Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.11 (CD10)
 9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sterol regulatory element binding proteins (SREBP-1 and SREBP-2) are the key transcription factors for the regulation of the cellular cholesterol level. To identify proteolytic enzymes for SREBPs, a fluorogenic peptide substrate, MOCAc-GRSVLSFK(Dnp)rr-NH2, was synthesized according to the proposed cleavage site of human SREBP-2. In microsome fractions from hamster liver, we found a peptidase activity inhibitable by the synthetic inhibitor Ac-GRSVL-aldehyde with an IC50 of 40 nM. This peptidase separated into three peaks of approximately 400 kDa, 60 kDa, and 30 kDa (Mp400, Mp60 and Mp30 respectively) upon gel permeation chromatography. Mp30 was purified to apparent homogeneity with an Mr of 32 kDa. The partial amino acid sequence of Mp30 possessed homology to cathepsin B (EC 3.4.22.1). A 109 kDa protein band on SDS-PAGE which corresponded to Mp400 exhibited homology to neprilysin (EC 3.4.24.11) in partial amino acid sequence. These findings suggest several degradative pathways for SREBP in liver microsome membranes.
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PMID:Characterization of cleavage enzymes for sterol regulatory element binding protein in hamster liver microsomes. 1032 22

We report a rare case of a half molecule 7S IgM (HM 7SIgM) consisting of a unique mu heavy chain and kappa light chain found in blood and urine samples from a patient with primary Waldenstrom macroglobulinemia. A 64kDa abnormal immunoglobulin was detected in serum and urine by immunoblot method, and purified by a two-dimensional SDS-PAGE after separation from IgG and albumin fractions on gel filtration. NH2-terminal amino acid sequence analysis of the heavy chain revealed that residues 1-20 were identical to those of the NH2-terminal region of kappa light chain derived from the same patient. This sequence was then followed by a sequence that could not be identified by a computer homology search on the protein database. Using polypeptide segments obtained from the unique mu chain by digestion with endopeptidase, we identified a sequence spanning from residue 127 in the variable region of the known mu chain to residue 19 in the known CH1 domain and a sequence spanning from residues 67-82 in the heavy chain variable region class II. From these results, we concluded that the 64 kDa protein was an abnormal half molecule 7S IgM consisting of a kappa light chain and a unique mu heavy chain of 35 kDa polypeptide in which the NH2-terminal 20 amino acids were replaced by 20 amino acids derived from the sequence of kappa light chain in the NH2-terminal region.
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PMID:Occurrence of heavy chain of 7S IgM half-molecule whose NH2-terminal sequence is identical with that of kappa light chain sequence in patients with Waldenstrom macroglobulinemia. 1034 Apr 36

Carboxyl group modification with DCCD and NCD-4 was employed to investigate the chemical environment of the side chains of archaeopsin-1 (aO-1) and bacterioopsin (bO). Some differences were observed between aO-1 and bO. Although DCCD or NCD-4 did not modify aO-1 in bleached membrane, they modified bO in bleached membrane and in mixed DMPC/CHAPS/SDS micelles at neutral pH, thereby affecting the opsin shift and the photocycle of the regenerated chromophore. On the contrary, after solubilization with SDS, aO-1 and bO were modified by DCCD and NCD-4, which decreased the chromophore regeneration. In particular, the reaction of aO-1 in SDS with NCD-4 proceeded in a 1:1 ratio at neutral pH. The fluorescence and CD spectra indicated that the modified site was located in the hydrophobic, asymmetrical region. Lysyl-endopeptidase digestion of NCD-4 modified aO-1 produced a fluorescent fragment and amino acid sequence analysis showed that Asp85 or Asp96 in helix C is a probable candidate for the modified residue at present. Kinetic CD measurements revealed that the introduction of N-acylurea at an Asp residue in helix C did not affect the formation of the transient intermediate but inhibited the side chain packing during refolding.
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PMID:The effect of carboxyl group modification on the chromophore regeneration of archaeopsin-1 and bacterioopsin. 1034 18

Dipeptidylpeptidase IV (DPP IV, CD26), a serine-type exo- and endopeptidase found in the cell surface membrane of many tissues, was employed as a model membrane glycoprotein to study the expression of sialoforms on cell surface glycoproteins. Native, enzymatically active DPP IV was purified from plasma membranes of kidney and liver by lectin affinity chromatography in conjunction with crown ether anion exchange chromatography. The enzyme was gradient-eluted in continuous fractions, all showing a single polypeptide band of about 100 kDa when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing, denaturing conditions. Analysis of the purified DPP IV by isoelectric focusing (IEF) showed that it consists of several polypeptides of different isoelectric points (IP) ranging from 5.5 to 7.0. In vitro- desialylation of the enzyme and subsequent isoelectric focusing revealed that the differences in isoelectric points were due to differences in the degree of sialylation. Differences in the degree of sialylation between the fractions were also demonstrated by SDS-PAGE under nonreducing and nondenaturing conditions. Increased sialylation of the enzyme as demonstrated by isoelectric focusing resulted in increased migration velocity in nonreducing and nondenaturing SDS-polyacrylamide gels. In vitro -desialylation of the enzyme and its resialylation confirmed that sialylation was responsible for this extraordinary migration behavior. The native enzyme was predominantly sialylated via alpha 2, 6-linkage, as shown by lectin affinity blotting employing Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA). These findings demonstrate that a distinct membrane glycoprotein may exist in various sialoforms, distinguished from each other by a different number of sialic acid residues. Moreover, these sialoforms can be individually purified by crown ether anion exchange chromatography.
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PMID:Sialoforms of dipeptidylpeptidase IV from rat kidney and liver. 1056 54

Insulin gene therapy requires that insulin secretion be coupled to metabolic requirements. To this end, we have developed an insulin transgene whose transcription is stimulated by glucose and inhibited by insulin. Glucose- and insulin-sensitive promoters were constructed by inserting glucose-responsive elements (GlREs) from the rat L-pyruvate kinase (L-PK) gene into the insulin-sensitive, liver-specific, rat insulin-like growth factor binding protein-1 (IGFBP-1) promoter. Glucose (5 to 25 mM) stimulated, and insulin (10-10 to 10-7 M) inhibited, luciferase expression driven by these promoters in primary cultured rat hepatocytes. The capacity of transfected hepatocytes to secrete mature, biologically active insulin was demonstrated using a human proinsulin cDNA (2xfur), modified to allow protein processing by endogenous endopeptidase activity. Medium conditioned by insulin-producing hepatocytes contained greater than 300 microU/ml immunoreactive insulin, while denaturing SDS-PAGE of an anti-insulin immunoprecipitate revealed bands with the mobilities of insulin A, and B chains. Biological activity of hepatocyte-produced insulin was demonstrated in a transfection assay, in which medium conditioned by insulin-producing hepatocytes exerted an effect similar to 10-7 M insulin. We then combined the glucose- and insulin-sensitive promoter with the modified human proinsulin cDNA to create a metabolically sensitive insulin transgene ((GlRE)3BP-1 2xfur). In both H4IIE hepatoma cells stably transfected with this construct, and normal rat hepatocytes (GlRE)3BP-1 2xfur-mediated insulin secretion increased in response to stimulation by glucose. Moreover, a capacity to decrease insulin production in response to diminishing glucose exposure was also demonstrated. We conclude that the transcriptional regulation of insulin production using these glucose- and insulin-sensitive constructs meets the requirements for application in a rodent model of insulin gene therapy. Gene Therapy (2000) 7, 205-214.
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PMID:Glucose regulated production of human insulin in rat hepatocytes. 1069 97

Three chromatographically distinct forms of a novel fibrinogen-clotting serine endopeptidase, TL-BJ1, 2 and 3, were purified from the venom of Bothrops jararaca by a combination of ammonium sulfate precipitation and chromatographic steps. The three forms of TL-BJ have similar amidolytic and plasma coagulating activities. TL-BJ 1, TL-BJ 2 and TL-BJ 3 cause the specific clotting of fibrinogen with release of fibrinopeptide A, the specific activities are 16.8 NIH U/mg (TL-BJ 1), 16.7 NIH U/mg (TL-BJ 2) and 20.8 NIH U/mg (TL-BJ 3). The most sensitive chromogenic substrates for measuring the amidolytic activity of TL-BJ 3 were D-Pro-Phe-Arg-pNA, D-Phe-pipecolyl-Arg-pNA and Z-D-Arg-Gly-Arg-pNA. The amidolytic and coagulant activities of TL-BJ were inhibited by phenylmethylsulfonyl fluoride but not by hirudin. Benzamidine derivatives, which are competitive inhibitors of trypsin-like serine endopeptidases, also inhibited the amidolytic activity of TL-BJ. In SDS/PAGE the main bands of TL-BJ 1, 2 and 3 showed molecular masses of 30 kDa, 31 kDa and 32 kDa. Upon incubation with N-glycosidase F only TL-BJ 3 remained unchanged, whereas TL-BJ 1 and TL-BJ 2 showed products with molecular masses around 23 kDa. Thus, TL-BJ 3 does not seem to be N-glycosylated. The N-terminal amino acid sequences of TL-BJ 2 and TL-BJ 3 are identical while TL-BJ 1 has five substitutions.
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PMID:A novel fibrinogen-clotting enzyme, TL-BJ, from the venom of the snake Bothrops jararaca: purification and characterization. 1074 51

Insulin-degrading enzyme (IDE) has been shown to degrade a number of biologically important peptides, including insulin and the amyloid-beta protein implicated in Alzheimer's disease. However, lack of a facile method to generate purified enzyme and related mutants has made it difficult to study the precise role of IDE in the clearance of these peptides. Therefore, we determined whether recombinant wild-type and mutant human IDEs can be overexpressed as functional enzymes in bacteria. Three vectors carrying cDNAs encoding N-terminally polyhistidine-tagged recombinant IDEs were constructed, and the proteins expressed in Escherichia coli were purified by metal affinity chromatography (final yield approximately 8 mg per liter of culture). The recombinant IDEs, like the endogenous mammalian enzyme, migrate with 110-kDa apparent molecular masses in SDS-polyacrylamide gels and as a approximately 200-kDa species in gel filtration. Further analysis by native PAGE indicates that IDE can form multimers of different complexities. The wild-type recombinant endopeptidase degrades insulin with an efficiency similar to that of the enzyme purified from mammalian tissues. Purified IDEs are stable at 4 degrees C for at least 1 month. Purified recombinant protein was used to raise specific polyclonal antibodies that can immunoprecipitate native mammalian IDE. Thus, the procedure described allows the rapid production of large amounts of purified IDE and demonstrates that IDE can be produced in an active form in the absence of other potential interacting mammalian proteins.
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PMID:Functional human insulin-degrading enzyme can be expressed in bacteria. 1083 95

An endopeptidase has been purified from sprouts of bamboo (Pleioblastus hindsii Nakai) to electrophoretic homogeneity by four purification steps. Its Mr was estimated to be 82 kDa by SDS-PAGE. Enzyme activity was inhibited strongly by diisopropyl fluorophosphate, and weakly by p-chloromercuriphenylsulfonic acid, but not at all by EDTA or pepstatin, indicating that it was a serine protease. The preferential cleavage sites for this protease were found to be large hydrophobic and amide residues at the P1 position. The specificity of the bamboo serine protease differed from that of cucumisin [EC 3.4.21.25], which cleaved the charged amino acid residues at the P1 position.
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PMID:Isolation and characterization of a serine protease from the sprouts of Pleioblastus hindsii Nakai. 1096 47

Cultured fibroblasts secrete an 88-kDa serine protease that cleaves insulin-like growth factor binding protein-5 (IGFBP-5). Because IGFBP-5 has been shown to regulate IGF-I actions, understanding the chemical identity and regulation of this protease is important for understanding how IGF-I stimulates anabolic functions. The protease was purified from human fibroblast-conditioned medium by hydrophobic interaction, lectin affinity, and heparin Sepharose affinity chromatography followed by SDS-polyacrylamide gel electrophoresis. An 88-kDa band was excised and digested with lysyl-endopeptidase. Sequencing of the high pressure liquid chromatography-purified peptides yielded the complement components C1r and C1s. To confirm that C1r/C1s accounted for the proteolytic activity in the medium, immunoaffinity chromatography was performed. Most of the protease activity adhered to the column, and the eluant was fully active in cleaving IGFBP-5. SDS-polyacrylamide gel electrophoresis with silver staining showed two bands, and IGFBP-5 zymography showed a single 88-kDa band. Amino acid sequencing confirmed that the 88-kDa band contained only C1r and C1s. C1r in the fibroblast medium underwent autoactivation, and the activated form cleaved C1s. C1s purified from the conditioned medium cleaved C(4), a naturally occurring substrate. The purified protease cleaved IGFBP-5 but had no activity against IGFBP-1 through -4. C1 inhibitor, a protein known to inhibit activated C1s, was shown to inhibit the cleavage of IGFBP-5 by the protease in the conditioned medium. In summary, human fibroblasts secrete C1r and C1s that actively cleave IGFBP-5. The findings define a mechanism for cleaving IGFBP-5 in the culture medium, thus allowing release of IGF-I to cell surface receptors.
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PMID:The complement component C1s is the protease that accounts for cleavage of insulin-like growth factor-binding protein-5 in fibroblast medium. 1098 4

A new plant endopeptidase was obtained from unripe fruits of Bromelia balansae Mez (Bromeliaceae). Crude extracts were partially purified by ethanol fractionation. This preparation (redissolved ethanol precipitate, REP) showed maximum activity at pH 8.8-9.2, was very stable even at high ionic strength values (no appreciable decrease in proteolytic activity could be detected after 24 h in 1 M sodium chloride solution at 37 degrees C), and exhibited high thermal stability (inactivation required heating for 60 min at 75 degrees C). Anion exchange chromatography allowed the isolation of a fraction purified to mass spectroscopy, SDS-PAGE, and IEF homogeneity, named balansain I, with pI = 5.45 and molecular mass = 23192 (mass spectrometry). The purification factor is low (2.9-fold), but the yield is high (48.3%), a common occurrence in plant organs with high proteolytic activity, where proteases represent the bulk of protein content of crude extracts. Balansain I exhibits a similar but narrower pH profile than that obtained for REP, with a maximum pH value approximately 9.0 and was inhibited by E-64 and other cysteine peptidases inhibitors but not affected by inhibitors of the other catalytic types of peptidases. The alanine and glutamine derivatives of N-alpha-carbobenzoxy-L-amino acid p-nitrophenyl esters was strongly preferred by the enzyme. The N-terminal sequence of balansain I showed a very high homology (85-90%) with other known Bromeliaceae endopeptidases.
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PMID:Purification of balansain I, an endopeptidase from unripe fruits of Bromelia balansae Mez (Bromeliaceae). 1099 73


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