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)

UDP-N-acetylglucosamine: beta-D-mannoside beta-1,4N-acetylglucosaminyltransferase III (GnT-III: EC 2.4.1.144) catalyzes the addition of N-acetylglucosamine in beta 1-4 linkage to the beta-linked mannose of the trimannosyl core of N-linked sugar chains. The enzyme has been purified over 153,000-fold in 1.5% yield from a Triton X-100 extract of rat kidney by fractionation procedures utilizing QAE-Sepharose, Cu(2+)-chelating Sepharose, and affinity chromatography on UDP-hexanolamine and substrate-conjugated Sepharose. The purified protein migrates as one major and one minor band with apparent molecular masses of 62 kDa and 52 kDa, respectively. The purified enzyme was digested with trypsin, and the amino acid sequences of four peptides were determined. Oligonucleotide primers were designed according to those amino acid sequences and used in the polymerase chain reaction. Screening for the cDNA for GnT-III was carried out by plaque hybridization using a rat kidney cDNA library (lambda gt10) and a polymerase chain reaction product as the probe. Rat kidney GnT-III has 536 amino acids and three putative N-glycosylation sites. There is no sequence homology to other previously cloned glycosyltransferases, but the enzyme appears to be a type II transmembrane protein like the other glycosyltransferases. The GnT-III activity in transiently transfected COS-1 cells was found to be about 500-3600-fold as compared to that in non- or mock-transfected cells.
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PMID:Purification, cDNA cloning, and expression of UDP-N-acetylglucosamine: beta-D-mannoside beta-1,4N-acetylglucosaminyltransferase III from rat kidney. 132 61

A serum-dependent and two serum-independent variants of the Bowes melanoma cell line, RPMI7272, were transfected with plasmids containing a geneticin-resistance (neo) gene transcribed by the HSV thymidine kinase promoter and an SV40 T antigen gene under control of the mouse metallothionein I promoter. T-antigen increased the cloning efficiency of the serum-dependent cell line in soft-agar more than 50-fold, but cloning efficiency of serum-independent lines was not increased. Trypsinization of serum-independent lines required 100 times lower concentrations of trypsin than serum-dependent cells. Human metal-inducible T-antigen-producing (HMT) melanoma cells supported replication of transfected plasmids containing an SV40 origin of replication. Transient expression of interferon or plasminogen activator from such plasmids was 40-fold higher than in untransformed melanoma cells and could be enhanced 30-fold more by stimulation of transcription of the T antigen gene with cadmium chloride. HMT cells can be grown in suspension and thus may represent an attractive alternative to monkey kidney COS cells.
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PMID:Transformation of Bowes melanoma cells with SV40 T antigen. 136 20

The existence of rat 18-kilodalton (kDa) prorelaxin, which has been postulated from the coding sequence of cloned cDNA and the results of cell-free translation studies, has been directly demonstrated in rat ovaries with antibodies against bacterially expressed rat prorelaxin. The peptide expressed in E. coli from a rat prorelaxin cDNA construct was comprised of the B- and A-chains of relaxin and a 105-amino acid connecting region. Immunoreactive bands of 18 and 16.5 kDa were shown in ovaries from day 20 pregnant rats. Partial amino acid sequence analysis of both peptides revealed that they had identical N-terminal sequences, corresponding to rat prorelaxin. Both 18- and 16.5-kDa bands were present only from midpregnancy until near term, when they declined sharply. These changes in the concentration of 18-kDa prorelaxin match changes in preprorelaxin mRNA levels, suggesting that relaxin synthesis is regulated at the transcriptional level and not by protein processing. Prorelaxin was transiently secreted by COS-1 cells transfected with preprorelaxin cDNA. Treatment of culture medium with trypsin resulted in the appearance of material corresponding in size to mature relaxin. Thus, correctly folded prorelaxin appears to be a suitable precursor for relaxin. The combined concentrations of 18- and 16.5-kDa peptides in ovaries on day 20 of pregnancy were considerably more than 30 times greater than that of relaxin, however, suggesting that prorelaxin might also be more than a precursor per se.
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PMID:Demonstration of relaxin precursors in pregnant rat ovaries with antisera against bacterially expressed rat prorelaxin. 154 14

Cyclosporin A (CsA) is an immunosuppressive agent that inhibits the synthesis of lymphokines by T lymphocytes at the level of transcription. A cytoplasmic protein, cyclophilin, is the most thoroughly studied CsA-binding protein, but its ubiquitous presence in cells of all types raises questions about its role in immunosuppression. In an attempt to ascertain the presence of a cell surface receptor, we synthesized two polyvalent macromolecular CsA derivatives, CsA-BBa-ovalbumin and CsA-BBa-aminodextran (CBD), from the product of the photochemical reaction of CsA and 4-benzoylbenzoic acid (CsA-BBa). (i) They inhibited the peptidylprolyl cis-trans isomerase activity of cyclophilin and the synthesis of interleukin 2 by phorbol ester-activated EL-4 cells. (ii) CBD also inhibited interleukin 2 secretion by Con A-activated T-cell-enriched mouse splenocytes. 4-Benzoylbenzoic acid (BBa)-aminodextran and aminodextran were inactive. (iii) Direct binding and competition studies with [3H]CsA indicated that CBD does not enter EL-4 cells (i.e., it acted at the surface). (iv) CBD caused agglutination of EL-4 cells, murine B and T lymphocytes, human thymocytes, and two T-cell hybridomas. Agglutination was inhibited by a monoclonal antibody to CsA and by CsA and CsA-BBa, but not by BBa. No agglutination was seen with BBa-aminodextran or aminodextran. HeLa cells, Vero (monkey kidney) cells, a mouse plasmacytoma, COS cells, and a poorly differentiated B-cell lymphoma were not agglutinated. (v) EL-4 cells failed to be agglutinated after treatment with trypsin or chymotrypsin. Specific agglutination was again possible after incubation for 5 h at 37 degrees C in the absence of enzyme. (vi) CBD covalently linked to crosslinked agarose beads inhibited interleukin 2 production by phorbol ester-stimulated EL-4 cells. No activity was seen if cell-to-bead contact was prevented by a 0.02-microns microporous filter that did not interfere with the passage of CBD. Our findings support the presence of a functional receptor on the surface of selected cells of the immune system.
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PMID:Evidence for a functional receptor for cyclosporin A on the surface of lymphocytes. 158 69

Rat renin cDNA was transfected into COS-7 and Chinese hamster ovary (CHO) cells and expressed under the control of the Simian Virus 40 early promoter. Conditioned media of the transfected cells showed renin activity only after trypsin treatment, suggesting prorenin was secreted into the medium. From the trypsinized serum-free culture of the transfected CHO cells active renin was purified to homogeneity by a simple three-step procedure. The active renin had similar specific activity, molecular weight, Km, pH optimum, and isoelectric point compared to native renin. The amino-terminal sequence was the same as that deduced from the renin cDNA. This suggests that the recombinant rat renin is similar to kidney renin in many respects, and is easily obtained by the present procedures.
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PMID:Expression of rat renin in mammalian cells and its purification. 160 Jun 38

We previously reported on patients with severe insulin resistance due to unprocessed insulin proreceptors. A structural change of the cleavage site from Arg-Lys-Arg-Arg to Arg-Lys-Arg-Ser due to G----T point mutation appeared to be the cause for failure to process the proreceptors. To determine whether the mutation of insulin proreceptors at the cleavage site was responsible for unprocessed insulin receptors and to elucidate the structural and binding characteristics of the proreceptors, we transfected cDNA with the mutation in COS 7 cells and examined the expressed insulin receptors. At 72 hours after transfection, insulin binding increased to the maximum in cells transfected with either normal or mutated cDNA, and insulin binding was 40 and 14 times higher than that of nontransfected cells, respectively. The declining rate of insulin binding after reaching the maximum was delayed in cells transfected with mutated cDNA. Affinity cross-linking and surface-labeling studies showed a 135-kilodalton (kD), normal alpha-subunit in the cells transfected with normal cDNA and a 210-kD proreceptor in the mutant cells. The proreceptors were cleaved by trypsin to yield normal-sized alpha- and beta-subunits. The sensitivity to trypsin was similar to that demonstrated in patients' cells, and the most effective concentration for the cleavage was 0.025%. Autophosphorylation resulted in decreased 32P incorporation into proreceptors of cells transfected with mutated cDNA at both basal and insulin-stimulated states, without a change in insulin sensitivity. Competitive binding studies with insulin, proinsulin, and miniproinsulin showed that the proreceptors had a lower relative affinity for proinsulin, but this characteristic disappeared after trypsin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of unprocessed insulin proreceptors in COS 7 cells transfected with cDNA with Arg735----Ser735 point mutation at the cleavage site. 164 Aug 58

Human renin is synthesized as a 406-amino acid preprorenin protein that is processed by a signal peptidase during secretion, to release prorenin as a 386-amino acid zymogen. The 46-amino acid "pro" domain is removed by a renin-processing enzyme, to produce enzymatically active renin, by cleavage at an Arg-Leu bond. The effects of the renin-processing enzyme can be mimicked by trypsin activation, where high concentrations of trypsin are incubated with prorenin for brief periods of time, followed by excess trypsin inhibitor to minimize secondary proteolytic processing by trypsin. In order to study the role of the pro segment in the secretion, folding, and activity of human renin, we engineered a construct where the pro domain from the preprorenin cDNA was deleted. This construct was introduced into mammalian cells and its expression was assayed in transient and stable systems. In COS-1 cells transfected with the prerenin expression vector pREN3, active renin was secreted with a specific activity of 1360 micrograms of angiotensin l/min/mg, compared with trypsin-activated prorenin, which has a specific activity of 818 micrograms of angiotensin l/min/mg. The active renin secreted in this system had a significantly reduced potency for the renin inhibitor SQ 32,970. These results demonstrate that the pro segment is dispensable for the folding and secretion of renin. A permanent cell line expressing the active form of renin was obtained by co-transfection of NRP cells with pREN3 and pHyg. A colony designated B/1 was identified, subcloned, and shown to secrete active renin (110 pg of renin/10(6) cells) optimally when maintained in both G418 and hygromycin.
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PMID:Stable expression, secretion, and characterization of active human renin in mammalian cells. 173 22

Deletions were constructed within a functional human immunodeficiency virus type 1 (HIV-1) proviral clone in order to assess the role of the envelope protein in virus particle formation. A graded exonuclease deletion technique was used to produce 12 clones with deletions of 175-308 nucleotides in the first conserved domain of envelope. This included 9 clones with frameshift deletions and 3 clones with in-frame deletions. Isogenic pairs of env deletion clones were produced with or without an additional deletion in the vif and vpr genes. Upon transfection, all clones produced virus particles, as determined by p24 antigen, reverse transcriptase, and sucrose gradient assays with conditioned media. Virus particles produced from clones with deletions in env or vif and vpr, or both regions, banded on sucrose gradients with a mobility similar to that of virus produced by the parental clone. The p24 gag capsid protein in the particles was resistant to trypsin, but the particles were disrupted by treatment with Triton X-100, suggesting the presence of a surrounding lipid bilayer. Furthermore, electron microscopic studies revealed both mature and immature virus particles derived from COS cells transfected with the env deletion clones. Cocultivation experiments with lymphoid cells and cells transfected with each of the env deletion clones demonstrated that the virus particles were noninfectious.
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PMID:Formation of noninfectious HIV-1 virus particles lacking a full-length envelope protein. 182 17

Lactase-phlorizin hydrolase (LPH) (EC 3.2.1.23/62) is a major intestinal microvillar membrane glycoprotein that digests lactose, the main carbohydrate of milk. To investigate structure/function relationships of LPH and to assess the impact of intracellular processing on the function of LPH and on its transport to the cell surface, we have expressed a full-length cDNA encoding LPH in mammalian COS-1 cells. Analysis of the expressed protein by immunoprecipitation with monoclonal anti-LPH antibodies and treatments with endo-beta-N-acetylglucosaminidase H and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two polypeptides with apparent molecular masses of 215 and 230 kDa, representing the mannose-rich (pro-LPHh) and complex (pro-LPHc) glycosylated forms of the precursor. By contrast to pro-LPH in human enterocytes, the expressed pro-LPH in COS-1 cells does not undergo intracellular proteolytic cleavage to generate a form similar to the mature enzyme of the brush-border membrane. Intracellular cleavage, however, is not essential for the molecule to acquire its enzymatic activity since pro-LPH in COS-1 cells is enzymatically as active as LPH isolated from intestinal brush-border membranes. Indirect immunofluorescent staining of transfected cells demonstrated that pro-LPH is expressed at the cell surface. This was further corroborated by the sensitivity of the complex glycosylated form (pro-LPHc) to trypsin in the medium. Our results provide the first conclusive evidence that pro-LPH is an enzymatically active molecule and that the intracellular proteolysis of pro-LPH is not essential for the generation of transport-competent forms of LPH.
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PMID:Expression of a full-length cDNA coding for human intestinal lactase-phlorizin hydrolase reveals an uncleaved, enzymatically active, and transport-competent protein. 190 19

The amyloid beta peptide (A beta P) is the major constituent of the amyloid deposits that accumulate extracellularly in the brain of patients with Alzheimer's disease. This peptide is obtained from transmembrane amyloid protein precursors (APP) which sometimes contain a Kunitz protease inhibitor (KPI) insert in their extracellular domain and therefore are able to inhibit serine proteases. Expression of the transmembrane and the secreted APP containing the KPI domain was obtained by transient transfection of COS-1 cells. The overexpressed proteins were detected in immunoblotting experiments and inhibition of trypsin was analyzed using reverse enzymography. Our results indicate that post-translational modifications including glycosylation improve the inhibition of trypsin by the APP containing the KPI domain.
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PMID:Glycosylation of the amyloid peptide precursor containing the Kunitz protease inhibitor domain improves the inhibition of trypsin. 212 Nov 36


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