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)

Trypsin-like and carboxypeptidase B-like proteinases are believed to play important roles in the conversion of proinsulin into insulin as well as in the intracellular processing of a variety of other precursor forms. To facilitate the study of these enzymes we have developed sensitive methods for their detection in tissue preparations and incubation media. Studies with rat islet homogenates indicate the presence of both trypsin-like and carboxypeptidase B-like activities with slightly acidic pH optima. The trypsin-like activity was activated by thiols and inhibited by several thiol reagents but the carboxypeptidase was inhibited only by chelating agents. These properties suggest that these enzymes are related to the tissue cathepsins. Additional experimental approaches to the problems of positively identifying and localizing converting enzymes at the subcellular level are briefly discussed.
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PMID:Carboxypeptidase B-like and trypsin-like activities in isolated rat pancreatic islets. 82 May 28

The derivative of the trypsin-kallikrein inhibitor (Kunitz), TKI+, was prepared with the reactive-site peptide bond Lys-15-Ala-16 hydrolyzed. This was achieved by selective borohydride reduction of the Cys-14-Cys-38 disulfide bond, followed by tryptic cleavage of the reactive-site peptide bond, air reoxidation of the half-cystine residues, and purification by ion-exchange chromatography. The derivative corresponds to the hypothetical 'modified' inhibitor TKI+, which so far could not be obtained from virgin inhibitor by a direct modification reaction (partial proteolysis). The derivative isolated was homogeneous as revealed by amino acid analysis, disc electrophoresis, inactivation by carboxypeptidase B, and inactivation by sodium borohydride reduction. The inhibitory activity of the sodium-borohydride-reduced inhibitor was fully recovered after air reoxidation. The site of cleavage in the inhibitor was confirmed by performic acid oxidation and subsequent isolation of the two corresponding peptides containing residues 1-15 and 16-58 of the entire polypeptide chain. From several aminopeptidases tested only aminopeptidase K rapidly cleaved Ala-16 and Arg-17 from the modified inhibitor and at a reduced rate Ile-18. Des-(Ala16,Arg17)-inhibitor and des-Ala16-inhibitor are both lacking a strong inhibitory activity against bovine trypsin. This indicates a decrease in the association constant by factor of at least 10(8)-10(10). The reactive-site-modified inhibitor is not subject to further enzymic breakdown and therefore is a permanent inhibitor of trypsin. However, the modified inhibitor forms the inactive complex much slower than virgin inhibitor. In the modified inhibitor the hydrolyzed peptide bone was resynthesized to yield virgin inhibitor by forming the complex with trypsin and subjecting the complex to kinetic control dissociation. This proves that the bond Lys-15--Ala-16 is at the reactive site of this inhibitor. Preparation of a modified and still active inhibitor (Kunitz) is in agreement with the general model proposed for the interaction of proteinase-inhibitor--proteinase interactions. This presents new evidence that this model is generally applicable.
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PMID:Preparation and characterization of the active derivative bovine trypsin-kallikrein inhibitor (Kunitz) with the reactive site lysine-15 -- alanine-16 hydrolyzed. 94 16

Carboxypeptidase B (peptidyl-L-lysine (-L-arginine) hydrolase, EC 3.4.12.3) has been isolated and purified to apparent homogeneity from activated extracts of human pancreas tissue. The purified enzyme has been shown to be a single polypeptide of 34 000 daltons. In this respect the enzyme from pancreatic tissue, designated native human carboxypeptidase B, differs from the two forms present in human pancreatic juice (fractions I and II), both of which are composed of two polypeptides of approximately 24 000 and 9000 daltons. In addition, the three forms of human carboxypeptidase B differ in electrophoretic mobility in polyacrylamide gel electrophoresis and in chromatographic behavior on DEAE-cellulose. Two immunological methods, micro-complement fixation and radioimmunoassay, have shown a high degree of structural similarity between the three forms of human carboxypeptidase B. Micro-complement fixation experiments indicate that the amino acid sequences of the three enzymes differ by less than one percent. In vitro digestion studies have indicated that trypsin alone is sufficient to convert native carboxypeptidase B to carboxypeptidase B II. However, no combination of trypsin, chymotrypsin, and/or elastase was capable of converting native carboxypeptidase B to carboxypeptidase B I in vitro.
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PMID:Human carboxypeptidase B. II. Purification of the enzyme from pancreatic tissue and comparison with the enzymes present in pancreatic secretion. 100 23

Highly purified kininogen preparation with the activity of 16-18 int. units per mg was isolated from rabbit blood serum. Its molecular weight was estimated to be 54 000 by gel filtration through Sephadex G-200. Leucine was identified as N-terminal amino acid by the dansylation method. Rabbit kininogen consists of 394 amino acid residues (except tryptophane). Amino acid composition of kininogen is characterized by a high content of dicarbonic amino acids, proline and by a low content of methionine. Kininogen molecule does not contain SH-groups. 13.1-13.5 SH-groups were found in kininogen after the reduction of S-S bonds with beta-mercaptoethanol in the presence of 8 M urea, thus indicating the presence of 6-7 S-S bonds in kininogen molecule. Kininogen group does not occupy C-terminal position in the molecule, because the treatment of the protein with carboxypeptidase B does not change the content of bradykinine in it. Purified kininogen preparation is a substrate for kallikrein from rabbit blood plasma, human saliva and trypsin. Unlike trypsin, kallikreines from human blood plasma and saliva release kinines from kininogen with reduced S-S bonds. Under spontaneous reoxidation of reduced S-S bonds up to 90%, substate properties of kininogen for tripsin recover only by 50%. Rabbit kininogen is similar to beef kininogen II in its molecular weight, amino acid composition and the number of S-S bonds.
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PMID:[Amino acid makeup, structural characteristics and substrate specificity of rabbit plasma kininogen]. 113 95

The seminal plasma and sperm of fresh and stored poultry semen were analyzed for the presence of eight peptide hydrolase enzymes. Five enzymes: carboxypeptidase A, carboxypeptidase B, chymotrypsin, glycylglycylglycine hydrolase and pepsin were not present in either plasma or sperm. An aminopeptidase-like and a cathepsin-like activity were found in seminal plasma and sperm while a trypsin-like activity was found in sperm only. There was a significant difference between full sib groups with respect to aminopeptidase-like activity in fresh and stored plasma, while storage for 24 hours resulted in a significant increase in trypsin-like activity of sperm. The aminopeptidase-like activity of fresh sperm was positively correlated with duration and percent fertility of fresh semen, while neither cathepsin-like activity nor trypsin-like activity were correlated with fertility of fresh or stored semen except for a positive correlation between the cathepsin-like activity of fresh plasma and percent fertility of fresh semen.
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PMID:The activity of some peptide hydrolase enzymes in fresh and stored poultry semen from full sib groups of males and their relationship to fertility. 118 12

We have investigated the possible role of the fetal pituitary and ACTH in the control of the synthesis and post-translational processing of the enkephalin precursor, proenkephalin A (proEnk A), in the fetal sheep adrenal gland in late gestation. Fetal hypophysectomy (n = 8) or sham operations (n = 4) were performed between 109 and 118 days of gestation. At 138-139 days, either ACTH(1-24) (10.5 micrograms/0.24 ml saline per h, n = 4) was infused intravenously for 72 h into hypophysectomized fetal sheep or 0.9% (w/v) NaCl alone (0.24 ml/h, n = 4) was infused for 72 h into hypophysectomized fetal sheep and sham-operated animals. At the end of the infusion the pregnant ewe was killed and left or right adrenal glands (n = 12) were collected from the fetal sheep that were intact and given saline (Intact + sal; n = 4), hypophysectomized and given saline (Hx + sal; n = 4) and hypophysectomized and given ACTH (Hx + ACTH; n = 4). Each adrenal was homogenized in acid (acetic acid (1 mol/l)/HCl (20 mmol/l)/2-mercaptoethanol (0.2%)). After centrifugation, the supernatant was loaded onto a Sephadex G-75 column (2.0 x 50 cm), eluted at 80 ml/24 h and fractions were collected (5 ml, n = 42). An aliquot of each fraction (2 ml) was dried down prior to enzymatic digestion (trypsin/carboxypeptidase B) and oxidation with H2O2, and assay for methionine-O-enkephalin (immunoreactive Met-O-Enk).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effect of fetal hypophysectomy with or without ACTH replacement on the molecular weight profile of enkephalin-containing peptides in the adrenal medulla of the fetal sheep. 132 54

Astrocytes were prepared from rats of 4 ages, embryonic day 20, postnatal days 3 and 8, and adult, in order to study the developmental time course of expression of enkephalin and somatostatin (SS). Glial fibrillary acidic protein (GFAP) content was constant in both cortical and cerebellar astrocytes prepared from all ages. SS mRNA and peptide decreased over this developmental time course in cerebellar astrocytes; the time course of changes in SS mRNA paralleled that for rat cerebellum. Proenkephalin (PE) mRNA increased about 3-fold in cerebellar astrocytes from embryonic day 20 to adult but remained constant in cortical astrocytes; in contrast, PE mRNA showed a 10- to 12-fold increase in rat cerebellum and cortex developmentally. For both cerebellar and cortical astrocytes, free met-enkephalin decreased from embryonic day 20 to adult, whereas total met-enkephalin (measured following trypsin-carboxypeptidase B digestion of the extracts) increased. These results suggest (1) that there is a developmental regulation of the expression of both enkephalin and SS peptides in astrocytes, and (2) that the regulation occurs at the level of transcription for SS but at the level of precursor processing for PE. Possible trophic functions for astrocyte-derived peptides early in CNS development are discussed.
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PMID:Developmental expression of the proenkephalin and prosomatostatin genes in cultured cortical and cerebellar astrocytes. 135 15

The effect of dietary protein on enzyme activity of pancreatic juice was studied in ten growing, castrated, Large White male pigs. Animals, fitted with permanent cannulas in the pancreatic duct and in the duodenum, were divided into two groups receiving either casein or rapeseed concentrate as a protein source. After a 15 d adaptation period to the experimental diet, the volume of pancreatic secretion was significantly higher, whereas the protein concentration was lower in the casein group compared with the rapeseed group. No statistical difference was observed in the daily protein output between groups. Total secreted activities of carboxypeptidase A (EC 3.4.17.1), and elastase (EC 3.4.21.36) were higher in the casein group during the nocturnal period, whereas total activities of trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), carboxypeptidase B (EC 3.4.17.2) and amylase (EC 3.2.1.1) in pancreatic secretions during the post-prandial periods were increased by the ingestion of the rapeseed diet. It is concluded that the pancreatic enzyme secretion is sensitive to the nature of the protein ingested.
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PMID:Effects of diets containing casein and rapeseed on enzyme secretion from the exocrine pancreas in the pig. 137 39

Recombinant human nerve growth factor (rhNGF) was expressed and secreted by Chinese hamster ovary cells and purified to homogeneity using ion-exchange and reversed-phase (RP) chromatography. The isolated product was shown to be consistent with a 120-amino-acid residue polypeptide chain by amino acid composition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), RP-HPLC, and mass spectrometry and with an N-terminal sequence consistent with that expected from the cDNA for human nerve growth factor. By size-exclusion chromatography, rhNGF behaves like a noncovalent dimer. Limited enzymatic digests of the 120-residue monomer produced additional species of 118 (trypsin, removal of the C-terminal Arg119-Ala120 sequence) and 117 (trypsin plus carboxypeptidase B, removal of the C-terminal Arg118-Arg119-Ala120 sequence) residues. Each of these species was isolated by high-performance ion-exchange chromatography and characterized by amino acid and N-terminal sequence analyses, SDS-PAGE, RP-HPLC, and mass spectrometry. All three species were present in the digests as both homodimeric and heterodimeric combinations and found to be equipotent in both the chick dorsal root ganglion cell survival and rat pheochromocytoma neurite extension assays.
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PMID:Biochemical characterization of recombinant human nerve growth factor. 140 13

E. coli DH 5 alpha cells harboring a plasmid pWR 590-BCA 4 for fused human proinsulin production were cultured. The fused human proinsulin was isolated from the fermented cells and then subjected it to cleavage with BrCN. The cleaved product was then converted to crude proinsulin-S-sulfonate using oxidative sulfitolysis. The isolation of human proinsulin-S-sulfonate was accomplished by ion exchange chromatography on QAE-sephadex A-25, followed by gel filtration on sephadex G-50. The purified human proinsulin-S-sulfonate was folded using a disulfide interchange method. The folding mixture was then chromatographed on sephadex G-50 and purified proinsulin was obtained. The proinsulin was then converted to human insulin and C-peptide by a combination cleavage with trypsin and carboxypeptidase B. The total yield of human insulin was about 5 mg/L The Zinc insulin crystals were obtained with amorphous human insulin using citrate method. The amino acid composition N-terminal sequences as well as C-terminal amino acid residues are in agreement with expected results. The hypoglycemic activity of purified human insulin is 26-27 U/mg, as judged by mouse convulsion assay, and the RIA activity is about 99% of that of porcine insulin.
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PMID:[Studies on genetic engineering of human insulin-purification and characterization of human proinsulin and insulin]. 141 26


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