Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Copious outgrowth of chondrocytes was obtained by explantation from each of three rabbit and one surgically-resected human articular cartilages pretreated briefly with trypsin. In lapine explants, ascorbate (40 micrograms/ml) increased DNA three-fold over control values and resulted in deposition of a chondroid matrix. It doubled radiosulfate incorporation by the outgrowths. Up to 56% of the sulfated glycosaminoglycan synthesized was located in the trypsin-digestible pericellular coat compared with about 15% in previous monolayer cultures. The collagens synthesized were characterized partially. In rabbit cell cultures, the alpha 1:alpha 2 ratio varied from 2.9 to 3.8. In human cultures, an unusual post-alpha 2 peak was observed. The findings suggest an uncoupling of the phenotypic expression of the major cartilaginous macromolecules in the cultures. There were no distinctive differences between chondrocytes derived from normal and fibrillated human cartilage of the same individual.
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PMID:Explant culture of human and rabbit articular chondrocytes. 15 89

Isolated adrenal cells from Vitamin E-deficient and control rats were prepared by a trypsin digestion method. Cyclic adenosine 3',5'-monophosphate (cyclic AMP) formation was studied in response to adrenocorticotropin (ACTH) in the presence and absence of ascorbate by measuring the conversion of prelabeled adenosine 5'-triphosphate [14C]ATP to cyclic [14C]AMP. Ascorbate (0.5 mM) inhibited ACTH-induced cyclic [14C]AMP formation in adrenal cells isolated from Vitamin E-deficient rats but had no effect in the control cells. The inhibitory effect of ascorbate on ACTH-induced cyclic AMP formation in Vitamin E-deficient rats decreased as the concentration of ACTH increased. In Vitamin E-deficient rats ascorbate inhibited ACTH-induced cyclic [14C]AMP formation after 30 min of incubation. There was no further significant accumulation of cyclic [14C]AMP at 60 min or 120 min although in the absence of ascorbate cyclic [14C]AMP continued to be formed. The in vitro addition of alpha-tocopherol reduced the inhibition of ACTH-induced cyclic [14C]AMP formation by ascorbate in Vitamin E-deficient rats. These studies suggest that alpha-tocopherol and ascorbate may affect ACTH-induced cyclic AMP formation through interaction with the membrane-bound enzyme adenylate cyclase.
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PMID:Effect of ascorbic acid on ACTH-induced cyclic AMP formation and steroidogenesis in isolated adrenal cells of vitamin E-deficient rats. 16 1

1. The uncoupler-stimulated ATPase activity of castor bean endosperm mitochondria and submitchondrial particles has been studied. The rate of ATP hydrolysis catalyzed by intact mitochondria was slow and little enhanced by addition of uncouplers at the concentration required for uncoupling the oxidative phosphorylation. ATP-ase activity was stimulated at higher concentrations of uncouplers. 2. 1-Anilinonaphthalene 8-sulfonate fluorescence was decreased when the mitochondria were oxidizing succinate. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone and antimycin reversed the succinate-induced fluorescence diminution. ATP did not induce the fluorescence response. 3. The addition of succinate, NADH or ascorbate/N,N,N'-N'-tetramethyl-p-phenylenediamine as electron donor induced high ATPase activity in the presence of low concentrations of uncouplers. Stimulating effect of uncouplers was completely abolished by further addition of antimycin. 4. Submitochondrial particles were prepared by sonication. The particles catalyzed a rapid hydrolysis of ATP and carbonylcyanide-p-trifluoromethoxyphenylhydrazone at 10-8 M did not stimulate the ATPase activity. Addition of succinate induced uncoupler-stimulated ATPase activity. The effect of succinate was completely abolished by further addition of antimycin. 5. The treatment of submitochondrial particles by trypsin or high pH also induced uncoupler-stimulated ATPase activity. 6. The above results were interpreted to indicate that ATPase inhibitor regulated the back-flow reaction of mitochondrial oxidative phosphorylation.
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PMID:Respiration-department uncoupler-stimulated ATPase activity in castor bean endosperm mitochondria and submitochondrial particles. 23 83

The effect of trypsin on the photosynthetic electron transport of spinach chloroplasts has been investigated by measurements of the flash-induced absorption changes, indicating chlorophyll a1 at 703 nm, chlorophyll aII at 690 nm and at 515 nm via electrochromism the electrical potential gradient across the thylakoid membrane, respectively, and of the fluorescence induction caused by moderate actinic light. It was found: (1) In the presence of benzyl viologen as electron acceptor and with water as natural electron donor trypsin, incubation leads to a complete suppression of the absorption changes of the electrochromic effect and of chlorophyll aI and chlorophyll aII. (2) Addition of System I electron donors (N-methylphenazonium sulfate plus ascorbate or 2,6-dichlorophenolindphenol plus ascorbate) fully restores the chlorophyll aI photoreaction, whereas the initial amplitude of the electrochromic absorption change at 515 nm amounts about 50% of the control value without trypsin. The chlorophyll aII inhibition remains uneffected by System I electron donors. (3) System II electron donors (benzohydroquinone plus ascorbate or TPB) are unable to overcome the inhibition of electron transport by trypsin. (4) The fluorescence induction curve in 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea-blocked chloroplasts is modified by trypsin. The level of maximal fluorescence is remarkably decreased, whereas the initial fluorescence remains constant. The rise in kinetics is slightly decelerated. From these results, it is concluded that in the linear electron transport from water to benzyl viologen, mild trypsin treatment specifically attacks System II at a site very close to the reaction center, either on the oxidizing or on the reducing side. The reaction center of System II itself is relatively stable against trypsin. Arguments are presented which argue in favor of the trypsin attack being primarily directed at the reducing side of System II.
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PMID:Studies on the nature of the inhibitory effect of trypsin on the photosynthetic electron transport of system II in spinach chloroplasts. 95 70

Oxidative damage to bovine serum albumin (BSA) was induced by hydroxyl radical (HO.) generating systems of xanthine oxidase (XO) + EDTA-Fe3+ and ascorbate + EDTA-Fe3+. Formation of bityrosine and loss of tryptophan were observed in the ascorbate + EDTA-Fe3+ system and carbonyl formation was induced by both systems. Mannitol and ethanol very strongly inhibited the carbonyl and/or bityrosine formation, indicating that the oxidative damage to BSA was due to HO(.). The sulfhydryl (SH) groups of BSA were very sensitive to the XO + EDTA-Fe3+ but not to the ascorbate + EDTA-Fe3+ system. Catalase but not hydroxyl radical scavengers or superoxide dismutase strongly inhibited the loss of SH groups, indicating that H2O2 is involved in their oxidation. Fragmentation of BSA was observed during exposure to the XO + EDTA-Fe3+ and ascorbate + EDTA-Fe3+ systems and the products presented a broad band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Little formation of amine groups was observed in these systems, indicating that little peptide bond cleavage occurred. BSA exposed to the ascorbate + EDTA-Fe3+ system was more readily degraded by trypsin than that exposed to the XO + EDTA-Fe3+ system. Elastase degraded BSA exposed to the ascorbate + EDTA-Fe3+ system but not to the XO + EDTA-Fe3+ system.
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PMID:Oxidative damage to bovine serum albumin induced by hydroxyl radical generating systems of xanthine oxidase + EDTA-Fe3+ and ascorbate + EDTA-Fe3+. 133 12

Microsomes from rat liver were used to investigate the mechanisms by which thiol compounds protect cellular membranes against damage from oxidants. Glutathione (GSH), dihydrolipoate and dithioerythritol, but not cysteine, ameliorated the loss of thiol groups of microsomal proteins attacked by Fe/ADP/NADPH or Fe/ADP/ascorbate prooxidant systems. The protection by GSH, but not dihydrolipoate or dithioerythritol, appeared to be enzymic since it was lost after microsomes were heated or treated with trypsin. The blocking of microsomal protein thiols with N-ethylmaleimide also diminished the protective effect of GSH. Lipid peroxidation, as assessed by chemiluminescence and vitamin-E loss, was inhibited in parallel with the protection of protein thiols. In microsomes lacking vitamin E, the protection of protein thiols by exogenous thiols was diminished. However, the GSH-dependent protection of vitamin E showed no preference for alpha-tocopherol over other tocopherol homologs. It is suggested that a GSH-dependent enzyme maintains protein thiols in the face of oxidative damage during microsomal peroxidation. A maintenance of protein thiols might not only protect important metabolic functions, but may also afford an antioxidant capacity to membranes, and account for one facet of the GSH-dependent inhibition of lipid peroxidation.
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PMID:Protection by glutathione and other thiol compounds against the loss of protein thiols and tocopherol homologs during microsomal lipid peroxidation. 144 67

The effect of glucose/Cu2+ incubation on (a) pure methionine oxidation, (b) the oxidation of active-site methionine in alpha 1-proteinase inhibitor (alpha 1PI) and (c) the resulting activity and structural changes of this inhibitor was investigated. While no methionine was oxidized during a 24 day, 37 degrees C incubation with 0.01 M EDTA and 100 mM glucose, 64.2% oxidation occurred in 6 days when 0.01 mM Cu2+ was added to the 100 mM glucose. The first-order rate constant for oxidation in 10 mM glucose, 0.01 mM Cu2+ was 0.0218 day-1. Oxidation was inhibited by catalase, but accelerated by ascorbate ion. The active-site methionyl residue of alpha 1PI was oxidized 71.3% after a 4 day incubation in 100 mM glucose, 0.01 mM Cu2+ (pH 7.45), 0.1 M phosphate buffer. The elastase and trypsin inhibiting activities were lowered to 3.1 and 1.5% of control samples during this incubation. The inclusion of 1 mM DETAPAC, a transition metal chelator, resulted in a 98 + % retention of activity. Intrinsic fluorescence (350 nm excitation, 415 nm emission) of alpha 1PI increased 576% over control for the sample incubated in 100 mM glucose, 0.01 mM Cu2+ and SDS-PAGE revealed protein fragment molecular weights of 44.4 and 39.8 kDa. These studies suggest that both methionine oxidation and free radical induced fragmentation contribute to loss of alpha 1PI activity during glucose/Cu2+ incubations.
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PMID:Methionine oxidation and inactivation of alpha 1-proteinase inhibitor by Cu2+ and glucose. 162 10

Interstitial collagenase either obtained from human neutrophils by phorbol myristate acetate (PMA) induced degranulation or isolated from human gingival crevicular fluid was found to be activated by addition of an oxidative agent, hypochlorous acid (HOCl). Collagenase released by PMA stimulated neutrophils was completely in latent form but underwent partial autoactivation during 16 h incubation at 22 degrees C in the presence of soy bean trypsin inhibitor. The partial autoactivation was potentiated to complete activation of released collagenase after addition of exogenous HOCl. Ascorbate prevented this activation of neutrophil collagenase. Isolated human gingival crevicular fluid collagenase represented an apparent Mr of 70 kD in completely latent form, whereas 70/54 kD enzyme species were detected for partially autoactive form of the enzyme. Western blot analysis of gingival crevicular fluid using a polyclonal antibody raised against purified human neutrophil collagenase revealed the same 70/54 kD molecular forms of the enzyme. The latent gingival crevicular fluid collagenase was also activated by HOCl and this activation could be prevented by ascorbate. Activation of the 70 kD latent collagenase by HOCl as well as by other non-proteolytic activators such as an organomercurial compound (phenylmercuric chloride) and a gold(I) compound (gold thioglucose) was not associated with detectable changes in apparent Mr, whereas trypsin activation resulted in fragmentation of 70 kD enzyme to 54 kD species. Our results provide further evidence for the neutrophil origin of gingival crevicular fluid collagenase and suggest that, in addition to proteolytic activation, oxidative and antioxidative agents seem to be able to regulate neutrophil collagenase activity.
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PMID:Hypochlorous acid induced activation of human neutrophil and gingival crevicular fluid collagenase can be inhibited by ascorbate. 166 35

NADPH-supported lipid peroxidation monitored by malondialdehyde (MDA) production in the presence of ferric pyrophosphate in liver microsomes was inactivated by heat treatment or by trypsin and the activity was not restored by the addition of purified NADPH-cytochrome P450 reductase (FPT). The activity was differentially solubilized by sodium cholate from microsomes, and the fraction solubilized between 0.4 and 1.2% sodium cholate was applied to a Sephadex G-150 column and subfractionated into three pools, A, B, and C. MDA production was reconstituted by the addition of microsomal lipids and FPT to specific fractions from the column, in the presence of ferric pyrophosphate and NADPH. Pool B, after removal of endogenous FPT, was highly active in catalyzing MDA production and the disappearance of arachidonate and docosahexaenoate, and this activity was abolished by heat treatment and trypsin digestion, but not by carbon monoxide. The rate of NADPH-supported lipid peroxidation in the reconstituted system containing fractions pooled from Sephadex G-150 columns was not related to the content of cytochrome P450. p-Bromophenylacylbromide, a phospholipase A2 inhibitor, inhibited NADPH-supported lipid peroxidation in both liver microsomes and the reconstituted system, but did not block the peroxidation of microsomal lipid promoted by iron-ascorbate or ABAP systems. Another phospholipase A2 inhibitor, mepacrine, poorly inhibited both microsomal and pool-B'-promoted lipid peroxidation, but did block both iron-ascorbate-driven and ABAP-promoted lipid peroxidation. The phospholipase A2 inhibitor chlorpromazine, which can serve as a free radical quencher, blocked lipid peroxidation in all systems. The data presented are consistent with the existence of a heat-labile protein-containing factor in liver microsomes which promotes lipid peroxidation and is not FPT, cytochrome P450, or phospholipase A2.
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PMID:Cholate solubilization of liver microsomal membrane components which promote NADPH-supported lipid peroxidation. 172 52

The peroxidation of rat liver microsomal lipids is stimulated in the presence of iron by the addition of NADPH or ascorbate and is inhibited by the addition of glutathione (GSH). The fate of GSH and the oxidative modification of proteins under these conditions have not been well studied. Rat liver microsomes were incubated at 37 degrees C under 95% O2:5% CO2 in the presence of 10 microM ferric chloride, 400 microM ADP, and either 450 microM ascorbic acid or 400 microM NADPH. Lipid peroxidation was assessed in the presence 0, 0.2, 0.5, 1, or 5 mM GSH by measuring thiobarbituric acid reactive substance (TBARS) and oxidative modification of proteins by measuring protein thiol and carbonyl groups. GSH inhibited TBARS and protein carbonyl group formation in both ascorbate and NADPH systems in a dose-dependent manner. Heat denaturing of microsomes or treatment with trypsin resulted in the loss of this protection. The formation of protein carbonyl groups could be duplicated by incubating microsomes with 4-hydroxynonenal. Ascorbate-dependent peroxidation caused a loss of protein thiol groups which was diminished by GSH only in fresh microsomes. Both boiling and trypsin treatment significantly decreased the basal protein thiol content of microsomes and enhanced ascorbate-stimulated lipid peroxidation. Protection against protein carbonyl group formation by GSH correlated with the inhibition of lipid peroxidation and appeared not to be due to the formation of the GSH conjugate of 4-hydroxynonenal as only trace amounts of this conjugate were detected. Ninety percent of the GSH lost after 60 min of peroxidation was recoverable as borohydride reducible material in the supernatant fraction. The remaining 10% could be accounted for as GSH-bound protein mixed disulfides. However, only 75% of the GSH lost during peroxidation appeared as glutathione disulfide, suggesting that some was converted to other soluble borohydride reducible forms. These data support a role for protein thiol groups in the GSH-mediated protection of microsomes against lipid peroxidation.
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PMID:Inhibition of protein carbonyl formation and lipid peroxidation by glutathione in rat liver microsomes. 173 26


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