Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.9 (enterokinase)
 675 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Twenty strains of Staphylococcus aureus from ATCC type cultures and strains found in clinical studies were cultivated, and their endopeptidase activity specific for glutamic acid was surveyed using benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. The activity was found in two of the strains, ATCC 12600 and ATCC 25923. A glutamic acid-specific proteinase, which we propose to call SPase, was purified from the culture filtrate of S. aureus strain ATCC 12600 by a series of column chromatographies on DEAE-Sepharose twice and on Sephacryl S-200. A single band was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified SPase. The molecular weight of the proteinase was estimated to be 34000 by SDS-PAGE. When synthetic peptides and oxidized insulin B-chain were used as substrates, SPase showed the same substrate specificity as V8 proteinase, EC 3.4.21.9, which specifically cleaves peptide bonds on the C-terminal side of glutamic acid and aspartic acid. Examination with p-nitroanilides of glutamic acid and aspartic acid as substrates, however, revealed that both proteinases are highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure of SPase, its gene was cloned from genomic DNA of S. aureus ATCC 12600, and the nucleotide sequence was determined. Taking the amino acid sequence of SPase from the NH2-terminus to the 27th residue into consideration, the clones encode a mature peptide of 289 amino acids, which follows a prepropeptide of 68 residues. SPase was confirmed to be a novel endopeptidase specific for glutamic acid, being different from V8 proteinase which consists of 268 amino acids.
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PMID:Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600. 159 45

The postnatal development of enteropeptidase activity has been examined on mucosal scrapping of the proximal part of the mouse small intestine. The activity was present at birth and remained low during the first 15 days of life. Then it rapidly increased reaching adult level within 2 days. Daily administration of cortisone acetate (25 micrograms X g body weight (bw)-1 X day-1), insulin (12.5 mU X g bw-1 X day-1), or epidermal growth factor (4 micrograms X g bw-1 X day-1) during 3 days to 8-day-old mice induced a premature increase of enteropeptidase. The maximal increase was observed with cortisone treatment, the enzymic activity representing 70% of the adult level. Thyroxine alone (1 microgram X g bw-1 X day-1) had no significant effect on enteropeptidase activity. Hormonal interactions have been evaluated by studying the effects of different hormonal combinations. Finally, cortisone acetate which has a major effect on this activity during suckling period was unable to influence adult small intestinal enteropeptidase activity.
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PMID:Development of enteropeptidase activity in mouse small intestine: influence of hormones. 389 38

Fasting reduced small intestinal length. It also decreased mucosal weight, DNA and protein content, and concentrations of enterokinase, maltase, and sucrase in both duodenal and jejunal segments. In contrast, the concentrations of lactase and leucine aminopeptidase were not affected. Concomitantly, serum insulin levels dropped to one-fifth of the control levels while serum glucose concentrations showed a lesser degree of reduction. Glucose supplementation alone raised the serum insulin level, prevented the decrease in DNA content, and showed a protective effect on mucosal protein, mucosal weight, mucosal thickness, and villus height. Glucose also protected the sucrase and maltase concentrations; more significantly for maltase in the jejunal segment. Insulin alone, although it increased the serum insulin level to that found with glucose supplementation alone, had no protective effect on the loss in protein, DNA, and most enzymes except for maltase concentration in the jejunal segment. Addition of insulin to glucose did not modify the glucose effect on the contents of DNA, protein, and concentrations of sucrase and maltase. These results suggest that the glucose effect on the mucosa is not mediated by insulin. In addition, the retention of both maltase and sucrase activities through only glucose supplementation suggests the loss of maltase and sucrase in fasting is due to nutrient rather than specific substrate restriction.
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PMID:Effect of glucose and insulin on small intestinal brush border enzymes in fasted rats. 640 48

Glucose-dependent insulinotropic polypeptide or gastric inhibitory polypeptide (GIP) is a 42 amino acid intestinal hormone, which exhibits several direct and indirect effects on fat and glucose metabolism. To determine the bioactive region(s) of the molecule, synthetic and proteolytic fragments of the hormone were generated and tested for their ability to induce a biological response in the isolated, perfused rat pancreas and stomach. A synthetic fragment corresponding to porcine GIP residues 1-30 retained strong insulinotropic activity in the isolated, perfused rat pancreas but greatly reduced somatostatinotropic activity in the isolated perfused rat stomach. A synthetic fragment corresponding to porcine GIP residues 15 to 30 was biologically inactive. However, enterokinase treatment of the synthetic 15-30 fragment restored partial insulinotropic activity in the isolated, perfused rat pancreas. The hypothesis that the restoration of biological activity was due to the enzymatic removal of the amino-terminal dipeptide (Asp-Lys) of GIP15-30 was supported by the observation that a synthetic fragment lacking these two residues was also insulinotropic. Further fractionation of the molecule generated a biologically active 19-30 fragment, suggesting that the residues necessary for the insulin response are contained within this region.
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PMID:The insulinotropic region of gastric inhibitory polypeptide; fragment analysis suggests the bioactive site lies between residues 19 and 30. 896 53

Recently, we cloned a fragment of the endogenous murine leukemia viral envelope gene from beta cell line (MIN6N8a) as a new autoantigen gene, whose product was reactive with nonobese diabetic (NOD) mice sera. As a result of determination of nucleotide sequence, this envelope protein had the CKS-17 peptide sequence having immunosuppressive activity. To investigate the role of our cloned transmembrane envelope protein in the pathogenesis of autoimmune insulin-dependent diabetes mellitus (IDDM) as an autoantigen or immunosuppressive modulator, a high amount of transmembrane envelope protein was essentially required. Therefore, the expression of our cloned retroviral transmembrane envelope gene was tried in Escherichia coli by a pET vector. However, the expression rate was very low (less than 2%) and most of the expressed protein was insoluble. To improve solubility, the hydrophobic transmembrane anchor domain of our envelope gene was deleted and then the expression of the hydrophilic transmembrane envelope protein was carried out by using the same pET expression system. The expressed protein was completely soluble and the expression yield was dramatically improved by around 25-fold increase. The hydrophilic transmembrane envelope protein was purified by one-step Ni-NTA affinity chromatography and then the fusion tag consisting of the six-histidine peptide and S peptide was removed by cleavage with enterokinase. The processed hydrophilic retroviral transmembrane envelope protein was still immune reactive with NOD sera and also showed immunosuppressive activity by down-regulating the Th1-type cytokine (interferon-gamma) and up-regulating the Th2-type cytokine (interleukin 10).
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PMID:Soluble expression in Escherichia coli of murine endogenous retroviral transmembrane envelope protein having immunosuppressive activity. 1033 56

Young castrated male goats (n = 8) were used to investigate the effect of long-term treatment with recombinant methionyl bovine somatotropin in a sustained release vehicle (bST; 100 mg at seven-day intervals in a 147-day experiment) and chronic culture (24 h) of omental adipose tissue in the presence of various hormones on lipogenic responses to catecholamines during acute incubation (2 h) in a sodium acetate supplemented glucose-free buffer. The rate of fatty acid synthesis in freshly-prepared adipose explants was low and did not differ from those cultured in the absence of hormones for 24 h. Hormonal combination of insulin (17 nmol.l(-1)) plus cortisol (138 nmol.l(-1)) or insulin plus recombinant enterokinase linker bST (4.5 nmol.l(-1) increased lipogenesis (P<0.05). Further addition of bST or cortisol decreased lipogenesis significantly (P<0.05) in the controls but not significantly in bST-treated animals. Cultured explants from either control or bST-treated animals showed significant inhibition of lipogenesis by both norepinephrine (10 micromol.l(-1)) and isoprenaline (10 micromol.l(-1)). BST treatment in vivo did not increase the responsiveness of cultured explants to norepinephrine in vitro, however, the responsiveness to isoprenaline(inhibition of lipogenesis) was greater in bST-treated animals than in the controls.
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PMID:Chronic effects of somatotropin treatment in vivo and in vitro on lipogenic activity of goat adipose tissue in a glucose-free buffer during acute incubation. 1047 Aug 65

IA-2 (insulinoma-associated protein 2) is a protein-tyrosine phosphatase receptor located in secretory granules of neuroendocrine cells. Initially, it attracted attention due to its involvement in the autoimmune response associated to diabetes. Later it was found that upon exocytosis, the cytoplasmic domain of IA-2 is cleaved and relocated to the nucleus, where it enhances the transcription of the insulin gene. A concerted functioning of the whole receptor is to be expected. However, very little is known about the structure and function of the transmembrane and extracellular domains of IA-2. To address this issue, we solved the x-ray structure of the mature ectodomain of IA-2 (meIA-2) to 1.30A resolution. The fold of meIA-2 is related to the SEA (sea urchin sperm protein, enterokinase, agrin)) domains of mucins, suggesting its participation in adhesive contacts to the extracellular matrix and providing clues on how this kind of molecule may associate and form homo- and heterodimers. Moreover, we discovered that meIA-2 is self-proteolyzed in vitro by reactive oxygen species, suggesting the possibility of a new shedding mechanism that might be significant in normal function or pathological processes. Knowledge of meIA-2 structure should facilitate the search of its possible ligands and molecular interactions.
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PMID:Structure of the mature ectodomain of the human receptor-type protein-tyrosine phosphatase IA-2. 1804 54

Exendin-4, a peptide analogue of glucagon-like peptide-1 (GLP-1), has been developed for treatment of type 2 diabetes. Herein, the secretive exendin-4 fusion protein, expressed by methanol induction in Pichia pastoris system, was purified to homogeneity by chromatography followed by enterokinase cleavage of the fusion protein and subsequent purification of the recombinant exendin-4. Purity of the recombinant exendin-4 was 95.6%. Bioactivity assay revealed that it had glucose-lowering and insulin-releasing action in vivo.
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PMID:Purification and bioactivity of exendin-4, a peptide analogue of GLP-1, expressed in Pichia pastoris. 1806 Mar 65

With the continuous improvement of the living standards of human society, the number of diabetics worldwide is growing rapidly. To date, the main effective therapy for diabetic is intravenous injection of insulin, which is accompanied by a lot of shortage such as high cost and side effects. To obtain long-term bioactive anti-diabetic drug for oral administration, we used human proinsulin (hpi) as a foreign gene to construct a recombinant plasmid pJS700-HPI with an enterokinase site Asp-Asp-Asp-Asp-Lys suitable for digestion. Plasmid pJS700-HPI was transformed into Bacillus subtilis by double cross-over and an amylase inactivated mutant was produced. After induction of spores formation, western blot was used to monitor HPI surface expression on spores. Oral administration to the silkworms with spores implied that the HPI protein displayed on recombinant spores may be digested and absorbed into the silkworm's hemolymph due to the resistant characters of spores and the addition of enterokinase site.
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PMID:Display of human proinsulin on the Bacillus subtilis spore surface for oral administration. 2338 Aug 2

Insulin-like growth factor (IGF)-I is a growth promoting hormone that exerts its actions through endocrine, paracrine and autocrine modes. Local IGF-I is essential for normal growth, whereas circulating IGF-I plays a crucial role in regulating the production and secretion of growth hormone (GH) by the pituitary gland. These actions of IGF-I are modulated by six insulin-like growth factor binding proteins (IGFBPs). In teleosts, two subtypes of each IGFBP are present due to an extra round of whole-genome duplication. IGFBP-1 is generally inhibitory to IGF-I action under catabolic conditions such as fasting and stress. In salmon, IGFBP-1a and -1b are two of three major circulating IGFBPs and assumed to affect growth through modulating IGF-I action. However, exact functions of salmon IGFBP-1 subtypes on growth regulation are not known due to the lack of purified or recombinant protein. We expressed recombinant salmon (rs) IGFBP-1a and -1b with a fusion protein (thioredoxin, Trx) and a His-tag using the pET-32a(+) vector expression system in Escherichia coli. Trx.His.rsIGFBP-1s were isolated by Ni-affinity chromatography, enzymatically cleaved by enterokinase to remove the fusion partners and further purified by reversed-phase HPLC. We next examined effects of rsIGFBP-1a and -1b in combination with human IGF-I on GH release from cultured masu salmon (Oncorhynchus masou) pituitary cells. Unexpectedly, IGF-I increased GH release and an addition of rsIGFBP-1a, but not rsIGFBP-1b, restored GH levels. The results suggest that IGFBP-1a can inhibit IGF-I action on the pituitary in masu salmon. Availability of recombinant salmon IGFBP-1s should facilitate further functional analyses and assay development.
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PMID:Production of recombinant salmon insulin-like growth factor binding protein-1 subtypes. 2866 56


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