopapain isoform II (73%) and CC-III (77%) from C. candamarcensis.     

Heat stable proteinase from Thermomonospora fusca. Characterization as a serine proteinase

An extracellular proteinase secreted by the thermophilic bacteria Thermomonospora fusca YX (YX-proteinase) is a serine proteinase as shown by its inactivation by the site specific reagents, phenylmethanesulfonyl fluoride, dansyl fluoride, and carbobenzoxy-L-phenylalanine chloromethyl ketone. This conclusion is further supported by the effect of various proteinase inhibitors on its activity. The activity of the proteinase toward small synthetic ester substrates shows that the enzyme has a primary specificity for the aromatic and hydrophobic amino acids. The amino acid composition and NH2-terminal sequence, as well as its size, suggest that the enzyme is related to the chymotrypsin-like microbial proteinase, alpha-lytic protease from Myxobacter 495 and protease A and B from Streptomyces griseus.     

The activating system of chitin synthetase from Saccharomyces cerevisiae. Purification and properties of the activating factor.

The yeast proteinase that causes activation of the chitin synthetase zymogen has been purified by a procedure that includes affinity chromatography on an agarose column to which the proteinaceous inhibitor of the enzyme had been covalently attached. The purified enzyme yielded a single band upon disc gel electrophoresis at pH 4.5 in the presence of urea. At the same pH, but without urea, a faint band was detected in coincidence with enzymatic activity, whereas at pH 9.5, either in the absence or in the presence of sodium dodecyl sulfate, no protein zone could be seen. From sedimentation and gel filtration data, a molecular weight of 44,000 was estimated. The proteinase was active within a wide range of pH values, with an optimum between pH 6.5 AND 7. Titraton of the activity with the protein inhibitor from yeast required 1 mol of inhibitor/mol of enzyme. A similar result was obtained with phenylmethylsulfonyl fluoride, an indication that 1 serine residue is required for enzymatic activity. The enzyme exhibited hydrolytic activity with several proteins and esterolytic activity with many synthetic substrates, including benzoylarginine ethyl ester and acetyltyrosine ethyl ester.A comparison of the properties of the enzyme with those of known yeast proteinases led to the conclusion that the chitin synthestase activating factor is identical with the enzyme previously designated as proteinase B (EC 3.4.22.9). This is the first time that a homogeneous preparation of proteinase B has been obtained and characterized.     

Purification of extracellular proteinases from B. subtilis SKB 256 by biospecific chromatography

Abstract-A simple and efficient method of preparing highly purified extracellular proteinases of B. subtilis B-1 (SKB 256) has been developed. A sorbent based on sorsilen impregnated with hemoglobin or cytochrome c has been synthesized for this purpose. A significant difference between the efficiency of hemoglobin and cytochrome c as biospecific ligands has been observed: the enzyme yield amounted to 40.6 and 65.6% of the total amount of enzyme adsorbed, respectively. The culture was shown to contain two major proteinase forms with different molecular masses that could be separated by chromatography on a Sephadex G-50 but gave only one band with MW 27 kDa upon denaturing electrophoresis in 12.5% PAG in the presence of 0.1% SDS. The influence of eluent pH, ionic strength and ethanol concentration on the sorption of the proteinases on the biospecific sorbent, as well as on the desorption from it, has been investigated. Positive influence of 20% ethanol on proteinase desorption has been demonstrated.     

Flavones are inhibitors of HIV-1 proteinase.

Substituted gamma-chromones were found to weakly inhibit HIV-1 proteinase, an important enzyme in the replication and processing of the AIDS virus. Chromones bearing hydroxyl substituents and a phenolic group at the 2-position (flavones) were the most active compounds and structure-activity relationships for a limited series of flavone inhibitors are presented. Dixon plots are reported and a possible mechanism for flavone-induced inhibition is proposed. The results are also compared with those for some structurally related non-peptidic inhibitors of HIV-1 proteinase. Since some flavonoid compounds have already been shown to have antiviral activity against AIDS, the present observations of anti-HIV-1 proteinase activity may be particularly significant.     

Drosophila cathepsin B-like proteinase: a suggested role in yolk degradation

A cysteine, cathepsin B-like proteinase activity has been found in Drosophila embryos. It appears associated with yolk granules and its activity during embryogenesis correlates well with the degradation of these organelles. In mature oocytes, the enzyme is found in an inactive form which may be activated by limited proteolysis by a serine proteinase also present in oocytes. In early embryos, when solubilized in vitro, the cathepsin B-like proteinase is found in a form of high molecular mass (approx 1000 kDa). This decreases with development down to about 39 kDa, likely the mass of the free proteinase. The heavy form apparently results from the tight association with a yolk protein complex. The proteinase has been found in vitro to degrade readily the yolk polypeptides. The proteinase activity increases during early embryogenesis in parallel with the decrease in molecular weight of the heavy form, and decreases to low values in late embryos. We have also found that ammonium chloride can inhibit in vivo the degradation of yolk and, in parallel, the developmental inactivation of the proteinase. The results altogether suggest that the cathepsin B-like proteinase is implicated in yolk degradation in Drosophila.     

Latent proteinase activity of gamma-glutamyl transpeptidase light subunit.

gamma-Glutamyl transpeptidase, which is composed of two unequal subunits, exhibits proteinase activity when treated with agents such as urea and sodium dodecyl sulfate. The heavy subunit is preferentially and rapidly degraded. The enzyme also degraded bovine serum albumin in the presence of urea; however, several other proteins and model proteinase substrates were not cleaved. Treatment of the enzyme with 6-diazo-5-oxo-L-norleucine, a gamma-glutamyl analog, results in parallel loss of transpeptidase and proteinase activities indicating that the site at which gamma-glutamylation of the enzyme occurs (presumably a hydroxyl group on the light subunit) is also involved in proteinase activity. The purified light subunit, but not the heavy subunit, exhibits proteinase activity even in the absence of urea. Results suggest that dissociation of the enzyme unmasks the proteinase activity of the light subunit involving the site at which gamma-glutamylation of the enzyme occurs, and that the heavy subunit may impose transpeptidase reaction specificity by contributing the binding domains for gamma-glutamyl substrates.     

The effects of geomagnetic storms on proteinase and glycosidase activities in fish intestinal mucosa

It has been demonstrated that the glycosidase activity of cyprinoid fishes (carp and crucian carp) exposed to a geomagnetic storm for up to 20 h considerably decreases; however, the proteinase activity is weakly altered (a statistically significant decrease in the enzyme activity has been observed only in fasting fish). An in vitro study of the effects of individual half hour intervals of the geomagnetic storm that correspond to the main and recovery phases on the same enzyme activities demonstrates the opposite trend. Independently of the experimental conditions, geomagnetic storms have been shown to influence the enzyme system of fasting fish negatively.     

The purification and characterization of a glomerular-basement-membrane-degrading neutral proteinase from rat mesangial cells.

A neutral proteinase, capable of degrading gelatin, has been found in both an active and a latent form in the medium from the culture of rat mesangial cells. The latent form had an Mr of 80,000-100,000 and could be activated with either 4-aminophenylmercuric acetate or prolonged incubation at neutral pH. The active form of the enzyme was extensively purified. The estimated Mr of the purified enzyme on gel filtration was approximately 200,000, indicating that the active enzyme formed aggregates. However, analysis by SDS/polyacrylamide-gel electrophoresis under reducing conditions showed two protein bands, with Mr 68,000 and 66,000. Both proteins were found to contain proteolytic activity when run on SDS/substrate gels. The enzyme was inhibited by EDTA and 1,10-phenanthroline, but not by inhibitors for cysteine, serine or aspartic proteinases. The enzyme did not digest fibronectin, bovine serum albumin, proteoglycan or interstitial collagen. The enzyme degraded pepsin-solubilized placental type V collagen at 31 degrees C, whereas similarly solubilized type IV collagen was only degraded at higher temperatures. In addition, the neutral proteinase degraded native soluble type IV collagen. It also had activity on insoluble type IV collagen of glomerular basement membrane. The above properties suggest that the mesangial neutral proteinase belongs to the gelatinase group of metalloproteinases and that it may play a role in the normal turnover of extracellular glomerular matrix.     

Cell-wall-bound proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: characterization and specificity for beta-casein

Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40 degrees C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes beta-casein mainly and alpha- and kappa-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from beta-casein, which have been identified.     

Activation of human platelet adenylate cyclase by a bovine sperm component

The latent cysteine proteinase present in ascitic fluid of patients with neoplasia and released from ascites cells in culture has been partially purified and the enzyme after pepsin activation was shown to be immunologically related to the lysosomal proteinase, cathepsin B. The latent form was characterized as a single chain of Mr 40 000 as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by Western blotting and immune staining with an antiserum to human cathepsin B. Using the same techniques the enzyme after pepsin activation gave a single band of Mr 33 000. Analysis by isoelectric focusing showed that the latent enzyme before and after pepsin treatment is composed of several acidic isoenzymes. These findings suggest that this latent proteinase represents a precursor form of cathepsin B which is released extracellularly rather than being processed and directed to the lysosome.     

Inhibition of an endogenous growth-related proteinase enhances the recovery of a negative growth regulator from cultured human cells.

The possibility that a growth-related proteinase may act by degrading a negative growth regulatory protein has been investigated. Proteinase inhibitors which inhibit the enzyme also enhance the accumulation of the growth regulator by human fibroblasts. The negative growth regulator shows a similar specificity of inhibition of cellular growth to inhibitors of the growth-related proteinase.     

A cysteine proteinase secreted from human breast tumours is immunologically related to cathepsin B.

The stable cathepsin B-like cysteine (thiol) proteinase secreted from human breast tumours in culture was shown to be destabilized by mercurial compounds. After such treatment the enzyme cross-reacts in a radioimmunoassay with a monospecific antiserum to human liver cathepsin B. It is suggested that the secreted enzyme may be a precursor form of lysosomal cathepsin B.