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At least 19 recordsLinked to original sources

PubMed1976

Proteolysis of canine apolipoprotein by acid proteases in canine liver lysosomes.

Canine liver lysosomes were purified by sucrose discontinuous density gradient centrifugation and then ruptured by sonication to obtain the soluble fraction. This soluble lysosomal fraction, which contained a 25-fold increase in acid phosphatase activity per mg of total protein when compared with the original homogenate, was incubated with a subfraction (1.110 less than d less than 1.210 g/cm3, HDL3) of canine high density lipoproteins (HDL) at pH 3.8. HDL3 proteolysis by lysosomal proteases, measured as the release of peptides and amino acids by the ninhydrin reaction, followed hyperbolic curves with straight lines (r = 0.99) obtained on Lineweaver-Burk plots. Km calculated from the Lineweaver-Burk plot was 635 mug of HDL3 protein per 0.5 ml of incubation mixture. Optimum HDL3 proteolysis was observed from pH 3.8 to 4.5. Incubation with the other subcellular organelle fractions did not result in HDL3 proteolysis. To evaluate the effects of enzyme inhibitors, iodoacetate, p-chloromercuribenzoate (both specific for the endopeptidase, cathepsin B (EC 3.4.22.1)) and pepstatin (specific for the endopeptidase, cathepsin D (EC 3.4.23.5) were tested. Iodoacetate and p-chloromercuribenzoate inhibited HDL3 proteolysis 100% and bovine serum albumin proteolysis 65%. Pepstatin inhibited HDL3 proteolysis 45% and bovine serum albumin proteolysis 70%. The in vitro data presented support the hypothesis that hepatic lysosomes play an important role in HDL3 catabolism in the dog. Furthermore, results obtained from enzyme inhibition studies suggest that a specific lysosomal endopeptidase, cathepsin B, may play the key role in HDL3 proteolysis.

Acid Phosphatase↗

PubMed1975

[Denaturation and enzymatic proteolysis in vitro of protein fractions of soya flour].

Soya, in spite of its high nutritional value and moderate cost, possesses certain undesirable qualities which limit its use in animal and human nutrition. The amendment of these qualities has resulted in much work. In this study the effects of technological treatments on the properties of certain protein fractions capable of being produced industrially were observed. Three fractions were prepared from defatted soybean flour of the "Harosoy 63" variety: an acid-precipitated fraction, a cold-insoluble fraction at 0-3 degrees C, and a water-soluble fraction. The properties of the fractions were studied both before denaturation and after denaturation by either heat or alcohol. The degree of proteolysis of each fraction by two digestive enzymes, pepsin and trypsin, was measured by the increase of non-protein nitrogen as a function of time. Several methods were used for electrophoretic analysis. The results showed that the thermal treatment at 100 degrees C and the treatment with varying concentrations of ethanol (from 10 to 100 p. 100) modified electrophoretic diagrams and the solubility of the proteins in trichloracetic acid. Moderate, moist heating of the protein fractions (100 degrees C, 20 mn) before proteolysis by pepsin and trypsin, in general, favored proteolysis. The most marked effect observed was in the acid-precipitated fraction (in which Kunitz inhibitor was concentrated treated by trypsin. Heating the fractions beyond thirty minutes had a negative effect on proteolysis: the level of proteolysis was the same, or in some cases, lower than before denaturation, especially on subsequent treatment with pepsin. The effects of the ethanol treatment were different from that of heat: the proteolysis was accelerated only with the acid-precipitated fraction.

Ethanol↗

PubMed1977

Fibrin and fibrinogen proteolysis products: comparison between gel filtration and SDS polyacrylamide electrophoresis analysis.

The proteolysis of purified human fibrinogen, stabilized and non-stabilized fibrin by plasmin were investigated by gel filtration analysis and SDS polyacrylamide electrophoresis of the reaction products. Plasmin proteolysis of fibrinogen followed the sequential steps previously reported and the two analytical methods yielded concordant results. Large molecular weight proteolysis products, of substantially greater molecular weight than native fibrinogen, were identified by gel filtration analysis following dissolution of stabilized and non-stabilized fibrin clots; with further incubation with plasmin, these proteolysis products gradually diminished in size. On the other hand, SDS polyacrylamide electrophoresis of these fibrin digests demonstrated that while non-stabilized fibrin yielded breakdown products similar in size to those obtained after proteolysis of fibrinogen, stabilized fibrin digests showed moieties of greater molecular size estimated to be of molecular weight 400,000 to 800,000. The final breakdown products of stabilized fibrin differed from those of fibrinogen and nonstabilized fibrin in that fragment D was present in the "double D" cross-linked form.

Antigens↗

PubMed1977

Substrate-induced conformational changes in lactate dehydrogenase. Proteolysis of the immobilized enzyme in the presence of specific substrates.

We report here a new approach to the study of the conformation of enzymes in the presence of specific substrates. Rabbit muscle lactate dehydrogenase was attached to CL-Sepharose via a cleavable spacer arm (-NH-(CH2)6NHCO(CH2)2SS(CH2)2CO-). The bound lactate dehydrogenase was digested with subtilisin BPN' in the presence of substrates of lactate dehydrogenase. The use of a flow system permits the maintenance of saturating levels of substrates. Proteolysis was followed by loss of activity of the enzyme column. The time course of proteolysis in the presence of either NADH, NAD+, or pyruvate alone did not differ from the control. However, when NADH and pyruvate were present simultaneously, the enzyme became more susceptible to proteolysis. The initial rate of proteolysis was increased by 40%. The abortive ternary complex (lactate dehydrogenase - NAD+ - pyruvate) also showed an increase in susceptibility to proteolysis. These findings clearly show that the productive ternary complex (lactate dehydrogenase - NADH - pyruvate) is conformationally different from the apoenzyme and binary complexes under optimal catalytic conditions.

Enzymes, Immobilized↗

PubMed1979

Macrophage protein turnover. Evidence for lysosomal participation in basal proteolysis.

1. Turnover of intracellular proteins in cultured mouse macrophages was found to be slightly accelerated by the omission of serum from the culture medium. Media containing 10% (v/v) or more of serum established basal degradation rates in the cultures. 2. Basal degradation rates varied considerably between experiments, probably as a result of variable activation in vivo of the macrophages. 3. The selective carboxyl proteinase inhibitor pepstatin, which appeared to enter the lysosomes of the cells by pinocytosis, gave a progressive inhibition of basal proteolysis up to a maximum of about 40%. Cellular cathepsin D was largely inhibited after 48h of cultivation with pepstatin (100 micrograms/ml). 4. Leupeptin and 7-amino-1-chloro-3-tosylamidoheptan-2-one are less selective proteinase inhibitors. They also induced 25--35% inhibition of degradation, but their actions may not have been restricted to lysosomes. 5. Several solutes and particles that are endocytosed by macrophages and stored in lysosomes induce some inhibition of basal proteolysis, whether or not they themselves are substrates for proteolysis. 6. Colchicine was without effect on protein degradation, but cytochalasin B and the local anesthetics lidocaine and procaine, all of which have effects on microfilaments, were significantly inhibitory. This inhibition may result from a decrease in the rate of autophagy, and thus of lysosomal proteolysis, due to prevention of microfilament action.

Animals↗

PubMed1976

Partial proteolysis of some cellulase components from Trichoderma viride and the substrate specificity of the modified products.

An endo-cellulase component [EC 3.2.1.4] or random type, F II, was obtained from "Cellulase Onozuka," a commercial product from Trichoderma viride, and was subjected to partial proteolysiats with a protease preparation of the same fungal origin. The resulting modified cellulase was fractioned by two steps of column chromatography, and the resulting patterns, together with the substrate specificity expressed in terms of the randomness of CMC hydrolysis and the immunological properties against anti-F II-rabbit se-um, were examined. The chromatographic patterns were very similar to those of cellulase subfractions without proteolytic treatment. Moreover, the immunological response of the modified cellulases from F II was mostly positive and their randomness of CMC hydrolysis was generally lower, compared with subfractions of F II which were not subjected to proteolysis. The subfractions of Peak III, which were obtained from F II by proteolysis, showed mostly negative immunological response and higher randomness of CMC hydrolysis compared with subfractions of Peak III which were not subjected to proteolysis. Thus, some limited proteolysis of cellulase components may, at least in part, be responsible for its multiplicity in vivo.

Amino Acids↗

PubMed1978

Modes of proteolysis of cystathionase and ornithine aminotransferase by serine protease from the rat small intestine.

The mechanisms of proteolysis of two apo-forms of pyridoxal enzymes, cystathionase [EC 4.2.1.15] and ornithine aminotransferase [EC 2.6.1.13] by serine protease from the rat small intestine were compared. The apo-forms of these two pyridoxal enzymes are susceptible to the serine protease, whereas the holo-forms of the enzymes are not. Pyridoxal phosphate, the coenzyme of these two enzymes, prevented their inactivation by the serine protease. The difference in susceptibility of the apo- and holo-forms to the serine protease was due to the difference in their conformations. The time course of inactivation of cystathionase by the protease was apparently biphasic. During the first phase of inactivation, disappearance of the band corresponding to the native protomer, with a molecular weight of 47,000, was accompanied by accumulation of new material with a molecular weight of 39,000. In the late stage of proteolysis, extensive degradation of the large molecular weight intermediate was observed. This large intermediate product was isolated and found to compete with intact cystathionase as a substrate for the protease. Proteolysis of cystathionase was accompanied by both dissociation of the enzyme molecule and loss of its antigenicity. Limited proteolysis of ornithine aminotransferase apoenzyme by the serine protease resulted in formation of a large molecular weight product similar to the native apoenzyme, but with nicks in the molecule. Dodecylsulfate-polyacrylamide gel electrophoresis showed that the apoenzyme is degraded to intermediate forms (molecular weight 41,500 and 15,000) and later, to stable forms (molecular weight 25,500 and 13,500).

Animals↗

PubMed1979

[Studies for proteolysis in malignant tumours (author's transl)].

It's well known that increased proteolytic activity in plasma as well as in interstitial fluids is often associated with malignant tumours. There are numerous reports that cell cultures of malignant origin contain plasminogen activators responsible for an increased proteolysis. Our studies showed that patients with a carcinoma excrete in their urine a significant higher amount of an acid-stable inhibitor than healthy patients. This urine inhibitor is a split product (MW: 30,000) of the Inter-alpha-trypsin inhibitor (MW: 180,000). We conclude that the increased amount of acid-stable split products is the result of increased proteolysis in patients with malignant tumours. In contrast to other authors we do not think that the activation of plasmin by plasminogen is responsible for the proteolytic cleavage of ITI. Otherwise proteolysis by Cathepsin G and granulocytic Elastase would be reasonable. So enhanced proteolysis seems to be a sign of general enzymatic change associated with transformation.

Carcinoma, Squamous Cell↗

PubMed1978

[Quaternary structure and proteolysis of the polynucleotide phosphorylase from C. perfringens].

This report describes structural studies on purified polynucleotide phosphorylase from C. perfringens. A method is described for the purification of the enzyme which yields a product equivalent in activity to the native polynucleotide phosphorylase from E. coli. These studies revealed a molecular heterogeneity arising from successive stages of proteolysis, to which this enzyme is especially sensitive; unusally, the enzyme is obtained as a mixture of variable proportions of the native and proteolysed forms. We found in all cases a trimeric basic structure composed of the native (alpha) or proteolysed (lapha) or proteolysed (alpha', alpha") catalytic sub-units, However, the enzyme is rather easily dissociated into its sub-units, a phenomenon which seems to accompany proteolysis (Table). Under the action of either endogenous proteases or trypsin, two enzymatic forms are obtained: their quaternary structures seem analogous, but they differ in their catalytic properties from each other and from the initial enzyme. With some care at each step of purification, the polynucleotide phosphorylase of E. coli can be obtained exclusively in its native form. The greater susceptibility to proteolysis of the enzyme from C. perfrigens and the relationship between such degradation and quaternary structure seem to be at the origin of the peculiar behavior of this polynucleotide phosphorylase.

Chemical Fractionation↗

PubMed1976

Some properties of proteolysis by polymorphonuclear leukocyte-granule extracts.

The extracts of granules of human polymorphonuclear leukocytes hydrolyzed a variety of proteins including human and bovine hemoglobin, human fibrinogen, human and bovine serum albumin, bovine elastin, and casein. The hydrolysis of all the proteins except fibrinogen and elastin was increased by addition of urea. Various inhibitors of trypsin, kallikrein, plasmin, Clr, Cls, and other proteolytic enzymes had no inhibitory effect. Slight inhibition was observed with polyanethol sulfonate and strong inhibition with normal human serum. Serum of patients with hereditary angioneurotic edema having no functional C1-esterase inhibitor was as effective in inhibiting the proteolysis as normal serum. The inhibitor was localized in 4S fractions of normal serum fractionated on Sephadex G-200. Fractionation of normal serum by ammonium sulfate precipitation, Sephadex G-200 filtration, and CM-Sephadex chromatography did not result in appearance of inhibitory activity in more than one protein peak, suggesting the possibility that only one inhibitor might be responsible. Since all fractions which contained the inhibitor of proteolysis also contained alpha1-antitrypsin, since sera of patients having low alpha1-antitrypsin levels contained less inhibitory activity, and since antibodies against alpha1-antitrypsin reversed the inhibition obtained from normal serum, the inhibition of proteolysis may be attributed to alpha1-antitrypsin.

Angioedema↗

PubMed1977

Analysis of the structure of T4 bacteriophage-modified valyl-tRNA synthetase by limited proteolysis and isoelectric focusing.

The new form of valyl-tRNA synthetase (EC 6.1.1.9) that appears immediately after infection of Escherichia coli with bacteriophage T4 was purified and subjected to mild proteolysis using five different proteases. The inactivation of aminoacylation activity was both more extensive and rapid than that obtained with valyl-tRNA synthetase purified from uninfected E. coli. The addition of bulk tRNA from E. coli B protected the phage-specific form of valyl-tRNA synthetase from proteolysis, but ATP and valine did not exhibit a similar protective effect. The characteristic property of phage-modified valyl-tRNA synthetase, resistance to denaturation by 4 M urea, remained unaffected during treatment with trypsin. This suggested that the phage-specific factor tau, known to be associated with the synthetase in phage-infected cells, was protected from proteolysis in the synthetase-tau complex. Comparison by isoelectric focusing of normal valyl-tRNA synthetase, the phage-specific form of this enzyme, and phage enzyme from which tau had been removed, revealed no differences in the isoelectric points of these three molecules. Based on these results a model was drawn for the structural changes occurring in valyl-tRNA synthetase after association with the phage factor tau.

Amino Acyl-tRNA Synthetases↗

PubMed1978

[Proteolysis of products of mitochondrial protein synthesis in isolated mitochondria of Saccharomyces cerevisiae yeasts].

Products of mitochondrial protein synthesis were specifically labeled with 3H-leucine in the presence of cycloheximide at the end of the exponential phase of yeast aerobic growth on glucose. The mitochondria isolated from these cells lost 37-40% of the label from the protein fraction during 60 min incubation at 35 degrees, which was accompanied by the accumulation of 3H-leucine in TCA-soluble fraction. This process was suppressed by phenyl-methyl sulfonyl fluoride and p-chloromercuriphenyl sulfonate, the inhibitors of proteases, and could thus be considered as the proteolysis of the products of mitochondrial protein synthesis. The proteolysis was ATP dependent and was stimulated by puromycine which is known to induce the removal of incomplete polypeptides from mitochondrial ribosomes. A body of indirect evidence allows a suggestion to be made that the observed proteolysis can hardly be due to the action of cytoplasmic proteinases.

Fungal Proteins↗

PubMed1978

Hormonal and metabolic control of proteolysis.

Proteolysis in heart muscle is regulated by hormonal and metabolic factors. In the absence of insulin, protein degradation accelerated, autophagic vacuoles appeared within the heart and latency of lysosomal enzymes decreased. Addition of insulin prevented or reversed these changes. Provision of leucine inhibited proteolysis in control hearts. Addition of the products of transamination and oxidative decarboxylation of leucine, isoleucine and valine had similar inhibitory effects. Protein degradation was inhibited in ischaemic or anoxic hearts, but latency of lysosomal enzymes was decreased. Inhibition of degradation in energy-poor tissue may represent both an energy-requirement for proteolysis and inhibition by accumulation of metabolic products, such as lactate.

Adenosylmethionine Decarboxylase↗

PubMed2026

Disruption of the ubiquitin-mediated proteolysis pathway: a study of seed aging in Saposhnikovia divaricata caused by UBC1 gene family suppression.

BACKGROUND: Saposhnikovia divaricata (Turcz.) Schischk. is a perennial herb whose seed aging during storage significantly reduces germination rates, limiting industrial-scale production. Reactive oxygen species (ROS)-induced oxidative damage is a key driver of seed aging, but the underlying mechanisms in Saposhnikovia divaricata remain unclear. RESULTS: Suppression of the UBC1 gene family reduces the activity of ubiquitin-conjugating enzymes, leading to dysfunction of the ubiquitin-mediated proteolysis pathway, which in turn decreases protein degradation efficiency and causes the accumulation of damaged proteins. Transcriptome analysis revealed predominant downregulation of genes crucial for seed physiological maintenance. By the fourth year of storage, germination dropped sharply to 30.67%, accompanied by embryo cavitation. Downregulation of ribosome pathway genes hindered ribosome assembly and protein synthesis, while suppression of endoplasmic reticulum protein processing genes led to unfolded/misfolded protein accumulation and intensified cellular stress, accelerating aging. Proteomic analysis showed increased total differential and antioxidant-related proteins. ROS content fluctuated with storage time: peroxyl radicals peaked in year two (5.68 RFU/mg), whereas hydroxyl radicals and hydrogen peroxide were highest in year four (0.0655 pg/mL and 0.0946 pg/mL, respectively), with significant differences across periods. Elevated membrane-related proteins, increased electrical conductivity, and malondialdehyde content (maximum 54.30 nmol/g at year four) confirmed oxidative membrane damage. ROS-induced stress promotes protein misfolding, and reduced UBC1 expression is associated with impaired clearance of misfolded proteins by the ubiquitin-mediated proteolysis pathway. CONCLUSIONS: This study provides the first integrated transcriptomic and proteomic insight into UBC1 deficiency-mediated seed aging in Saposhnikovia divaricata. The findings enhance molecular understanding of seed aging and offer new directions for improving seed storage and viability.

Ubiquitin-Conjugating Enzymes↗

PubMed1978

Proteolysis and flash photolysis of bacteriorhodopsin in purple membrane fragments.

Pronase treatment of aqueous suspensions of purple membrane fragments from H.halobium leads to the cleavage of bacteriorhodopsin. The protein fragments remaining in the membrane after treatment with relatively small concentrations of enzyme (2% w/w) in normal daylight range in molecular weight from 20,000--21,000 daltons, indicating that cleavage occurs mainly near the extremities of the protein chain. At higher enzyme concentrations the relative amounts of protein fragments having smaller molecular weight increase. Generally, the relative loss of retinal chromophore is larger than that of protein and thus the retinal binding site seems to be located near one of the chain ends that is cleaved off by enzyme. Irradiation with white light during the time of proteolysis (at both low and high enzyme concentrations) results in extensive cleavage, so that under certain conditions no high molecular weight components can be detected in SDS-polyacrylamide gels. It, therefore, appears that parts of the bacteriorhodopsin chain become more exposed to enzyme digestion when the purple membrane is illuminated. Enzyme treated aqueous purple membrane fragment suspensions still show photocycle activity. The main consequence of proteolysis is a pronounced appearance of biphasicity in the decay of M412 and the regeneration of bR570. Simultaneously the yield of O660 is reduced. As with untreated purple membrane, the correlation between the rates of decay of M412 and regeneration of bR570 is greatest when the yield of O660 is lowest.

Bacterial Proteins↗

PubMed1979

Limited proteolysis of coupling factor-latent ATPase from Mycobacterium phlei. Effects of different enzymes and modifying agents.

The activation of the coupling factor-latent ATPase enzyme by tryptic proteolysis may resemble the activation of many proenzymes by limited proteolysis. The beta (53 000 dalton) subunit of solubilized coupling factor-latent ATPase from Mycobacterium phlei was selectively lost in some trypsin-treated samples. Since a concomitant loss of ATPase activity was not observed, the beta subunit may not be essential for ATPase catalytic activity. Treatment of solubilized coupling factor with chymotrypsin rapidly produced an A'-type (61 000 dalton) species from the native alpha (64 000 dalton) subunits with partial activation of the APTase enzyme. Secondary chymotryptic cleavage yielded an A"-type (58 000 dalton) species and a less-active enzyme. Storage of fresh coupling factor samples at -20degreeC in the presence of 4 mM MgCl2 with several freeze-thaw cycles resulted in loss of ATPase activity without apparent change in alpha subunit structure. Storage at 4 degrees C in the presence or absence of MgCl2 both decreased ATPase activity and generated A'-type alpha subunit species. Since presence was suspected. The peptide bonds first cleaved by trypsin, chymotrypsin, and the unknown protease are all apparantly located within the same small segment of alpha subunit polypeptide chain.

Adenosine Triphosphatases↗

PubMed1975

Proteolysis detection in milk. IV. Starch-gel electrophoresis and formol titration.

Starch-gel electrophoresis (SGE) and formol titration methods for detecting proteolysis in cold-stored raw milk have been studied to establish their value as quality indices. When examined by SGE, the first evidence of proteolysis in raw milks stored at 5 degrees C was the formation of para-k-casein. However, this fraction could not be detected on the starch gels until the total bacterial count (TBC) exceeded 10-minus 7/ml. The SGE method appeared more reliable than the previously discussed tyrosine value method. Formol titration did not appear to have any application in the screening of cold-stored raw milks with TBC less than 10-minus 7/ml, but would detect some milks of poorer bacteriological quality.

Animals↗

PubMed1979

Fragmentation of the human transplantation antigen heavy chain by limited proteolysis, acid cleavage, and cyanogen bromide treatment.

Highly purified, papain-solubilized HLA-A, -B, and -C antigens comprising a mixture of a great number of allelic forms from at least three loci have been fragmented by limited proteolysis, acid cleavage, and cyanogen bromide treatment. Limited proteolysis of 125I-labeled HLA-A, -B, and -C antigens with trypsin, chymotrypsin, thermolysin, and pepsin resulted in the production of two large fragments. One fragment was associated with beta 2-microglobulin and contained all of the carbohydrate. The other fragment, which had a molecular weight of about 13,000, is most probably derived from the COOH-terminal part of the heavy chain. Acid cleavage of the HLA antigen heavy chain gave rise to two main fragments with molecular weights of 22,000 and 11,000. Both fragments contained disulfide bonds. Two minor components, representing further cleavage products of the 22,000-dalton fragment, were also observed. Cleavage of the HLA antigen heavy chain at methionyl residues gave rise to one carbohydrate-containing, cysteine-free 14,000-dalton fragment and one 20,000-dalton fragment that contained all cysteines but no carbohydrate. NH2-terminal amino acid sequence analyses demonstrated that the 22,000-dalton acid cleavage fragment and the 14,000-dalton cyanogen bromide fragment were derived from the NH2-terminal part of the HLA antigen heavy chain.

Amino Acid Sequence↗