Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A protein closely resembling the purple membrane protein pre-exists in the cell membrane of H. halobium prior to the appearance of functional bacteriorhodopsin. It is associated with a differentiated membranous structure which has been isolated on a sucrose gradient and appears to be a precursor of the purple membrane. The identity of the precursor protein as a form of the purple membrane protein was established in different ways: (1) The cell proteins were labelled in vivo with 14C-proline during dark aerobic growth, the label was "chased", and the cells transferred to the illuminated near-anaerobic conditions under which purple membrane is optimally synthesised (induction conditions). Cell lysates were fractionated on sucrose gradients at different times after induction. Label first found in the precursor fraction appeared within 24 h in the purple membrane fraction. (2) SDS-urea-acrylamide gel electrophoresis of the purple membrane protein and the precursor showed only one protein band whose migration coincided with that of the purple membrane band. (3) The amino-acid analysis of the purified precursor was very similar to that of the purple membrane. The absorption spectrum of the precursor showed little of the characteristic absorption of bacteriorhodopsin at 570 nm. A major band appears at 412 nm, the exact nature of which is not known. The difference spectrum (reduced versus oxidised) of a purified fraction showed only traces of cytochrome. Thin-layer chromatography of an acetone-soluble lipid extract indicated the presence of retinal and beta-carotene. Cells grown in the presence of nicotine did not develop purple membrane after induction: the species absorbing at 412 nm was much less abundant than in non-inhibited cells, but a new fraction was present with a sharp peak at 345 nm consisting mainly of lycopene.
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PMID:Biogenesis of the purple membrane of Halobacterium halobium. 87 Jan 1

The 3C proteinase from the hepatitis A virus (HAV) was cloned into a multicopy expression vector in Escherichia coli under control of the tac promoter. The resulting plasmid construction produced 3C proteinase as a soluble and active enzyme constituting approximately 10% of total cellular proteins. The enzyme was purified to apparent homogeneity as judged by SDS gel electrophoresis and HPLC reversed-phase and FPLC ion-exchange chromatography. A colorimetric assay was developed, and synthetic peptides derived from the predicted cleavage sites of the HAV polyprotein were tested for proteolysis of the enzyme. The peptide representing the 2B/2C cleavage site was cleaved most efficiently with a Km and kcat of 2.1 +/- 0.5 mM and 1.8 +/- 0.1 s-1, respectively. Site-directed mutagenesis was then used to identify the cysteine at position 172 as the active site nucleophile. Finally, the purified enzyme showed the expected endoproteinase activity on the P1 precursor protein generated by in vitro transcription/translation.
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PMID:Expression and characterization of recombinant hepatitis A virus 3C proteinase. 131 94

The human gonadotropin-releasing hormone precursor protein, pHGnRH (Met-23-Ile69) (preproGnRH), and three of its fragment peptides, pHGnRH (Asp14-Ile69) (gonadotropin-releasing hormone associated peptide--GAP), pHGnRH (Phe38-Ile69), and pHGnRH (Ser47-Ile69), were assembled in a stepwise solid-phase cosynthesis employing Boc/Bzl tactics and an optimized acylation schedule which included recoupling steps with hexafluoro-2-propanol to help overcome the aggregation of the pendant peptide chains of the peptidoresin during difficult couplings. Reversed-phase high-performance liquid chromatography (HPLC) purification yielded products which were characterized by analytical reversed-phase HPLC, ion-exchange chromatography, capillary zone electrophoresis, SDS-polyacrylamide gel electrophoresis, and ion-spray mass spectrometry to reveal a high degree of homogeneity. Biological characterization demonstrated that only GAP stimulated luteinizing hormone and follicle-stimulating hormone release from primary cultures of rat anterior pituitary cells, while GAP, pHGnRH (Phe38-Ile69), and preproGnRH all inhibited prolactin release, with the latter being the most potent at concentrations comparable to bromocryptine. However, only GAP and pHGnRH (Phe38-Ile69) were able to displace a labeled gonadotropin-releasing hormone agonist from binding to rat pituitary membrane preparations. This first demonstration of significant biological activity with a precursor protein also suggests that the gonadotropin-releasing and prolactin release-inhibiting functions of GAP are not mediated through the same pituitary receptors.
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PMID:Total solid-phase synthesis and prolactin-inhibiting activity of the gonadotropin-releasing hormone precursor protein and the gonadotropin-releasing hormone associated peptide. 139 Jun 67

Previous studies on mitochondrial targeting presequences have indicated that formation of an amphiphillic helix may be required for efficient targeting of the precursor protein into mitochondria, but the structural details are not well understood. We have used CD and NMR spectroscopy to characterize in detail the structure of a synthetic peptide corresponding to the presequence for the beta-subunit of F1-ATPase, a mitochondrial matrix protein. Although this peptide is essentially unstructured in water, alpha-helix formation is induced when the peptide is placed in structure-promoting environments, such as SDS micelles or aqueous trifluoroethanol (TFE). In 50% TFE (by volume), the peptide is in dynamic equilibrium between random coil and alpha-helical conformations, with a significant population of alpha-helix throughout the entire peptide. The helix is somewhat more stable in the N-terminal part of the presequence (residues 4-10), and this result is consistent with the structure proposed previously for the presequence of another mitochondrial matrix protein, yeast cytochrome oxidase subunit IV. Addition of increasing amounts of TFE causes the alpha-helical content to increase even further, and the TFE titration data for the presequence peptide of the F1-ATPase beta-subunit are not consistent with a single, cooperative transition from random coil to alpha-helix. There is evidence that helix formation is initiated in two different regions of the peptide. This result helps to explain the redundancy of the targeting information contained in the presequence for the F1-ATPase beta-subunit.
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PMID:Conformational analysis of a mitochondrial presequence derived from the F1-ATPase beta-subunit by CD and NMR spectroscopy. 139 Sep 13

Dimethylglycine dehydrogenase (Me2GlyDH), an enzyme of choline catabolism specifically expressed in the mammalian liver, was analyzed in rat hepatocytes in culture. This mitochondrial enzyme carries the FAD cofactor covalently attached to the polypeptide chain by its riboflavin 8 alpha position to N pi of histidine [Cook, R., Misono, K.S. & Wagner, C. (1980) J. Biol. Chem. 259, 12475-12480]. Subcellular fractionation of [14C]riboflavin-labelled hepatocytes and immunoprecipitation with Me2GlyDH-specific antiserum identified a [14C]riboflavin-labelled polypeptide of the size of mature Me2GlyDH only in the mitochondrial fraction. Immunoprecipitation of extracts from [35S]Met-labelled hepatocytes revealed a putative precursor protein to the mature Me2GlyDH in the cytoplasmic fraction. These Me2GlyDH polypeptides were not expressed in cells of the rat hepatoma cell line FAO. A Me2GlyDH cDNA clone of apparent full length was isolated from a rat liver cDNA bank constructed in the plasmid vector pcD-X [Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T. & Arai, K. (1987) Methods Enzymol. 154, 3-28]. The nucleotide sequence of the cDNA contains an open reading frame encoding a protein of 96059 Da. This molecular mass agrees well with the migration on SDS/PAGE of the assumed Me2GlyDH precursor immunoprecipitated from the cytoplasm of [35S]Met-labelled cells. Proteolytic cleavage at the putative mitochondrial processing protease-recognition site Arg(-2)-Ala(-1)-Glu(+1) would lead to the formation of a protein of 91391 Da, which is in good agreement with the estimated 90 kDa of mature Me2GlyDH [Wittwer, A.J. & Wagner, C. (1981) J. Biol. Chem. 256, 4102-4108], and a 43-amino-acid leader peptide. The N-terminus of Me2GlyDH contains a conserved amino acid sequence which forms the dinucleotide-binding site in many enzymes with noncovalently bound FAD. Close to the modified histidine there is an amino acid sequence resembling a sequence conserved in thymidylate synthases and shown in these enzymes to be involved in the binding of the pteroyl polyglutamate cofactor.
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PMID:Rat liver dimethylglycine dehydrogenase. Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone. 171 Sep 85

Core I and core II proteins are the largest nuclear-encoded subunits of the mitochondrial ubiquinol-cytochrome-c reductase (bc1 complex) lacking redox prosthetic groups. cDNA clones of the two bovine core proteins have been isolated by the screening of lambda ZAP cDNA libraries either with an oligonucleotide probe based on the sequence of an internal peptide or with a polymerase-chain-reaction-amplified fragment. The core I precursor protein consists of 362 amino acids with a 34-amino-acid presequence typical for mitochondrial targeting signals. The mature protein migrates in SDS/polyacrylamide gels with an apparent molecular mass of 47 kDa, which does not correspond to the actual molecular mass of the protein of 35.8 kDa deduced from the cDNA sequence. The core II precursor protein is composed of 453 amino acids having a 14-amino-acid presequence as a targeting sequence. Comparison of the core I amino acid sequence with sequences of the newly discovered protein family [Schulte, U., Arretz, M., Schneider, H., Tropschug, M., Wachter E., Neupert, W. & Weiss, H. (1989) Nature 339, 147 - 149] comprising the processing enhancing protein (PEP), matrix processing peptidase (MPP), and core I and II proteins from Neurospora crassa and Saccharomyces cerevisiae, revealed a remarkable identity of 39% and a high similarity of 49% to N. crassa PEP, which in this fungus is identical to core I. Core II protein is only a distant relative of this protein family. Based on these sequence comparisons and data obtained by genomic Southern blots, we anticipate that the bovine core I subunit, like the N. crassa core I protein, is bifunctional, being responsible for the maintenance of electron transport and processing of proteins during their import into the mitochondrial matrix. The analysis of the primary structure of the two core proteins completes the set of primary structures of all subunits of bovine ubiquinol-cytochrome-c reductase.
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PMID:Core I protein of bovine ubiquinol-cytochrome-c reductase; an additional member of the mitochondrial-protein-processing family. Cloning of bovine core I and core II cDNAs and primary structure of the proteins. 171 95

Prosaposin is the precursor protein for saposins, which are small lysosomal proteins required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. Prosaposin, in addition to generating the saposins in the lysosomes, also exists as an unprocessed approximately 70-kDa protein in many tissues and secretory fluids. In this study, we isolated prosaposin from human milk. Milk was fractioned by ammonium sulfate precipitation, then chromatographed with DEAE-Sephacel and G-3000 SW gel permeation-HPLC. A fraction containing prosaposin was finally purified with the anti-saposin C IgG attached affinity column. The protein staining of the purified preparation on SDS-PAGE and the Western blotting showed a single band. The sequence of the initial 10 amino acids from N-terminus of the purified protein was identical to the sequence of prosaposin deduced from cDNA. Although prosaposin itself showed beta-glucosidase activator activity at a slight degree, the activity increased much after trypsin treatment. Western blotting of the trypsin-treated sample confirmed the formation of small saposin-like bands from prosaposin by the action of trypsin.
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PMID:Isolation and characterization of prosaposin from human milk. 195 98

A highly purified fraction obtained from scrapie (263-K strain)-infected hamsters' brains by an alternative procedure without proteinase K treatment contained a protease-resistant form of the scrapie precursor protein (PrPSc) and infectivity of 9.9 +/- 0.7 log LD50/ml. Polyclonal antibodies produced against hamster scrapie amyloid protein (PrP27-30) and used in a neutralization test diminished infectivity of the PrPSc preparations by 1.6 log after intracerebral inoculation and by 1 log after intraperitoneal inoculation. PrPSc was subjected to size-exclusion HPLC; greater than or equal to 60% of the eluted infectious units were recovered from the peak with an apparent mass of 30.4 +/- 0.6 kDa. Characterization by UV absorption spectra, SDS/PAGE, immunoblots, N-terminal amino acid sequence, and neutral sugar and amino sugar analyses demonstrated homogeneity of the infectious units. The neutral sugar and amino sugar compositional analyses revealed high mannose, glucosamine, fucose, and sialic acid content. This demonstrated an extensive posttranslational modification by the complex type of N-linked glycosylation and glycane core of C-terminal glycolipid of PrPSc. The results correspond to the predicted size, composition, and sequence of PrPSc and indicate that this protein may be the only component of scrapie infectious unit or the infectious form of scrapie precursor.
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PMID:Molecular mass, biochemical composition, and physicochemical behavior of the infectious form of the scrapie precursor protein monomer. 197 20

The subcellular distribution of enkephalin (EK) precursor proteins was investigated to clarify the intracellular site of biosynthesis of EK in rat dental pulp tissue. The contents of met-EK-like peptides in nuclear, microsomal, and supernatant fractions of the pulp tissue were markedly increased after sequential digestion with trypsin and carboxypeptidase B, indicating the enrichment of the precursors in these fractions. Sephadex G-100 gel filtration showed a common peak of the precursor proteins in the homogenate and its microsomal and supernatant fractions, and the molecular weight was determined to be about 58,000 by SDS polyacrylamide gel electrophoresis. Both the partially purified precursor protein from the supernatant fraction and N alpha-benzoyl-DL-arginine-beta-naphthylamide (BANA) were used as substrates for a lysosomal enzyme separated by Sephadex G-75 gel filtration. The major peak of EK-producing activity of the enzyme was identical with that of BANA-degrading activity of the enzyme. These results demonstrate the EK-producing activity of lysosomal proteinase, and also indicate the usefulness of the two substances as substrates for the enzyme.
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PMID:Subcellular distribution of enkephalin precursor proteins in rat dental pulp and the usefulness as a substrate for enkephalin-producing enzymes. 199 Feb 37

A total of 81 cell clones persistently infected with the LAV-1 or HTLV-IIIB strain of human immunodeficiency virus type 1 (HIV-1) was isolated from cells which were obtained by serial passage of some proliferating MT-4 cells after a drastic cytolysis of most cells by HIV-1-infection. These cell clones were classified into 8 types (I to VIII) in terms of the expression of HIV-1 antigens, syncytium formation capacity, and reverse transcriptase activity and infectivity of virus particles in the culture fluid. Type I cell clones were producers of infectious HIV-1 particles, while types II to VIII cell clones did not produce infectious HIV-1 or were producers of uninfectious defective HIV-1 particles. Immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis (PAGE) showed that the gag precursor protein in L-2 cell clone (type IV) was not cleaved to mature gag proteins, while the env precursor protein on L-3 cell clone (type III) was not cleaved to mature env protein. H-7 cell clone (type VIII) did not express any HIV-1 antigen. All these cell clones after the superinfection with infectious HIV-1 synthesized intact gag and env proteins, which were, at least in part, related to the HIV-1 genome persistently present in the cell clones before the superinfection, resulting in production of infectious HIV-1. For example, it was found that L-2 cell clone contained a single copy of the LAV-1 genome per haploid cell and produced doughnut-shaped particles. On the other hand, the cell clone isolated from the L-2 cell clone superinfected with infectious HTLV-IIIB contained the integrated HTLV-IIIB genome in addition to the LAV-1 genome present before the superinfection, and produced intact HIV-1 particles in addition to doughnut-shaped particles from a single cell. These results indicate that complementation and/or genetic recombination events in the superinfected cells may account for the production of infectious intact HIV-1 virions.
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PMID:Production of infectious particles from defective human immunodeficiency virus type 1 (HIV-1)-producing cell clones by superinfection with infectious HIV-1. 200 Nov 75


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