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)

The kidney and several other thyroid hormone-responsive tissues contain a NADP-regulated thyroid hormone (TH)-binding protein (THBP), with an apparent molecular mass of 36 kDa on SDS-PAGE, responsible for most of the intracellular high-affinity T3 and T4 binding. THBP was purified to homogeneity from human kidney cytosol and used to generate proteolytic peptides. Microsequencing of four peptides revealed identity to amino acid sequences deduced from a human cDNA homolog to a cDNA encoding kangaroo mu-crystallin. This protein is a major structural kangaroo lens protein with no known function in other species. A full-sized cDNA (TH5.9) was isolated by 5'- and 3'-rapid amplification of cDNA ends using a human brain cDNA library and gene-specific PCR primers, confirming identity to the previously cloned human cDNA. The TH5.9 cDNA encodes a 314-residue protein (theoretical mol wt = 33,775) with significant homologies (40 to 60%) with two bacterial enzymes: lysine cyclodeaminase and ornithine cyclodeaminase. The TH5.9 cDNA was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Purified GST fusion protein, but not GST, bound T3 specifically with high affinity [dissociation constant (Kd) = 0.5 nM] in the presence of NADPH, and was labeled by UV-driven cross-linking of underivatized [(125)I]T3. T3 binding and photoaffinity labeling of GST fusion protein were activated by NADPH [activation constant (K[act]) = 10(-8) M], but not by NADH. The expressed protein displays the appropriate binding properties, indicating that TH5.9 cDNA encodes the NADP-regulated THBP characterized in human tissues.
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PMID:Purification, molecular cloning, and functional expression of the human nicodinamide-adenine dinucleotide phosphate-regulated thyroid hormone-binding protein. 932 54

Sequencing of polymerase chain reaction-amplified cDNAs from cultured cells of three patients with mevalonate kinase deficiency revealed a G --> A transversion at nucleotide 1000 of the coding region, converting alanine to threonine at position 334 (A334T). To characterize this defect, we expressed wild-type and mutant cDNAs in Escherichia coli as the glutathione S-transferase fusion proteins, with purification by affinity chromatography. SDS-polyacrylamide gel electrophoresis analysis for wild-type and mutant fusion proteins indicated an expected molecular mass of 42-43 kDa. Kinetic characterization of the wild-type fusion protein yielded Km values of 150 +/- 23 and 440 +/- 190 microM (mean +/- S.E.) for substrates (RS)-mevalonate and ATP, respectively. Expressed wild-type mevalonate kinase (MKase) had a maximum velocity of 13.6 +/- 1.4 units/mg of protein (n = 22, +/-S.E.), whereas the A334T mutation yielded an enzyme with average Vmax of 0.26 +/- 0.02 unit/mg of protein (n = 6, +/-S.E.), representing a decrease to 1.4% of control Vmax. Restriction digestion with HhaI, in conjunction with direct sequencing of cDNAs, revealed that two patients were homozygous and one heterozygous for the A334T allele, establishing autosomal recessive inheritance within families. Although the A334T enzyme had a normal Km for ATP of 680 +/- 226 microM (n = 3, +/-S.E.), the Michaelis constant for (RS)-mevalonate was increased >30-fold to 4623 +/- 1167 microM (n = 4, +/-S.E.) under standard assay conditions. Comparable kinetic results were obtained using extracts of lymphoblasts, which were homozygous for the A334T allele. Alanine 334 is invariant in MKase from bacteria to man and located in a glycine-rich region postulated to have homology with ATP-binding sequences. Our results indicate that the bacterial expression system for human MKase will provide a useful model system in which to analyze inherited mutations and identify the first active site residue in MKase associated with stabilization of mevalonate binding.
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PMID:Identification of an active site alanine in mevalonate kinase through characterization of a novel mutation in mevalonate kinase deficiency. 933 62

Phospholamban (PLN) was expressed in Escherichia coli as a protein fusion with glutathione S-transferase (GST). GST-PLN was mostly present in the insoluble protein fraction and accounted for approximately 50% of total insoluble protein. Attempts to suppress inclusion body formation or to use GST as an affinity-purification tag failed. A successful purification method is based on preparative SDS/PAGE and electrodialysis. From 1 g cells we typically purified 13.5 mg fusion protein with a PLN content of 2.8 mg. We genetically inserted an enterokinase (EK) protease site just in front of the PLN sequence and demonstrated the proteolytical liberation of PLN from the carrier protein. The approach described represents a substantial advancement in PLN expression and purification.
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PMID:Purification of the cardiac sarcoplasmic reticulum membrane protein phospholamban from recombinant Escherichia coli. 934 33

The Hck tyrosine kinase, a member of Src family, is predominantly expressed in myeloid cells. In this report we have analyzed interaction of cellular proteins with Src homology 3 (SH3) domain of Hck. For this purpose we used various GST-Hck fusion proteins comprising a part of unique region, complete unique region and/or complete SH3 domain of Hck, and glutathione S-transferase (GST). When these fusion proteins (or GST), immobilized on glutathione-agarose beads were incubated with [35S] methionine labelled cell extracts, multiple proteins which interact specifically with SH3 domain of Hck were detected by SDS-PAGE followed by autoradiography. The Hck interacting proteins could also be detected by a tandem blot binding assay in which the blot was incubated with purified fusion protein (or GST) and then the interacting proteins were identified by using antibody against GST. When a part of or complete unique domain was present along with SH3 domain, the interaction of some specific proteins was reduced several fold. These results raise the possibility of unique domain altering the properties of SH3 domain, thus modulating or restricting the interaction of SH3 domain with specific cellular proteins. This modulatory effect of unique domain was localized to 28 amino acids upstream of SH3 domain. SH3 interacting proteins were associated with serine/threonine and tyrosine kinase activities towards exogenous substrates. Most of the SH3 binding proteins were soluble in Triton X-100. Differentiation of promyelocytic leukemia cell line HL-60 into macrophage like cells resulted in appearance of novel SH3 binding proteins. Hck was detected in the eluate of WGA-Sepharose column, suggesting that it interacts with WGA binding glycoprotein (s). A rat spleen cDNA library was screened for the SH3 binding proteins by protein interaction cloning. Sequence analysis of the clones showed the presence of proline rich regions containing PPXP motifs.
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PMID:Interaction of SH3 domain of Hck tyrosine kinase with cellular proteins containing proline-rich regions: evidence for modulation by unique domain. 934 26

Fourteen isoforms of glutathione S-transferase (GST) have been separated and purified from mullet (Mugil cephalus) liver by scaling up an automatic analytical method based on anionic exchange chromatography. The activity of each isoenzyme with several substrates was determined. Dimeric combinations of six subunits make up this heterogeneous isoenzyme population. Five of these were resolved by reverse phase chromatography; four of them, named a, b, c and d, were present in more than one isoform, had the same apparent molecular mass (25.2 kDa) by SDS-PAGE, and were immunochemically related to plaice GST-A and possibly to rat GST-5 but not to plaice GST-B or any other rat GST subunit; they would belong to the theta class. Subunit e was only present in isoenzyme I which was basic, had an apparent molecular mass of 23.4 kDa and would belong to the alpha class, since it was recognized by antibodies towards plaice GST-B and rat GST-1 and GST-8 and less intensely by anti-(rat)GST-2. Another subunit, named f, with 25.2 kDa apparent molecular mass that could not be distinguished by reverse phase chromatography, was detected immunochemically by positive reaction with antibodies to rat GST-1 and GST-2 in addition to reaction with anti-(plaice)GST-A. As suggested by these results we discuss the existence of genetic polymorphism, the differential expression and the evolutionary relationships of mullet GSTs.
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PMID:Purification and characterization of multiple glutathione transferase isoenzymes from grey mullet liver. 936 73

Our earlier studies reported the identification of a rat testicular protein of 24 kDa with significant similarity at the N-terminus with Mu class glutathione S-transferases (GSTs). Treatment of goat sperm with antisera against this protein identified immunoreactive sites on the spermatozoa and inhibited in vitro fertilization of goat oocytes by the antibody-treated sperm. The above observations indicated the presence of GST-like molecule(s) important for fertility related events on goat spermatozoa. In this study, we report the purification of goat sperm GSTs (GSP1) which were purified by glutathione affinity chromatography and were enzymically active towards 1-chloro-2,4,-dinitrobenzene, a general GST substrate, and ethacrynic acid, a substrate for Pi class GSTs. GSP1 resolved into three major components on reverse-phase HPLC: peaks 1 and 2 with molecular masses of 26.5 kDa and peak 3 with a molecular mass of 25.5 kDa, as determined by SDS/PAGE. Multiple attempts to obtain N-terminal sequences of the first two peaks failed, indicating N-terminal block; however, they reacted to specific anti-Mu-GST antisera on Western blots and ELISA, and not to anti-Pi-GST antisera, which provides evidence for the presence of Mu-GST-reactive sites on peaks 1 and 2. The third component showed 80% N-terminal similarity with human and rat GSTP1-1 over an overlap of 15 amino acids, and reacted to anti-Pi-specific antisera in ELISA. Sperm labelled with antibodies against a 10-mer and an 11-mer peptide, designed from the N-terminal sequences of Mu and Pi class GSTs respectively, showed the presence of both Mu- and Pi-GST on goat sperm surface at distinct cellular domains. Selective inhibition of Pi class GST by the Pi-specific antisera, either at 0 h or at 3 h after initiation of sperm capacitation, leads to a reduction in fertilization rates. In contrast, the inhibition of Mu class GST by specific antisera at 0 h does not inhibit fertilization, although such treatment at 3 h after the initiation of capacitation reduces fertilization rates. The results indicate that both Pi- and Mu-GSTs are involved in fertilization, but the Mu-GST sites essential for fertilization are exposed only after 3 h of capacitation. The enzymic activity of GSP1 or live spermatozoa is not inhibited by the two antisera. The inability of the antibodies to cause such inhibition indicates that the reduction in fertilization rates and acrosome reaction caused by the antibodies is through a mechanism which does not interfere with the catalytic activity of the molecule. Therefore we established the presence of Pi and Mu class GST on goat sperm, their localization and their possible function in fertility-related events.
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PMID:Studies on glutathione S-transferases important for sperm function: evidence of catalytic activity-independent functions. 942 4

A 996 bp Aspergillus nidulans cDNA encoding the ASPND1 immunodominant antigen was cloned and expressed in Escherichia coli as a fusion protein with the enzyme glutathione S-transferase (GST) from Schistosoma japonicum. The GST-ASPND1 fusion protein was purified from isolated bacterial inclusion bodies by preparative SDS-PAGE. After cleavage with thrombin, the ASPND1 recombinant antigen (ASPND1r) and the GST protein were separated by SDS-PAGE and immunoblotted with a number of different human sera. The sera from 22 (88%) of 25 patients with an aspergilloma recognized the ASPND1r recombinant antigen on immunoblots. Forty-nine normal human sera and 14 sera from patients with other infections were unreactive. The ASPND1r expressed in E. coli could therefore be used, in combination with previously reported recombinant antigens, as a standardized antigen for serological and clinical diagnosis of Aspergillus-associated diseases.
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PMID:The recombinant antigen ASPND1r from Aspergillus nidulans is specifically recognized by sera from patients with aspergilloma. 949 92

Nine glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, and gL) have been identified in bovine herpesvirus 1 (BHV-1). gM has been identified in many other alpha-, beta-, and gammaherpesviruses, in which it appears to play a role in membrane penetration and cell-to-cell fusion. We sought to express BHV-1 open reading frame U(L)10, which encodes gM, and specifically identify the glycoprotein. We corrected a frameshift error in the published sequence and used the corrected sequence to design coterminal peptides from the C terminus. These were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The fusion protein containing the 63 C-terminal amino acids from the corrected gM sequence engendered antibodies that immunoprecipitated a 30-kDa protein from in vitro translation reactions programmed with the U(L)10 gene. Proteins immunoprecipitated by this antibody from virus-infected cells ran at 36 and 43 kDa in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 43 and 48 kDa in nonreducing SDS-PAGE. Only the larger of the pair was present in virions. A 7-kDa protein was released from gM by reducing agents. The 7-kDa protein was not recognized in Western blots probed with the anti-gM antibody but reacted specifically with antibodies prepared against BHV-1 U(L)49.5, previously reported to be a 9-kDa protein associated with an unidentified 39-kDa protein (X. Liang, B. Chow, C. Raggo, and L. A. Babiuk, J. Virol. 70:1448-1454, 1996). This is the first report of a small protein covalently bound to any herpesvirus gM. Similar patterns of hydrophobic domains and cysteines in all known gM and U(L)49.5 homologs suggest that these two proteins may be linked by disulfide bonds in all herpesviruses.
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PMID:Bovine herpesvirus 1 glycoprotein M forms a disulfide-linked heterodimer with the U(L)49.5 protein. 952 25

The class Alpha glutathione S-transferase (GST) subunit A5 is expressed in the livers of young male and female rats. After sexual maturation, this protein is no longer detectable in the livers of male rats, but is still expressed in female rats. We have previously demonstrated that the sexually dimorphic secretion of growth hormone regulates the levels of certain class Mu GSTs in rat liver, and this study was designed to investigate the hormonal regulation of GSTA5. Control and hypophysectomized rats of both sexes were used to study the role of growth hormone in the regulation of hepatic GSTA5; and the influence of testosterone on the expression of this same subunit was investigated in intact females and castrated males. Liver cytosols were subjected to SDS/PAGE and immunoblotting using antibodies directed towards rat (r)GSTA5, and to affinity purification on glutathione-Sepharose followed by reverse-phase HPLC in order to quantify the relative levels of rGSTA1, A2, A3, A4, M1 and M2 subunits. These analyses revealed that the expression of rGSTA5 is, indeed, regulated by both growth hormone and testosterone.
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PMID:Growth hormone- and testosterone-dependent regulation of glutathione transferase subunit A5 in rat liver. 962 Aug 80

In adipocytes, insulin stimulates the translocation of the glucose transporter, GLUT4, from an intracellular storage compartment to the cell surface. Substantial evidence exists to suggest that in the basal state GLUT4 resides in discrete storage vesicles. A direct interaction of GLUT4 storage vesicles with the plasma membrane has been implicated because the v-SNARE, vesicle-associated membrane protein-2 (VAMP2), appears to be a specific component of these vesicles. In the present study we sought to identify the cognate target SNAREs for VAMP2 in mouse 3T3-L1 adipocytes. Membrane fractions were isolated from adipocytes and probed by far Western blotting with the cytosolic portion of VAMP2 fused to glutathione S-transferase. Two plasma membrane-enriched proteins, p25 and p35, were specifically labeled with this probe. By using a combination of immunoblotting, detergent extraction, and anion exchange chromatography, we identified p35 as Syntaxin-4 and p25 as the recently identified murine SNAP-25 homologue, Syndet (mSNAP-23). By using surface plasmon resonance we show that VAMP2, Syntaxin-4, and Syndet form a ternary SDS-resistant SNARE complex. Microinjection of anti-Syndet antibodies into 3T3-L1 adipocytes, or incubation of permeabilized adipocytes with a synthetic peptide comprising the C-terminal 24 amino acids of Syndet, inhibited insulin-stimulated GLUT4 translocation to the cell surface by approximately 40%. GLUT1 trafficking remained unaffected by the presence of the peptide. Our data suggest that Syntaxin-4 and Syndet are important cell-surface target SNAREs within adipocytes that regulate docking and fusion of GLUT-4-containing vesicles with the plasma membrane in response to insulin.
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PMID:Syndet, an adipocyte target SNARE involved in the insulin-induced translocation of GLUT4 to the cell surface. 966 52


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