Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate biosynthesis and intracellular transport of 5'-nucleotidase, we purified this enzyme from rat liver and prepared antibodies. In immunoblot analysis, 5'-nucleotidase from the plasmalemmal, Golgi, and tritosomal fractions migrated as a single band of 72 kDa. The immunoreactive 68-kDa band was detected in the rough microsomal fraction and only the 72-kDa polypeptide contained sialic acids. The single polypeptide of 61 kDa immunoprecipitated from the translation products with the membrane-bound polysomal RNAs by the cell-free system converted to the 68-kDa form associated with membranes, when translated in the presence of microsomal vesicles. 5'-Nucleotidase from cultured primary hepatocytes pulse-labeled with [35S]methionine for 20 min migrated as a 68-kDa polypeptide. Within 45 min of chase, the 68-kDa form was converted to the 72-kDa form. Upon treatment with tunicamycin, a new immunoreactive polypeptide of 59 kDa was obtained. These results suggest that 5'-nucleotidase is translated on the membrane-bound polysomes as a 61-kDa precursor and that the cleavage of the 2-kDa signal peptide and the core glycosylation co-translationally convert the precursor to the 68-kDa form, which is subsequently processed in the Golgi complex to the 72-kDa form.
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PMID:Biosynthesis and intracellular transport of rat liver 5'-nucleotidase. 300 3

p60src of wild-type Rous sarcoma virus is myristylated at its N-terminal glycine residue. We have shown previously that this myristylation is necessary for p60src membrane association and for cell transformation by using src mutants with alterations within the N-terminal 30 kilodaltons of p60src. In this study we analyzed the process of p60src myristylation in wild type- and mutant-infected cells. All myristylated src proteins examined lack the initiator methionine, but two mutant src proteins lacking the initiator methionine are not myristylated, indicating that removal of the initiator methionine and myristylation are not obligatorily coupled. Analysis of the kinetics of myristylation and the association of p60src with cellular proteins p50 and p90 indicated that myristylation occurs before p60src becomes membrane associated and that transient association with p50 and p90 occurs regardless of myristylation. Myristylation is required for stable association of p60src with the plasma membrane but is not sufficient for membrane association. A mutant with an src deletion of amino acids 169 through 264 has an src protein that is myristylated but not membrane bound, remaining stably associated with p50 and p90. This mutant is transformation defective. Several N-terminal deletion mutants possessing tyrosine kinase activity have myristylated and membrane-bound src proteins but are not fully active in cell transformation, suggesting that additional N-terminal functional domains exist.
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PMID:Processing of p60v-src to its myristylated membrane-bound form. 301 13

The synthesis and secretion of apolipoprotein B-100 (apoB-100) have been studied in a human hepatoma cell line, the Hep G2 cells. The time needed for the synthesis of apoB-100 was estimated to be 14 min, which corresponds to a translation rate of approximately 6 amino acids/s. ApoB-100 was compared with albumin and alpha 2-macroglobulin as to the distribution between the membrane and the luminal content in the endoplasmic reticulum (ER) and the Golgi apparatus. The results suggested that apoB-100 approximately followed the distribution of these secretory proteins in the Golgi, while the ratios between the percent membrane-bound apoB-100 and percent membrane-bound albumin or alpha 2-macroglobulin were 3-4:1 in the ER. This may suggest that apoB-100 occurs in a membrane-associated form in ER prior to the integration in the lipoproteins. Pulse-chase studies combined with subcellular fractionation was used to investigate the kinetics for the intracellular transfer of apoB-100. A 3-min pulse of [35S]methionine was followed by an increase in apoB-100 radioactivity in the ER during the first 10-15 min of chase. The following 10-15 min of chase were characterized by linear decrease in apoB-100 radioactivity with a decay rate of approximately 6%/min. The residence kinetics for apoB-100 in the ER differed from that of transferrin and probably also from that of albumin. By comparing the time for the pulse maximum in ER with that in the denser Golgi fractions the time needed for the transfer between ER and Golgi could be estimated to be 10 min. The time needed for the secretion of newly synthesized apoB-100 was estimated to be 30 min. This indicates that the transfer of the protein through the Golgi apparatus to the extracellular space requires 20 min.
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PMID:Pulse-chase studies of the synthesis and intracellular transport of apolipoprotein B-100 in Hep G2 cells. 302 51

Enzyme IIImtl is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, we report the isolation of IIImtl from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of IIImtl with [32P]PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase Glu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp- Asp. The corresponding peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which we assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the IIImtl proteins was found to be 15,000. We have also determined the N-terminal sequence of both proteins. Comparison of the IIImtl peptide sequences and the C-terminal part of the enzyme IImtl of Escherichia coli reveals considerable sequence homology, which supports the suggestion that IImtl of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II. In particular, the homology of the active-center peptide of IIImtl of S. aureus and S. carnosus with the enzyme IImtl of E. coli allows one to predict the N-3 histidine phosphorylation site within the E. coli enzyme.
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PMID:Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme IIImtl of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme IImtl of Escherichia coli. 306 11

Three classes of activators of human neutrophils that induce the intracellular translocation of protein kinase C from the cytosol to the particulate fraction were compared for their effects on the properties of the particulate (membrane-bound) enzyme. In cells stimulated with 10 ng/ml of phorbol-12-myristate-13-acetate (PMA) the particulate enzyme is almost fully active in the absence of added Ca2+ or phospholipids and this activity is not released by the Ca2+-chelator EDTA. In contrast, binding of protein kinase C to the particulate fraction in cells treated with the chemotactic factor f-Met-Leu-Phe (fMLF) or with the ionophore A-23187 plus Ca2+ is observed only when the cells are lysed in the presence of 1 mM Ca2+. With these stimuli the particulate enzyme retains a nearly absolute requirement for Ca2+ and phospholipids. Thus only the full intercalation of protein kinase C caused by PMA, which is resistant to removal by chelators stabilizes an active form of protein kinase C in the neutrophil membrane. In confirmation of this conclusion, in isolated plasma membranes loaded with partially purified protein kinase C by incubation with 5 microM Ca2+ further incubation with PMA, but not with fMLF, caused a significant fraction of the bound PKC to become resistant to removal by chelators, and to be nearly fully active in the absence of added activators.
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PMID:Differential mechanisms of translocation of protein kinase C to plasma membranes in activated human neutrophils. 308 66

A membrane-bound aminopeptidase able to remove methionine from haemoglobin nascent peptides is described. The enzyme also hydrolyses methionine from methionyl-lysyl-bradykinin but not lysine from lysyl-bradykinin. The tripeptide Met-Ala-Ser is poorly hydrolysed. This aminopeptidase also splits amino acid 2-naphthylamides, being, however, less specific with respect to these synthetic substrates.
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PMID:Removal of N-terminal methionine from haemoglobin nascent peptides by a membrane-bound rat liver methionine aminopeptidase. 308 45

Phorbol ester (12-O-tetradecanoyl-phorbol 13-acetate) stimulates the secretion of tissue-type plasminogen activator by the melanoma cell line, Bowes. This effect is associated with increased levels of mRNAs for both tissue-type plasminogen activator and a 48 kDa-protein. Labelling of melanoma cells with L-[35S]methionine allowed to identify an intracellular protein which, by 3 criteria, was identical with the in vitro translation product of the 48kDa-protein mRNA: a Mr of 48,000 on electrophoresis in the presence of sodium dodecyl sulphate; inducibility by phorbol ester and failure of reducing agents to affect electrophoretic mobility. As detectable by L-[35S]methionine labelling, the protein was mainly localized in the cytosol. In vitro phosphorylation reactions, carried out on subcellular fractions revealed a membrane-associated protein which also had the three characteristics of the aforementioned 48 kDa-protein. Phosphorylation did not require Ca2+-ions. Addition of phorbol ester to the reaction mixtures increased the phosphorylation. Reconstitution experiments between membrane and cytosol fractions of phorbol ester-treated and untreated cells showed that the 48kDa protein occurs in a cytosolic, unphosphorylated and a membrane-bound, phosphorylated form and that the former is converted to the latter by a phorbol ester activated, membrane-associated protein kinase.
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PMID:Phorbol ester stimulates the synthesis and phosphorylation of a 48 kDa-intracellular protein in plasminogen activator secreting melanoma cells. 308 56

We have prepared polyclonal antiserum against sheep seminal vesicle prostaglandin H synthase (also termed cyclooxygenase) which cross-reacted with human cyclooxygenase, thereby enabling us to directly determine the synthetic rate of cyclooxygenase protein and its modulation by the monokine interleukin-1 (IL-1). Cultured human dermal fibroblast cells were labeled with [35S]methionine, and the membrane-bound cyclooxygenase was solubilized and immunoprecipitated 35S-labeled fibroblast cyclooxygenase migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular size of approximately 73,000 daltons, similar to that of native sheep cyclooxygenase and of cyclooxygenase covalently labeled by [3H]aspirin, i.e. [3H]acetylcyclooxygenase. Additional validation of the immunoprecipitated 35S-labeled cyclooxygenase band indicated that it was specifically displaced by unlabeled sheep cyclooxygenase. N-terminal amino acid radiosequence analysis of [3H]proline-labeled cyclooxygenase revealed [3H]proline residues in positions 3, 6, and 8, consistent with the previously reported N-terminal sequence of sheep cyclooxygenase. Endoglycosidase H treatment of 35S-labeled fibroblast cyclooxygenase caused a decline in apparent molecular size (due to removal of mannose residues) which was similar to that seen with the native sheep cyclooxygenase. [35S]Methionine pulse-chase experiments indicated a half-life of 1 h for fibroblast cyclooxygenase. The monokine interleukin-1 stimulated fibroblast cyclooxygenase synthesis in a time- and dose-dependent fashion; as little as 0.03 unit/ml of IL-1 produced significant stimulation of 35S-labeled cyclooxygenase synthesis. Maximum stimulation was 3-10-fold after preincubation of the cells with 0.3 unit/ml of IL-1 for 12-16 h. IL-1 treatment of cells yielded parallel dose-response curves for stimulation of prostaglandin E2 formation, increased cellular cyclooxygenase activity, and increased synthetic rate of newly formed cyclooxygenase, suggesting that the IL-1 effect is mediated mainly, if not solely, via induction of cyclooxygenase synthesis.
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PMID:Regulation of fibroblast cyclooxygenase synthesis by interleukin-1. 312 73

We have isolated overlapping cDNA clones representing the full-length transcript (4038 base pairs) for murine beta-1,4-galactosyltransferase. The coding sequence predicts a membrane-bound glycoprotein with 3 distinct structural features: 1) a large, potentially glycosylated COOH-terminal domain (355 amino acids) which is positioned within the Golgi lumen and contains both the catalytic and alpha-lactalbumin binding site; 2) a single transmembrane domain (20 amino acids); and 3) a short NH2-terminal domain containing 2 Met residues, separated by 12 amino acids. The gene for murine beta-1,4-galactosyltransferase is unusual in that it specifies 2 mRNA transcripts which differ in length by about 200 base pairs. The longer transcript contains both Met residues found in the NH2-terminal domain; the shorter transcript contains only the downstream Met. These results predict that 2 related forms of beta-1,4-galactosyltransferase of 399 and 386 amino acids are synthesized as a consequence of alternative translation initiation. Both forms of the enzyme are identical in primary structure with the exception that the long form has an NH2-terminal extension of 13 amino acids which, in part, potentially encodes a cleavable signal sequence. The structural implications, topological distribution and potential biological significance of the 2 forms of the enzyme are discussed.
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PMID:Evidence for two forms of murine beta-1,4-galactosyltransferase based on cloning studies. 314 31

The yeast KEX2 protease is the only enzyme that has a proven role in the activation of polypeptide hormones through cleavage at pairs of basic residues. The enzyme that fulfils this role in higher eukaryotes has yet to be unequivocally identified. In this investigation, a KEX2-like calcium-dependent protease has been identified in rat hepatic microsomes. The enzyme is membrane-bound, has a pH optimum of 5-6 and converts proalbumin to albumin. More importantly, like the KEX2 protease, it meets two other exacting criteria defined by specific mutations in humans. Namely, it does not process proalbumin Christchurch (-1 Arg----Gln) which lacks one of the requisite basic residues and, whilst not itself a serine protease, it is inhibited by the reactive center variant, alpha 1-antitrypsin Pittsburgh (358 Met----Arg) but not by normal alpha 1-antitrypsin.
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PMID:Calcium-dependent KEX2-like protease found in hepatic secretory vesicles converts proalbumin to albumin. 327 32


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