Effects of glucose and nitrogen source on the levels of proteinases, peptidases, and proteinase inhibitors in yeast.

In Saccharomyces cerevisiae harvested from early exponential growth on glucose-containing media, the specifc activities of proteinases A and B, carboxypeptidase Y, and the inhibitors IA, IB, IC of these three proteinases, respectively, are found to be 10-30% of the specific activities observed in media without glucose, containing acetate as a carbon source; the activities of two aminopeptidases in glucose-grown cells were 30-50% of those in acetate-grown cells. In contrast to fructose-biphosphatase, phosoenolpyruvate carboxykinase, and cytoplasmic malate dehydrogenase, which are inactivated after the addition of glucose to derepressed cells, the proteinases and inhibitors are not inactivated after glucose addition, but appear to be repressed. Growth of the yeast on poor nitrogen sources or starvation for nitrogen results in 2-3 fold increases in the levels of most proteinases and peptidases, but this effect is not observed with glucose as the carbon source.     

A study on the identities of the three species of chromatin-associated proteinases in a mutant of Saccharomyces cerevisiae which lacks four major vacuolar proteinases

Using mutant strain ABYS1 of Saccharomyces cerevisiae lacking four main vacuolar proteinases, proteinase A, proteinase B, carboxypeptidase Y, and carboxypeptidase S, we examined the identities of chromatin-associated proteinases, ruling out possible contamination of the chromatin fraction by them. The chromatin of strain ABYS1 showed three peaks of proteolytic activity at pH 4, 7, and 11, and these activities were found to be derived from three species of proteinases, the aspartic, serine neutral, and serine alkaline ones. As these chromatin-associated proteinases of strain ABYS1 were identical in both quality and quantity to those of wild-type strain of yeast, we suggest that the yeast chromatin contains three species of specific proteinases as essential components.     

Aminopeptidases as potential targets for the control of the Australian sheep blowfly, Lucilia cuprina.

A series of experiments were carried out to investigate the role of proteinase enzymes in the growth of larvae of the sheep blowfly, Lucilia cuprina. First, instar larvae were incubated on an artificial growth media in the presence of various concentrations of inhibitors of all the major proteinase classes. Inhibitors of serine proteinases and aminopeptidases were found to cause significant growth inhibition and in some cases death of the larvae within 24 h, suggesting that these enzymes were the major classes involved in protein digestion in the gut of the insect. A second group of experiments analysed the effects of two inhibitors from the same or different proteinase classes in the growth media. Synergistic inhibition of larval growth was observed with the incorporation of inhibitors of serine proteinases and aminopeptidases. The results suggest that these classes of proteinases are both central to protein digestion in this insect, probably in the gut, and that the inhibition of both types of activity leads to an almost complete blockade of digestion. Testing in vivo gave similar results with infections on sheep skin inhibited by either serine proteinase or aminopeptidase enzyme inhibitors and the combination of both stopped the infection process. The role of aminopeptidases in larval metabolism and as potential targets for blowfly control agents is examined.     

The study of two alkaliphilic thermophile bacteria of the <Emphasis Type="Italic">Anoxybacillus</Emphasis> genus as producers of extracellular proteinase

Two strains of alkaliphilic thermophile bacteria of the genus Anoxybacillus from hydrothermal vents of Lake Baikal were detected and characterized. It was demonstrated that proteinases secreted by these bacteria had wide substrate specificity, hydrolyzed proteins and n-nitroanilide substrates, and showed maximal activity at pyroglutamyl-alanine-alanine-leucine n-nitroanilide hydrolysis. We determined maximal activity of the proteinases at alkaline pH values (10.0–10.5), the enzymes were thermostable and were characterized by a wide thermal optimum (55–70°C). The results of inhibitor analysis and substrate specifity examination of extracellular enzymes demonstrated their belonging to the subtilisin-like serine proteinases.     

Digestive peptidases in Tenebrio molitor and possibility of use to treat celiac disease

Digestion in Tenebrio molitor larvae occurs in the midgut, where there is a sharp pH gradient from 5.6 in the anterior midgut (AM) to 7.9 in the posterior midgut (PM). Accordingly, digestive enzymes are compartmentalized to the AM or PM. Enzymes in the AM are soluble and have acidic or neutral pH optima, while PM enzymes have alkaline pH optima. The main peptidases in the AM are cysteine endopeptidases presented by two to six subfractions of anionic proteins. The major activity belongs to cathepsin L, which has been purified and characterized. Serine post‐proline cleaving peptidase with pH optimum 5.3 was also found in the AM. Typical serine digestive endopeptidases, trypsin‐like and chymotrypsin‐like, are compartmentalized to the PM. Trypsin‐like activity is due to one cationic and three anionic proteinases. Chymotrypsin‐like activity consists of one cationic and four anionic proteinases, four with an extended binding site. The major cationic trypsin and chymotrypsin have been purified and thoroughly characterized. The predicted amino acid sequences are available for purified cathepsin L, trypsin and chymotrypsin. Additional sequences for putative digestive cathepsins L, trypsins and chymotrypsins are available, implying multigene families for these enzymes. Exopeptidases are found in the PM and are presented by a single membrane aminopeptidase N‐like peptidase and carboxypeptidase A, although multiple cDNAs for carboxypeptidase A were found in the AM, but not in the PM. The possibility of the use of two endopeptidases from the AM – cathepsin L and post‐proline cleaving peptidase – in the treatment of celiac disease is discussed.     

Activities of some peptidases and proteinases in germinating kidney bean, Phaseolus vulgaris

The activities of aminopeptidase (EC 3.4.11), dipeptidase (EC 3.4.13), carboxypeptidase (EC 3.4.16), naphthylamidase (EC 3.4.11) and proteinases (EC 3.4.21) were assayed in extracts from the cotyledons and the axial tissues of resting and germinating kidney beans ( Phaseolus vulgaris L. cv. Processor).
The activities of the alkaline peptidases (aminopeptidase hydrolyzing Leu-Tyr at pH 9.2 and dipeptidase acting on Ala-Gly at pH 8.5) and naphthylamidases (hydrolyzing Leu-β-naphthylamide at pH 6.4) were high in the cotyledons of resting seeds, but decreased during germination. This decrease was faster than the loss of the total nitrogen. On the contrary, the activities of carboxypeptidase (hydrolyzing carbobenzoxy-Phe-Ala at pH 5.9) and proteinases (acting on haemoglobin at pH 3.7 and on casein at pH 5.4 and 7.0) were low in the resting seeds, but increased during germination reaching their maximal values when the mobilization of nitrogen was highest. It has been suggested that the breakdown of storage proteins is initiated inside the protein bodies by acid proteinases and carboxypeptidases. Although the activities of the alkaline peptidases and naphthylamidases decreased during germination, these were still relatively high and enough for the completion of the proteolytic breakdown. Thus, it is suggested that, as a final step in a chain of events, the main role for the alkaline peptidases in the cotyledons of germinating seeds is to provide amino acids for the growth of the seedling.     

Proteolytic enzymes in human leukemic lymphoid cells. III. Aminopeptidases, angiotensin-converting enzyme, and its inhibitor in cells of different immunological phenotype

Activities of plasma membrane proteinases such as angiotensin-converting enzyme (ACE), aminopeptidases, and dipeptidyl peptidase IV (DPP-IV) were determined in lymphoid cells of various immunological phenotype which were obtained from 30 patients with lymphoproliferative diseases. The enzyme activities significantly varied depending on the immunological phenotype and stage of cell differentiation, but no correlation was found between activities of ACE, DPP-IV, and aminopeptidases in the cells of different type. The cell lysates studied contained at least two classes of aminopeptidases: metal- and sulfhydryl-dependent enzymes. A sulfhydryl-dependent aminopeptidase with activity optimum at pH 8. 5-9.0 was found for the first time and is suggested to be from a poorly studied aminopeptidase family. In addition to ACE, lysates of leukemic T- and B-cells were found to contain an inhibitor of ACE which was not previously described for these cells.     

Compartmentation of inhibitors of proteinases A and B and carboxypeptidase Y in yeast

After centrifugal fractionation at 40,000 × g of a metabolic lysate from yeast spheroplasts proteinases A and B, and carboxypeptidase Y were found exclusively in the sediment, whereas inhibitors of these proteinases were present only in the supernatant. Immunoprecipitation with an antiserum prepared against the pure heat-stable proteinase B-inhibitor occured in the supernatant but not in the extract of the particulate fraction.     

Characterization of proteinases from Antarctic krill (Euphausia superba)

Fractions of three trypsin-like proteinases, TL I, TL II, and TL III, a chymotrypsin-like proteinase, CL, two carboxypeptidase A enzymes, CPA I and CPA II and two carboxypeptidase B enzymes, CPB I and CPB II, from Antarctic krill (Euphausia superba) have been characterized with respect to purity by the means of capillary electrophoresis, CE, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The masses of the trypsin-like and chymotrypsin-like proteinases were determined to be 25,020, 25,070, 25,060, and 26,260Da for TL I, TL II, TL III, and CL, respectively. The masses of the CPA enzymes are likely 23,170 and 23,260Da, whereas the CPB enzyme masses likely are 33,730 and 33,900Da. The degradation efficiency and cleavage pattern of the trypsin-like proteinases were studied with native myoglobin as a model substrate using CE, MALDI-TOF-MS, and nanoelectrospray mass spectrometry (nESI-MS). The degradation efficiency of the trypsin-like proteinases was found to be approximately 12 and 60 times higher compared to bovine trypsin at 37 degrees C and 1-3 degrees C, respectively. All three fractions of trypsin-like proteinases showed a carboxypeptidase activity in combination with their trypsin activity.     

Proteolytic enzymes in sea urchin eggs: characterization, localization and activity before and after fertilization

The pH versus proteinase activity curve (casein or hemoglobin plus urea substrate) for homogenates of unfertilized Lytechinus eggs reveals two regions of maximum activity: one between pH 3.5 and 4.3, and another of far greater magnitude from pH 8.0 to 11.0. The two classes of proteinases can be separated on a sucrose density gradient. Both the acid and alkaline proteinases in homogenates prepared in isotonic monovalent salt solutions are remarkably stable at pH 7.4 and 0°C. Using synthetic peptide substrates, an enzyme with the specific esterase activity of chymotrypsin was demonstrated; this enzyme accounts for the major part of the proteinase activity at alkaline pH. In addition, an enzyme with specific esterase activity of trypsin was shown to be present, but of low activity. The proteinase activity at acid pH is largely due to an enzyme resembling cathepsin D. The data also suggest the presence of cathepsin B and cathepsin IV (or catheptic carboxypeptidase). When eggs are homogenized in isotonic NaCl plus KCl at pH 7.4, 0.02 M tris buffer at 0°C, all of the alkaline proteinase, and 85–90% of the acid proteinase activity is sedimented at 10,000 g. The presence of any proteinase activity in the supernatant phase represents an artifact of the preparative procedures used. The granules which possess the proteinase activity are contained entirely in the yolk fractions; and the acid proteinase is contained in a population of granules which sediment more readily than those which contain the alkaline proteinase. The acid proteinase resembles the lysosomal acid hydrolases in that it is readily released from the particulates; in contrast, the alkaline proteinase is bound relatively firmly. In contradistinction to reports in the literature, no changes in proteinase activity nor intracellular distribution could be detected following fertilization.     

Substrate Specificity and Salt Inhibition of Five Proteinases Isolated from the Pyloric Caeca and Stomach of Sardine

To elucidate the mechanism of hydrolysis of fish muscle proteins by fish proteinases in fish sauce production, each pure preparation of three alkaline proteinases and two acid proteinases from sardine was tested for its ability to hydrolyze various proteins and its stability in the presence of 0 to 25% of NaCl. Each of the alkaline proteinases hydrolyzed casein more rapidly than other proteins. A major alkaline proteinase (III) hydrolyzed sarcoplasmic protein from sardine 5-times faster than other alkaline proteinases. Each of two acid proteinases hydrolyzed hemoglobin and myoglobin more rapidly than the other proteins. After preincubation with 25% NaCl, an alkaline proteinase (III) and an acid proteinase (II) were stable although the other proteinases became unstable. The two proteinases, alkaline proteinase III and acid proteinase II, were also stable for three months after the beginning of fish sauce production. The proteolytic activity of each of alkaline and the acid proteinases was strongly inhibited by more than 15% NaCl; however, minimum inhibition was observed when sardine muscle proteins were used as the substrate.     

Enzymatic digestibility of peptides cross-linked by ionizing radiation

p6gestibility by proteolytic enzymes of peptides cross-linked by ionizing radiation was investigated. Small peptides of alanine and phenylalanine were chosen as model compounds and aminopeptidases and carboxypeptidases were used as proteolytic enzymes. Peptides exposed to gamma-radiation in aqueous solution were analysed by high-performance liquid chromatography before and after hydrolysis by aminopeptidase M, leucine aminopeptidase, carboxypeptidase A and carboxypeptidase Y. The results obtained clearly demonstrate the different actions of these enzymes on cross-linked aliphatic and aromatic peptides. Peptide bonds of cross-linked dipeptides of alanine were completely resistant to enzymatic hydrolysis whereas the enzymes, except for carboxypeptidase Y, cleaved all peptide bonds of cross-linked peptides of phenylalanine. The actions of the enzymes on these particular compounds are discussed in detail.     

Amino acids at the N- and C-termini of human glutamate carboxypeptidase II are required for enzymatic activity and proper folding.

Human glutamate carboxypeptidase II (GCPII) is a co-catalytic metallopeptidase and its putative catalytic domain is homologous to the aminopeptidases from Vibrio proteolyticus and Streptomyces griseus. In humans, the enzyme is expressed predominantly in the nervous system and the prostate. The prostate form, termed prostate-specific membrane antigen, is overexpressed in prostate cancer and is used as a diagnostic marker of the disease. Inhibition of the form of GCPII expressed in the central nervous system has been shown to protect against ischemic injury in experimental animal models. Human GCPII consists of 750 amino acids, and six individual domains were predicted to constitute the protein structure. Here, we report the analysis of the contribution of these putative domains to the structure/function of recombinant human GCPII. We cloned 13 mutants of human GCPII that are truncated or extended at one or both the N- and C-termini of the GCPII sequence. The clones were used to generate stably transfected Drosophila Schneider's cells, and the expression and carboxypeptidase activities of the individual protein products were determined. The extreme C-terminal region of human GCPII was found to be critical for the hydrolytic activity of the enzyme. The deletion of as few as 15 amino acids from the C-terminus was shown to completely abolish the enzymatic activity of GCPII. Furthermore, the GCPII carboxypeptidase activity was abrogated upon removal of more than 60 amino acid residues from the N-terminus of the protein. Overall, these results clearly show that amino acid segments at the N- and C-termini of the ectodomain of GCPII are essential for its carboxypeptidase activity and/or proper folding.     

Neutral proteinases of human spleen. Purification and criteria for homogeneity of elastase and cathepsin G.

1. Human spleen was found to contain proteinases active against azo-casein at neutral and alkaline pH values. 2. The activity was stimulated by high ionic strength and some detergents. 3. Optimal extraction of the proteinases from the tissue was achieved with 1.0M-NaCl containing 0.1% Brij 35 and 0.1% trisodium EDTA. 4. The proteinases were efficiently adsorbed to insoluble material in the absence of salt in the initial stages of purification. 5. Two distinct proteinases were separated by chromatography on DEAE-cellulose, an elastase and a chymotrypsin-like enzyme designated cathepsin G. 6. Both enzymes were highly purified by further column chromatography. 7. The molecular weights of the enzymes were estimated by gel chromatography and sodium dodecyl sulphate-gel electrophoresis. 8. It was shown by isoelectric focusing and gel electrophoresis that both enzymes are cationic proteins that occur in multiple forms.     

Peptidohydrolases of Soybean Root Nodules : IDENTIFICATION, SEPARATION, AND PARTIAL CHARACTERIZATION OF ENZYMES FROM BACTEROID-FREE EXTRACTS

Nodule extracts prepared from Glycine max var Woodworth possessed endopeptidase, aminopeptidase, and carboxypeptidase activities. Three distinct endopeptidase activities could be resolved by disc-gel electrophoresis at pH 8.8. According to their order of increasing electrophoretic mobility, the first of these enzymes hydrolyzed azocasein and n-benzoyl-l-Leu-beta-naphthylamide, while the second hydrolyzed n-benzoyl-l-Arg-beta-naphthylamine (Bz-l-Arg-betaNA), n-benzoyl-l-Arg-p-nitroanilide (Bz-l-Arg-pNA), and azocasein. The third endopeptidase hydrolyzed Bz-l-Arg-betaNA, Bz-l-Arg-pNA, and hemoglobin. Fractions of these enzymes extracted from electrophoresis gels were shown to have pH optima from 7.5 to 9.8. All of the endopeptidases were completely inhibited by diisopropylphosphorofluoridate, demonstrating that they were serine proteases.Aminopeptidase activity was measured using amino acyl-beta-naphthylamides. Electrophoresis of nodule extracts at pH 6.8 resolved the aminopeptidase activity of nodule extracts into at least four fractions based on mobility and on activities toward amino acyl-beta-naphthylamides. The major activity of two of the aminopeptidases was directed toward l-Leu- and l-Met-beta-naphthylamide, while the other two aminopeptidases exhibited broader specificity and were capable of hydrolyzing a large number of amino acyl-beta-naphthylamides. Two of the aminopeptidases extracted from electrophoresis gels were classified as thiol type enzymes, and all four aminopeptidases had neutral to basic pH optima.     

Characterization and function of intracellular proteinases and proteinase inhibitors from yeast.

Data on the proteinase inhibitors IA, IB and IC from yeast and their possible intracellular interaction with the proteinases A and B and carboxypeptidase Y are presented. A role of proteolysis in "catabolite inactivation" is discussed.     

Detection of proteolytic enzymes in polyacrylamide gels

Aminopeptidases (1), dipeptidyl aminopeptidases (2), pyrrolidonyl peptidases (3), and carboxypeptidases (4,5) can be detected in polyacrylamide gels with appropriate-β-naphthylamide or carbonaphthoxyamino acid substrates while dipeptidases, tripeptidases (6), carboxypeptidases (7), and aminopeptidases can be detected by the coupled l-amino acid oxidase-peroxidase method of Lewis and Harris (6).In contrast, fewer methods are available for the detection of proteinases in gels. Trypsin-like (8,9) and chymotrypsin-like (5,10) proteinases can be detected with chromogenic β-naphthylamide and β-naphthol ester substrates, but proteinases such as thermolysin (11) and other bacterial neutral metal chelator-sensitive proteinases (12) cannot. For these latter proteinases, whose specificities are directed towards the amino acid residue containing the amino group of the bond to be hydrolyzed, and for proteinases, whose specificities remain to be determined, other methods of detection have to be employed.Uriel and Avrameas (13) detected proteinases in agarose gels by overlaying these gels with a second agarose gel mixture containing the substrate and a suitable pH indicator. However, the method suffers from interference by gel buffers and the instability of the pattern developed. Another procedure is to bring the gel in contact with a gelatinous layer of film material (14,15). This has been done successfully with tissue sections (16), paper electrophoretograms (17) and agarose gel separations (18).The most suitable approach is to diffuse an appropriate protein substrate into the gel after electrophoresis and detect the proteinase activity directly. Several variations of this method have been published (19–22), each with its own advantages and disadvantages. In this report a simple, sensitive method using cytochrome c as substrate, and requiring no staining, is described. This report describes its application to the detection of thermolysin and trypsin in anionic and cationic gel systems, respectively. The method has also been routinely used to locate bacterial and insect proteinases after electrophoresis.     

Alkaline serine proteinases D and E of Streptomyces griseus K-1.

Two DFP-sensitive alkaline proteinases with strong esterase activity toward Ac-(Ala)3-OMe, designated as alkaline serine proteinases D and E, were purified pronase, a protease mixture from St. griseus K-1. Each was shown to be homogeneous by acrylamide disc gel electrophoresis. The molecular weights of these enzymes were estimated to be about 27,000 be gel filtration. Studies on their actions on acyl-tl-amino acid methyl or ethyl esters indicated that proteinases D and E both exhibited a broad substrate specificity and hydrolyzed the ester bonds of esters containing Trp, Tyr, Phe, Leu, and Ala. The esterase activities of both enzymes toward Ac-(Ala)3-OMe were the highest among proteinases so far isolated from various sources. Proteinases D and E also lacked cystine residues in their molecules, being entirely different from alkaline serine proteinases A, B, and C in pronase. Some differences were , however, observed between them as regards pH stability, behavior on CM-cellulose, mobility on polyacrylamide electrophoresis, and amidase activity toward Suc-(Ala)3-pNA.     

Comparative analysis of proteinase activities of Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis (Lepidoptera: Crambidae)

Proteinase activities were compared in soluble and membrane fractions of guts obtained from larvae of Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis. Overall, serine proteinases from soluble fractions of the susceptible strain were more active than those of the resistant strain. The soluble trypsin-like proteinase activity of the resistant strain was approximately half that of the susceptible strain. The number and relative molecular masses of soluble and membrane serine proteinases were different. However, there were no significant differences in the activities of serine proteinases and aminopeptidases extracted from midgut membranes of the two strains. Cry1Ab protoxin hydrolysis by soluble proteinase extracts of the resistant strain was reduced approximately 20-30% relative to that of the susceptible strain. Reduced protoxin processing due to decreased activities of Bt protoxin activation proteinases may be associated with resistance to Bt toxin in this resistant strain of O. nubilalis.