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)

Acinetobacter glutaminase-asparaginase was chemically modified by succinylation and glycosylation with glycopeptides from human fibrin and gamma-globulin. These modifications markedly prolonged the half-lives of the enzyme in mice, rats, and rabbits. The plasma half-life in mice increased with decreasing isoelectric point. Glycosylation caused greater prolongation in rodents than succinylation. The kinetic properties of the modified enzymes were unchanged. Succinylation protected the enzyme from trypsin digestion. Glycosylated preparations had less heat inactivation than native and succinylated enzyme. Sedimentation equilibrium studies on a succinylated preparation showed reversible dissociation to a dimer (71, 400 g/mol) with an association constant of 1.3 times 10-6 liters/mol. This dissociation was identical with native enzyme, except for a 3% increase in molecular weight due to succinate groups. Sedimentation equilibrium studies on glycosylated preparations showed mixtures of molecular weight from 60, 000 to over 180, 000. Gel filtration and active enzyme sedimentation showed active polymers, but no active species smaller than tetramer.
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PMID:Biologic and physical properties of succinylated and glycosylated Acinetobacter glutaminase-asparaginase. 112 47

Primary cultures of rat renal proximal tubular epithelial cells were used to characterize the biosynthesis and processing of the mitochondrial glutaminase. When the cells were labeled with [35S]methionine in the presence of 20 microM carbonylcyanide m-chlorophenylhydrazone, only a 72-kDa peptide, which co-migrates with the primary translation product of the glutaminase mRNA, was immunoprecipitated. At lower concentrations of carbonylcyanide m-chlorophenylhydrazone, the 68- and 65-kDa peptides that are characteristic of the mature glutaminase and a 71-kDa peptide were synthesized. Pulse-chase experiments suggest that the 72-kDa cytosolic precursor could be quantitatively chased to generate the mature mitochondrial species. The observed kinetics indicate that the 71-kDa species is an intermediate in the import pathway. In addition, the 65-kDa glutaminase peptide was synthesized more rapidly than the 68-kDa peptide, and the two peptides were produced in a final ratio of 3:1, respectively. These results suggest that one subunit of the tetrameric glutaminase may be subject to covalent modification. In vitro processing was also characterized by incubating isolated rat liver mitochondria with the glutaminase precursor that was produced by in vitro translation of acidotic rat renal poly(A+) RNA. This system produced an identical sequence of processing reactions. The in vitro formation of the 71-kDa intermediate required a transmembrane potential. Both the intermediate and the mature forms of the glutaminase were recovered in the mitochondria and were resistant to trypsin digestion. Thus, the glutaminase precursor is rapidly translocated across the inner mitochondrial membrane and initially processed to yield an intermediate. The intermediate is subsequently processed to yield the two peptides that constitute the mature enzyme.
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PMID:Biosynthesis and processing of renal mitochondrial glutaminase in cultured proximal tubular epithelial cells and in isolated mitochondria. 221 60

Elastase, V8 protease, subtilisin, trypsin, and chymotrypsin all cleaved the 1462-residue polypeptide of rat carbamyl phosphate synthetase I in segment C 160-180 residues from the COOH-end. Its activator N-acetylglutamate (AcGlu) increased the rate of cleavage approximately ninefold, presumably by binding preferentially to the conformation in which C is exposed. ATP/Mg2+ prevented proteolysis both +/- AcGlu. Kd,app for AcGlu (66 microM) and ATP (4.2 microM with AcGlu and 5 mM Mg2+) was estimated from the pseudo-first-order rate constants for inactivation caused by cleavage with elastase at C. Chymotrypsin and trypsin also hydrolyzed the enzyme, independent of AcGlu, at site D within less than 20 residues of the COOH-end. D was protected by ATP only in the presence of AcGlu and K+, and enzyme hydrolyzed exclusively at D had greater than 30-fold higher Km's for AcGlu and ATP. Digestion by trypsin at a third site (B) approximately 530 residues upstream from C appeared to occur subsequent to hydrolysis at C. Slow cleavage by elastase at an additional site (A) to give 360- and 1100-residue peptides was unaffected by AcGlu and ATP, and caused only modest loss of activity. These peptides were isolated by chromatography on DEAE-cellulose. Assignment of the smaller one to the NH2-end on the basis of its cysteine content places site A in the junction between the segments homologous to the small glutaminase and large synthetase subunits of Escherichia coli carbamyl phosphate synthetase II. Neither peptide alone was active; maximal regain of activity (approximately 25%) occurred on combining them in equimolar proportions. The sizes of the peptides produced by further digestion of the site A digest gave the approximate locations of the other sites. Sites A (Ala-417) and B (Arg-787) have recently been identified by NH2-terminal sequencing (S. G. Powers-Lee and K. Corina (1986) J. Biol. Chem. 261, 15349-15352). Reasons for the low value of KAcGlu,app are examined, and protection by ATP is discussed in relation to previous models for the conformational equilibria of the enzyme.
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PMID:Proteolysis as a probe of ligand-associated conformational changes in rat carbamyl phosphate synthetase I. 328 64

We have examined the domain organization, and the locations of the sites phosphorylated by the cyclic-AMP-dependent protein kinase, in the multifunctional polypeptide of the pyrimidine-biosynthetic protein, CAD. Fragments produced after limited proteolysis by elastase or trypsin were separated by SDS/polyacrylamide gel electrophoresis and transferred onto nitrocellulose. The blots were probed with antibodies raised against the core aspartate carbamoyltransferase (ACTase) and dihydroorotase (DHOase) fragments to locate fragments containing these domains, and we also examined the locations of the phosphorylation sites by complete tryptic digestion of blotted, 32P-labelled fragments, followed by analytical isoelectric focussing. Our results are consistent with the domain order glutaminase(GLNase)-carbamoyl-phosphate synthetase-(CPSase)-DHOase-ACTase, as suggested by recently reported homologies between the predicted amino acid sequence for the Drosophila rudimentary gene product, and monofunctional CPSases/ACTases/DHOases. In particular, the finding of a 95-kDa elastase fragment which cross-reacted with both anti-DHOase and anti-ACTase antibodies rules out the previously suggested domain order: DHOase-GLNase-CPSase-ACTase. Phosphorylation by cyclic-AMP-dependent protein kinase accelerates cleavage of native CAD by both elastase and trypsin, and abolishes the protective effect of UTP. Site 1 is located close to the C-terminal end of the 160-kDa GLNase/CPSase region. Comparison with the predicted amino acid sequence of the Drosophila rudimentary gene revealed a strong homology between the tryptic peptide containing site 1 from hamster CAD, and a region at the extreme C-terminal end of the CPSase II domain of the Drosophila enzyme. Alignment of the Drosophila sequence and that of rat liver CPSase I, which is not phosphorylated by cyclic-AMP-dependent protein kinase, revealed that this putative site 1 region is missing in CPSase I. Site 2 could not be located with certainty, either from the limited proteolysis data, or from comparison of the sequence around this site and the sequence of the rudimentary gene. There were also one or more previously undetected minor phosphorylation site(s) located in the protease-sensitive hinge region between the DHOase and ACTase domains.
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PMID:Mapping of catalytic domains and phosphorylation sites in the multifunctional pyrimidine-biosynthetic protein CAD. 334 46

The structural and functional domains of Escherichia coli carbamoyl phosphate synthetase (CPS) have been identified by limited proteolysis. Incubation of CPS with several proteases, including trypsin, chymotrypsin, subtilisin and endoproteinase Asp-N, under native conditions, causes a time-dependent loss of enzymatic activity and the generation of a common fragmentation pattern. Amino-terminal sequencing studies demonstrated that the initial cleavage event by trypsin occurred at the carboxy-terminal end of the large subunit. The ultimate fragments produced in most of the proteolysis studies, 35- and 45-kDa peptides, were derived from areas corresponding to the putative ATP binding regions. Substrate protection studies showed that the addition of ligands did not affect the final fragmentation pattern of the protein. However, ornithine and UMP were found to significantly reduce the rate of inactivation by inhibition of proteolytic cleavage. MgATP and IMP provided modest protection whereas bicarbonate and glutamine showed no overall effect on proteolysis. Limited proteolysis by endoproteinase Asp-N resulted in the production of a fragment (or multiple fragments) which contained enzymatic activity but had lost all regulation by the allosteric ligands, UMP and ornithine. The small subunit has been shown to be protected from proteolysis by the large subunit. Proteolysis of the isolated small subunit resulted in the generation of a stable 31-kDa species which contained 10% of the original glutaminase activity. These studies demonstrate that a portion of the C-terminal end of the large subunit can be excised without entirely destroying the ability of CPS to catalyze the formation of carbamoyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mapping the structural domains of E. coli carbamoyl phosphate synthetase using limited proteolysis. 764 1

The intramitocondrial localization of the phosphate-activated glutaminase from Ehrlich cells has been examined by a combination of techniques, including: mitochondria subfractionation studies, chemical modification with sulfhydryl group reagents of different permeability, enzymatic digestion in both sides of the inner mitochondrial membrane, and immunological studies. Using alkaline extraction at high ionic strength, hypoosmotic shock and freezing-thawing cycle techniques, the enzyme was found in the particulate fraction. On the contrary, glutaminase activity was labile when subfractionation was carried out by digitonin/lubrol method; Western blot analysis localized the inactive enzyme in the matrix fraction. In addition, glutaminase was fully inactivated when mitoplasts were incubated with phospholipase A2 and phospholipase C. The enzyme also showed a non-linear Arrhenius plot with a break at 24 degrees C. The membrane-impermeant thiol reagents mersalyl and p-chloromercuriphenylsulfonic acid do not inhibit glutaminase activity in freeze-thawed mitochondria and mitoplasts, but N-ethylmaleimide, which is membrane permeant, strongly inhibited the enzyme. However, mersalyl and p-chloromercuriphenylsulfonic acid were effective inhibitors when the alkylation was performed on the matrix side of mitoplasts or using detergent-solubilized enzyme. Furthermore, trypsin digestion of mitoplasts was only effective inactivating glutaminase when the proteolysis was carried out on the matrix side of the vesicles. Enzyme-linked immunosorbent assay of the soluble and membrane fractions obtained in the preparation of submitochondrial particles, revealed that most of the enzyme was solubilized, but in the inactive form. Phase separation with Triton X-114 rendered most of the protein in the aqueous phase. These results taken together discard a transmembrane localization for the protein, whereas they are consistent with anchorage of glutaminase on the matrix side of the inner mitochondrial membrane, the matrix portion of the enzyme being relevant for its function.
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PMID:Submitochondrial localization and membrane topography of Ehrlich ascitic tumour cell glutaminase. 904 41

Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed mutagenesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coli CTP synthase that were highly susceptible to proteolysis. Lys187 is located at the CTP/UTP-binding site within the synthase domain, and cleavage at this site destroyed all synthase activity. Nucleotides protected the enzyme against proteolysis at Lys187 (CTP > ATP > UTP > GTP). The K187A mutant was resistant to proteolysis at this site, could not catalyse CTP formation, and exhibited low glutaminase activity that was enhanced slightly by GTP. K187A was able to form tetramers in the presence of UTP and ATP. Arg429 and Lys432 appear to reside in an exposed loop in the glutamine amide transfer (GAT) domain. Trypsin-catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, and nucleotides did not protect these sites from cleavage. The R429A and R429A/K432A mutants exhibited reduced rates of trypsin-catalyzed proteolysis in the GAT domain and wild-type ability to catalyse NH3-dependent CTP formation. For these mutants, the values of kcat/Km and kcat for glutamine-dependent CTP formation were reduced approximately 20-fold and approximately 10-fold, respectively, relative to wild-type enzyme; however, the value of Km for glutamine was not significantly altered. Activation of the glutaminase activity of R429A by GTP was reduced 6-fold at saturating concentrations of GTP and the GTP binding affinity was reduced 10-fold. This suggests that Arg429 plays a role in both GTP-dependent activation and GTP binding.
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PMID:Limited proteolysis of Escherichia coli cytidine 5'-triphosphate synthase. Identification of residues required for CTP formation and GTP-dependent activation of glutamine hydrolysis. 1275 39

Salt-tolerant glutaminase (Micrococcus glutaminase, with an apparent molecular mass of 48.3 kDa, intact glutaminase) from the marine bacterium Micrococcus luteus K-3 was digested using protease derived from M. luteus K-3. The digestion products were a large fragment (apparent molecular mass of 38.5 kDa, the glutaminase fragment) and small fragments (apparent molecular mass of 8 kDa). The digestion was inhibited by phenylmethanesulfonyl fluoride (PMSF). Digestion of intact glutaminase by serine proteases including trypsin, elastase, lysyl endopeptidase, and arginylendopeptidase also produced the glutaminase fragment. The N-terminus of the glutaminase fragment was the same as that of intact glutaminase. The N-termini of two small fragments were Ala394 and Ala396, respectively. The enzymological and kinetic properties of the glutaminase fragment were almost the same as those of intact glutaminase except for salt-tolerant behavior. The glutaminase fragment was a higher salt-tolerant enzyme than the intact glutaminase, suggesting that Micrococcus glutaminase is digested in the C-terminal region by serine protease from M. luteus K-3 to confer salt tolerance on glutaminase.
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PMID:Digestion by serine proteases enhances salt tolerance of glutaminase in the marine bacterium Micrococcus luteus K-3. 1529 Mar 24

Cytidine 5'-triphosphate synthase (CTPS) catalyzes the ATP-dependent formation of CTP from UTP using either NH3 or L-glutamine as the source of nitrogen. To identify the location of the ATP-binding site within the primary structure of E. coli CTPS, we used the affinity label 2',3'-dialdehyde adenosine 5'-triphosphate (oATP). oATP irreversibly inactivated CTPS in a first-order, time-dependent manner while ATP protected the enzyme from inactivation. In the presence of 10 mM UTP, the values of k(inact) and K(I) were 0.054 +/- 0.001 min(-1) and 3.36 +/- 0.02 mM, respectively. CTPS was labeled using (2,8-3H)oATP and subsequently subjected to trypsin-catalyzed proteolysis. The tryptic peptides were separated using reversed-phase HPLC, and two peptides were identified using N-terminal sequencing (S(492)GDDQLVEIIEVPNH(506) and Y(298)IELPDAY(K(306)) in a 5:1 ratio). The latter suggested that Lys 306 had been modified by oATP. Replacement of Lys 306 by alanine reduced the rate of oATP-dependent inactivation (k(inact) = 0.0058 +/- 0.0005 min(-1), K(I) = 3.7 +/- 1.3 mM) and reduced the apparent affinity of CTPS for both ATP and UTP by approximately 2-fold. The efficiency of K306A-catalyzed glutamine-dependent CTP formation was also reduced 2-fold while near wild-type activity was observed when NH3 was the substrate. These findings suggest that Lys 306 is not essential for ATP binding, but does play a role in bringing about the conformational changes that mediate interactions between the ATP and UTP sites, and between the ATP-binding site and the glutamine amide transfer domain. Replacement of the nearby, fully conserved Lys 297 by alanine did not affect NH3-dependent CTP formation, relative to wild-type CTPS, but reduced k(cat) for the glutaminase activity 78-fold. Our findings suggest that the conformational change associated with binding ATP may be transmitted through the L10-alpha11 structural unit (residues 297-312) and thereby mediate effects on the glutaminase activity of CTPS.
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PMID:The role of lysine residues 297 and 306 in nucleoside triphosphate regulation of E. coli CTP synthase: inactivation by 2',3'-dialdehyde ATP and mutational analyses. 1642 16

Because consumption of whey protein hydrolysates is on the increase, the possibility that prolonged ingestion of whey protein hydrolysates affect the digestive system of mammals has prompted us to evaluate the enzymatic activities of pepsin, leucine-aminopeptidase, chymotrypsin, trypsin, and glutaminase in male Wistar rats fed diets containing either a commercial whey isolate or a whey protein hydrolysate with medium degree of hydrolysis and to compare the results with those produced by physical training (sedentary, sedentary-exhausted, trained, and trained-exhausted) in the treadmill for 4 weeks. The enzymatic activities were determined by classical procedures in all groups. No effect due to the form of the whey protein in the diet was seen in the activities of pepsin, trypsin, chymotrypsin, and leucine-aminopeptidase. Training tended to increase the activity of glutaminase, but exhaustion promoted a decrease in the trained animals, and consumption of the hydrolysate decreased it even further. The results are consistent with the conclusion that chronic consumption of a whey protein hydrolysate brings little or no modification of the proteolytic digestive system and that the lowering of glutaminase activity may be associated with an antistress effect, counteracting the effect induced by training in the rat.
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PMID:Prolonged ingestion of prehydrolyzed whey protein induces little or no change in digestive enzymes, but decreases glutaminase activity in exercising rats. 2048 82


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