Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

From the cytosol fraction (supernatant fluid at 105,000 g) of chicken liver, 3 alpha-hydroxysteroid dehydrogenase was purified to an apparently homogeneous state by differential precipitation with ammonium sulfate, followed by column chromatographies with DE 51, DEAE-Toyopearl, and Sephadex G-100. Finally the dehydrogenase was purified 103-fold on the basis of the cytosol fraction. Polyacrylamide gel electrophoretic analysis in the presence of sodium dodecyl sulfate (SDS) revealed that molecular weight of the purified enzyme was 66 kDa, while that of the native dehydrogenase in the absence of SDS was estimated as 660 kDa or more from the peak of the enzyme in elution profile from Sephacryl S-200 column chromatography. The dehydrogenase required NADPH specifically for reduction of 3-oxo group of 5 beta-androstanedione (Km = 1.6 microM). Optimal temperature for 3-oxo reduction was 50 C in incubation for 10 min.
J Steroid Biochem Mol Biol 1990 Nov 30
PMID:Purification and characterization of 3 alpha-hydroxysteroid dehydrogenase from chicken hepatic cytosol. 227 41

The DNA sequence containing the start of the Escherichia coli nirB gene is reported. The N-terminal amino acid sequence of purified NADH-dependent nitrite reductase coincided with that predicted from the DNA sequence, confirming that nirB is the structural gene for nitrite reductase apoprotein and identifying the translation start point. Using nuclease S1 mapping, the sole transcription startpoint for the nirB gene was found 23 or 24 base-pairs upstream from the ATG initiation codon. By subcloning successively smaller DNA fragments into a beta-galactosidase expression vector plasmid, we located the promoter within a sequence bounded by a TaqI site at +14 with respect to the transcription startpoint and a HpaII site at -208. Measurements in vivo of beta-galactosidase expression and RNA levels due to nirB promoter activity showed that this promoter was activated during anaerobic growth. Optimal activity was found only after anaerobic growth in the presence of nitrite. The sequence of the nirB promoter is compared with sequences found at other anaerobically activated promoters.
J Mol Biol 1987 Aug 20
PMID:Location and sequence of the promoter of the gene for the NADH-dependent nitrite reductase of Escherichia coli and its regulation by oxygen, the Fnr protein and nitrite. 244 93

VL30 elements are a multigene family within the class of retroviruses and retrotransposons. We have characterized the response of normal rat fibroblasts to anoxia, in which endogenous VL30 element expression is strongly induced. Optimal induction up to 500-fold occurs under complete anoxia, although a lesser response is seen under atmospheres up to 2% oxygen. Phorbol esters and diacylglycerol, which induce mouse VL30 RNA approximately eightfold, show no effect on the rat VL30 system. The hypoxic conditions optimal for rat VL30 induction represent a mild cellular stress, with no reduction in cell viability during the induction period. Although the precise physiological role of this fibroblast response to temporary anoxia is unknown, it may occur during wound healing. The induction of VL30 by anoxia provides a unique model system wherein a member of the mammalian retrovirus/retrotransposon family is highly responsive to a common physiological signal.
J Mol Biol 1989 Feb 20
PMID:Retrotransposon-like VL30 elements are efficiently induced in anoxic rat fibroblasts. 246 7

Heterologous radioreceptor assays, commonly using ovine prolactin, may generate inconsistent results since prolactin (PRL) from one species may be recognized as growth hormone in another. Homologous radioreceptor assays (RRA) are most similar to the in vivo hormone-tissue receptor environment; however, lactogenic homologous RRAs have been reported only for mouse hepatic membranes. In this study, an assay system was developed to investigate homologous binding for porcine PRL in porcine uterine tissue. The pig does not produce a decidual PRL or a placental lactogen; yet, PRL affects uterine physiology during reproductively important events. Optimal binding conditions established for porcine PRL homologous RRA include 150 micrograms membrane, 45,000 cpm labeled porcine PRL and 500 microliters sodium phosphate buffer pH 7.6, incubated at 25 degrees C for 24 h. Binding of porcine PRL tracer is very low (less than 3%); however, when tissue is treated with the chaotropic agent, MgCl2 (4 M), binding increases from 3 to 28%. Dissociation kinetics show a rate of 3.79 X 10(-6)/s initially, and then 1.63 X 10(-6)/s. Competition for labeled PRL on binding sites with unlabeled porcine PRL results in 80% displacement with unlabeled porcine prolactin (NSB) of 7% at 1000 ng. Affinity constant generated from homologous inhibition assays is 0.326 X 10(8) M-1. Porcine growth hormone (GH), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) do not displace porcine PRL tracer. These data describe a lactogenic homologous RRA for porcine endometrial membranes similar to that previously reported for murine hepatic tissue. Homologous RRAs may allow elucidation of PRL receptor characteristics with more similarity to the in vivo hormone-receptor milieu.
Mol Cell Endocrinol 1989 Jul
PMID:Porcine endometrial prolactin receptors detected by homologous radioreceptor assay. 250 73

Certain toxic effects of phenytoin are thought to result from its cytochrome P-450-catalyzed bioactivation to a reactive arene oxide intermediate that binds covalently to proteins. Using an in vitro system, we examined an alternative hypothesis based upon the cooxidation of phenytoin to a reactive free radical intermediate by prostaglandin synthetase (PGS), horseradish peroxidase, or thyroid peroxidase. Microsomes from hepatic, thyroid, seminal vesicular, or pulmonary tissues, or PGS or horseradish peroxidase, were incubated with the appropriate enzymatic cofactors to study activities of cytochromes P-450 (NADPH), PGS (arachidonic acid), thyroid peroxidase (guiaicol, H2O2), and horseradish peroxidase (H2O2). The production of potentially teratogenic, reactive phenytoin intermediates during in vitro incubations was estimated by the amount of radiolabeled phenytoin bound covalently to microsomal protein or bovine serum albumin and by the detection of a free radical intermediate using ESR spectrometry. Arachidonic acid-dependent bioactivation of phenytoin was demonstrated for purified PGS and ram seminal vesicles (RSV), as well as for liver, lung, and kidney. Optimal arachidonate concentrations varied substantially for different tissues. Arachidonate-dependent binding of phenytoin with PGS and RSV was reduced to baseline levels by coincubation with the cyclooxygenase inhibitor indomethacin. Hydrogen peroxide-dependent covalent binding of phenytoin was observed with thyroid peroxidase and horseradish peroxidase, and binding was significantly reduced in these systems and in PGS and RSV by coincubation with the peroxidase inhibitor methimazole. Glutathione, the antioxidants caffeic acid and butylated hydroxyanisole, and the free radical trapping agent alpha-phenyl-N-t-butylnitrone (PBN) all significantly reduced arachidonate-dependent phenytoin binding. Oxygen uptake was increased in a dose-dependent manner by the arachidonate-dependent bioactivation of phenytoin by PGS. ESR spin-trapping techniques using PBN indicated the generation of a free radical intermediate during the metabolism of phenytoin by PGS. These results suggest that the hydroperoxidase component of PGS, as well as thyroid peroxidase and other peroxidases, can bioactivate phenytoin to a reactive free radical intermediate, which may be toxicologically relevant.
Mol Pharmacol 1989 Apr
PMID:In vitro bioactivation of phenytoin to a reactive free radical intermediate by prostaglandin synthetase, horseradish peroxidase, and thyroid peroxidase. 253 58

The protein-avidin-biotin capture (PABC) system was developed to decrease the adsorption-induced loss of antigen capture capacity (AgCC) of capture antibodies (CAb) used in sandwich ELISAs. This system involves immobilization of biotinylated CAbs through linkage by streptavidin (SA) to biotinylated carrier proteins adsorbed on polystyrene. Studies reported here describe the stoichiometry of the system and the influence of biotinylation of different carrier proteins and CAbs on the reaction stoichiometry and the AgCC of CAbs. Because of the widespread use of sandwich ELISAs to measure the concn of multivalent protein antigens, the AgCCs of monoclonal and polyclonal CAbs to pig IgG in the PABC system were compared with the AgCCs of these Abs immobilized on the plastic by direct adsorption. Optimal assay conditions for the carrier were obtained when 1 microgram/ml of the biotinylated protein was added to the polystyrene solid phase. An increasing degree of biotin substitution in three carrier proteins was paralleled by an increasing AgCC until a constant maximum was reached. Under conditions of maximal AgCC, 120 ng of the carrier rabbit gamma globulin (RGG; i.e. RGG25biot) was bound to polystyrene, which in turn yielded the maximum amount (i.e. 100 ng) of bound streptavidin (SA; Bdngmax) when 20 micrograms/ml of SA was added. Under conditions giving the Bdngmax for SA, CAb12biot yielded a higher Bdngmax than did CAb25biot or CAb2biot. When the AgCC of equal amounts of differentially biotinylated CAbs were compared, the following order of AgCC was observed: CAb12biot greater than CAb12biot greater than CAb25biot. Hence, while the maximal amount of CAb is immobilized on SA when CAb12biot is used, optimal AgCC is achieved with CAb2biot. The carrier:SA:CAb2biot ratio was 1:2:1 while that for carrier:SA:CAb12biot was 1:2:2. The same ratio was obtained using IgG2biot from four different species. Monoclonal antibodies to swine IgG showed a 5-6-fold increase in Bd%max when immobilized as CAbs using the PABC system versus when adsorbed on polystyrene. Plots of these data suggest that the differences result from a loss of functional affinity. On the contrary, no significant differences in Bd%max and hence functional affinity were observed when a polyclonal antibody to pig IgG was compared using the two assay configurations. Furthermore, when the globulin fraction of the anti-pig polyclonal was adsorbed on plastic, it behaved nearly as well as its affinity-purified counterpart immobilized by the PABC system. The PABC system appears to offer significant advantages for sandwich ELISAs utilizing monoclonal antibodies as the CAb, and may offer some advantages in other s
Mol Immunol 1989 Mar
PMID:The immunochemistry of sandwich ELISAs--III. The stoichiometry and efficacy of the protein-avidin-biotin capture (PABC) system. 270 73

The intercysteine loop sequence (93-100) in the beta-subunit has been postulated to be important for receptor binding and specificity in the glycoprotein hormones, LH and human CG (hCG). To demonstrate this directly, and to characterize the structural features essential for activity, we prepared a series of synthetic peptides and analogs incorporating this determinant loop region. Peptides were assayed for inhibition of labeled hCG binding to ovarian membrane receptors and stimulation of testosterone production in Leydig cells. Peptides with the native (93-100) sequence from hCG and hLH inhibited hCG binding half maximally at 2.18 and 2.62 x 10(-4) M, respectively, while the sequence from FSH was inactive. Isosteric substitution of Ala for Cys resulted in an inactive peptide, indicating that the (93-100) disulfide bridge is essential for activity. Optimal binding activity requires at least one net positive charge among the side chains, as shown by loss of activity in hybrid analogs with neutral or negative charges conferred by progressive replacement of Arg by Asp at 94 and 95 or by introduction of Asp at 96 and 97. Despite binding to receptors, the native sequence did not promote testosterone production at doses up to 10(-2) M. This contrasts with a second receptor binding sequence, beta (38-57) that activates testosterone production. There are differences between the (93-100) and (38-57) loop sequences in their chemical and physical properties, biological activity and antigenicity. While the cumulative evidence suggests that they associate with counterpart sites in alpha-subunit to form a topographical binding domain in the whole hormone, our results suggest that each sequence may contribute in different ways to activation of postreceptor events.
Mol Endocrinol 1989 Mar
PMID:Role of the beta 93-100 determinant loop sequence in receptor binding and biological activity of human luteinizing hormone and chorionic gonadotropin. 274 59

The human alpha-1-antitrypsin (A1AT) gene expressed in Escherichia coli as a full-length, non-fusion gene product accumulates to a relatively low level approaching less than or equal to 0.1% of total cellular protein. In contrast, deletion of the first 5, 10 or 15 codons leads to production of truncated A1AT derivatives at levels between 10 and 30% of total cellular protein. The protein with the largest truncation was insoluble and inactive following solubilization by chaotropic agents. In contrast, the two derivatives with the smaller truncations were found to be soluble, and exhibit identical specific activities in both trypsin and elastase inhibition assays to authentic human A1AT. The expression of the full-length A1AT was also optimized by making silent third position mutations within its first 15 codons. These mutations were chosen to optimize codon usage and minimize the possibility of RNA secondary structure formation in this region. Via this approach, expression of full-length, authentic, fully active A1AT was increased at least 20-fold to 2% of total cellular protein. Optimal expression was obtained using as few as three silent mutations in the first five codons, confirming the importance of this 5'-terminal region as had been defined by our deletion mutants. Both the full-length derivatives as well as the small N-terminal deletion derivative can be readily purified from bacterial extracts in fully active form suitable for the examination of their potential therapeutic application.
Mol Biol Med 1987 Oct
PMID:High-level production of fully active human alpha 1-antitrypsin in Escherichia coli. 282 66

The binding to catecholamines to the beta-adrenergic receptors on human polymorphonuclear leukocytes rapidly inhibits cell responses stimulated by chemoattractant ligands. As a first step in understanding the mechanism of the inhibition, we investigated the number of beta-receptors required to optimally block superoxide anion production, a response that is measured kinetically by a convenient spectrophotometric assay for the reduction of cytochrome c. We found that after blockade of 50-60% of the beta-adrenergic receptors with an irreversible antagonist, maximal inhibition of the response was still elicited by isoproterenol (ISO). Next we investigated the kinetics with which superoxide generation is inhibited. We found that half-maximal inhibition was observed at 3 x 10(-8) M ISO, which approximates the Kd because many of the receptors are involved. Cell responsiveness recovered when propranolol was added between the time of ISO and chemoattractant addition. From the recovery we estimated that the half-time for ISO dissociation is less than 10 sec. Finally, we examined the rate at which cell responses decay following ISO administration after chemoattractant. Optimal rates of inhibition, turning off oxidant production in seconds, occur at ISO concentrations greater than or equal to 3 x 10(-7) M. Taken together, these observations are consistent with an association rate constant for ISO estimated to be greater than or equal to 10(8) M-1min-1 and a dissociation rate constant greater than or equal to 4 min-1. These results are discussed in terms of the available data concerning the binding of agonists to beta-adrenergic receptors.
Mol Pharmacol 1988 Sep
PMID:The potency and kinetics of the beta-adrenergic receptors on human neutrophils. 290 65

Quantitative analysis of nitrocellulose filter binding data by the method of Clore, Gronenborn and Davies [(1982) J. Mol. Biol. 155, 447-466] has been used to show that lambda integration protein (Int) exhibits cooperativity in binding to specific recognition sites within the attachment site region (lambda attP) of bacteriophage lambda DNA. Optimal values of the equilibrium constant obtained were 3.0(+/- 1.0) X 10(10) M-1 for the P' site using a model of three sites with equal affinity and 1.9(+/- 0.4) X 10(10) M-1 for the P1 site on a two-site model. The value of the cooperativity parameter alpha is 172(+106)(-66) in all cases. The occurrence of a consensus recognition sequence is necessary but not sufficient for strong binding; cooperative interaction between Int molecules binding to adjacent members of an array of binding sites is also essential. The occurrence of binding site arrays distinguishes lambda attP very clearly from other DNA sequences containing single recognition sites by chance.
 ...
PMID:Cooperative DNA binding by lambda integration protein--a key component of specificity. 302 82


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