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S Ray

Publications and source records attributed to S Ray.

At least 19 recordsLinked to original sources
PubMed1992

Cloning, expression, and crystallization of recoverin, a calcium sensor in vision.

Recoverin, a recently discovered 23-kDa calcium-binding protein, activates retinal rod guanylate cyclase when the calcium level is lowered in the submicromolar range. We report here the cloning and sequencing of a cDNA for recoverin from a bovine retinal expression library. The recoverin coding sequence was inserted into a pET-11a expression vector under control of the T7 phage promoter. A second expression system, in which the coding sequence was placed under control of the lambda phage PR promoter, gave 10-fold higher yields (10 mg of purified recoverin per liter of Escherichia coli culture). The finding that retinal recoverin is myristoylated at its amino terminus led us to coexpress the recombinant protein and N-myristoyltransferase (EC 2.3.1.97). Myristoylated recombinant recoverin formed in this way in E. coli is like retinal recoverin in exhibiting a large calcium-induced shift in its tryptophan fluorescence emission spectrum. The availability of abundant protein enabled us to crystallize unmyristoylated recombinant recoverin and initiate x-ray studies. The space group of tetragonal crystals obtained from 75% saturation ammonium sulfate is I4 with unit cell dimensions a = 85.1 A and c = 59.8 A. These crystals of the calcium-bound form of the protein diffracted to a resolution of 2.2 A. The expression systems described here open the door to high-resolution x-ray crystallographic and nuclear magnetic resonance studies of this new member of the EF-hand superfamily and to the elucidation of its precise mode of action as a calcium switch.

Amino Acid Sequence↗
PubMed1992

Synthesis of 18F-labeled fluconazole and positron emission tomography studies in rabbits.

[4-18F] 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-l-yl)-2-propanol [( 4-18F] fluconazole) was synthesized from its amino precursor. Fieldel-Crafts acylation of 3-fluoroacetanilide with chloroacetyl chloride produced 2'-fluoro-4'-acteamido-2-(1H-1,2,4-triazole-1-yl) acetophenone in 12% yield. Sequential reaction with (1) dimethylsulphoxonium methylide and (2) 1,2,4-triazole followed by in situ hydrolysis resulted in 2-(2-fluoro-4-aminophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)-2-propan ol in 19% yield. A modified Schiemann reaction on this product resulted in [4-18F]fluconazole with a radiochemical yield of 1.0-2.0% (EOS) within 2 h. [4-18F]Fluconazole was used to measure the pharmacokinetics of fluconazole in rats by measurement of radioactivity in excised tissues and in rabbits by PET. In both species, there was rapid equilibration of [4-18F]fluconazole to a relatively uniform distribution of radioactivity in most organs.

Animals↗
PubMed1992

Functional characterization of temperature-sensitive mutants of simian virus 40 large T antigen.

We investigated the molecular properties of eight temperature-sensitive mutants of simian virus 40 large T antigen (tsA mutants). The mutants have single amino acid substitutions that block DNA replication at 39 to 41 degrees C in vivo. In vitro, five of the mutant proteins were highly sensitive to a brief heat shock at 39 degrees C, while the three remaining proteins were only partially sensitive at 41 degrees C. We characterized the five most defective mutant proteins, using a variety of biochemical assays for replication functions of T antigen. Heat shock of purified T antigen with a mutation at amino acid 422 significantly impaired the oligomerization, origin-binding, origin-unwinding, ATPase, and helicase functions of T antigen. In contrast, substitution of amino acid 186, 357, 427, or 438 had more selective, temperature-sensitive effects on T-antigen functions. Our findings are consistent with the conclusion that T antigen functions via a hierarchy of interrelated domains. Only the ATPase activity remained intact in the absence of all other functions. Hexamer formation appears to be necessary for core origin-unwinding and helicase activities; the helicase function also requires ATPase activity. All five tsA mutants were impaired in functions important for the initiation of DNA replication, but three mutants retained significant elongation functions.

Adenosine Triphosphatases↗
PubMed1992

Effects of progesterone on some brain neurotransmitters in intact rats.

Effects of progesterone on four neurotransmitters (viz, noradrenaline, 5-HT, dopamine and histamine) of brain were seen in rats with intact ovaries. It was found that progesterone lowers the noradrenaline concentration in medulla, pons, midbrain, hypothalamus, thalami and pituitary, uniformly, when the rats were killed within 4 hours of progesterone injection. At longer intervals (48 hrs) effects of progesterone were seen when progesterone in heavy dose was administered to rats pretreated with estrogen. It is likely that one of the modes of action of the oral contraceptives may be the reduction of noradrenaline content in selected areas of brain, by progesterone. It is also suggested, therapeutic usage of progesterone carries the risk of development of depression in the user.

Animals↗
PubMed1992

Microenvironment at the substrate binding subsite of the active site of UDPglucose 4-epimerase from Kluyveromyces fragilis using a fluorescent analog of UMP.

A chromophorics and fluorescent analog of uridine 5'-monophosphate (UMP), a known competitive inhibitor of UDPglucose 4-epimerase was synthesised. This analog, namely 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) uridine 5'-monophosphate, was found to be a powerful reversible inhibitor of UDPglucose 4-epimerase indicating its interaction with the substrate binding site of the enzyme. The extreme sensitivity of the fluorescence emission spectrum of this analog to solvent polarity makes it an excellent probe for the study of the environment at the active site of the enzyme. We report here the effective use of this UMP analog to demonstrate that the hydroxyl groups of the ribose moiety of UMP and presumably the substrates (UDPgalactose and UDPglucose) do not reside in a hydrophobic milieu.

Binding Sites↗
PubMed1991

Aminoacetone synthase from goat liver. Involvement of arginine residue at the active site and on the stability of the enzyme.

The arginine-specific reagents phenylglyoxal and butane-2,3-dione inactivated goat liver aminoacetone synthase with pseudo-first-order kinetics, with the rate dependent on modifier concentration. Phenylglyoxal and butane-2,3-dione appeared to react with one arginine residue per enzyme molecule. The inactivated enzyme could be re-activated by Tris, suggesting additional evidence of modification of the arginine residue. Acetyl-CoA, one of the substrates, completely protected the enzyme from inactivation. Glycine gave partial protection. Protection by substrates against inactivation by phenylglyoxal and butane-2,3-dione suggested the presence of an essential arginine residue at the substrate-binding region. Experiments with [7-14C]phenylglyoxal in the presence of acetyl-CoA showed that only the arginine residue at the active site could be modified by phenylglyoxal. The stability of the enzyme is dependent on the presence of both EDTA and Mg2+.

Acetyl Coenzyme A↗
PubMed1991

Recoverin: a calcium sensitive activator of retinal rod guanylate cyclase.

Vertebrate retinal photoreceptors recover from photoexcitation-induced hydrolysis of guanosine 3', 5'-monophosphate (cyclic GMP) by resynthesizing cyclic GMP, which reopens cation channels that have been closed by light. Activation of guanylate cyclase by light-induced depletion of cytosolic calcium is a key event in this recovery process. This cyclase has now been shown to be regulated by a 23-kilodalton calcium binding protein. The protein is present in both rod and cone photoreceptors and was named recoverin because it promotes recovery of the dark state. The amino acid sequence of recoverin exhibits three potential calcium binding sites (EF hands). That recoverin binds calcium was confirmed with calcium-45 and by observing calcium-induced changes in its tryptophan fluorescence. Recoverin activated guanylate cyclase when free calcium was lowered from 450 to 40 nM, an effect that was blocked by an antibody to recoverin. Thus, guanylate cyclase in retinal rods is stimulated during recovery by the calcium-free form of recoverin. A comparison of recoverin with other calcium binding proteins reveals that it may represent, along with the protein visinin, a family of proteins that are regulated by submicromolar calcium concentrations.

Amino Acid Sequence↗
PubMed1991

Synthesis and pregnancy-inhibiting activity of 7-substituted androst-5-ene derivatives.

Synthesis of 7-aryl/allyl-substituted androstene derivatives 3a through 3g has been carried out by Grignard reaction on 3 beta,17 beta-diacetoxyandrost-5-en-7-one (2) with aryl/allyl magnesium bromide. Isomeric mixture of products 3b and 3c/3e and 3f/3h was separated by column chromatography. Stereochemical assignment at C-7 has been made on the basis of 13C nuclear magnetic resonance studies and chemical considerations. Compounds 6a and 6b were synthesized by alkylation of compound 5 with beta-(N,N-diethylamino)ethyl chloride hydrochloride and 1-(2-chloroethyl)pyrrolidine hydrochloride, respectively. Compound 3g (isomeric mixture) prevented pregnancy in 60% of rats at 10 mg/kg daily dose administered orally on days 1 to 7 of pregnancy; however, its only isolable 7 beta-hydroxy isomer, 3h, was inactive at this dose.

Androstenes↗
PubMed1991

Sequence variation between alleles reveals two types of copy correction at the 27-kDa zein locus of maize.

In many inbred lines of maize, two 27-kDa storage protein (zein) genes are found within tandem duplications of 12 kb. Both genes of the duplicated allele from the maize inbred line A188 were sequenced and compared to a similar duplicated allele in another inbred line, W22, and to a single-copy allele in the inbred line W64A. The comparisons reveal interesting patterns in the distribution of sequence changes between these alleles. Differences between the two duplicated alleles that are conserved between the two genes of each allele are found exclusively in the 5' region. In contrast, differences between the individual genes of each allele in the 3' region are conserved between the two alleles. The first case is indicative of an intraallelic copy correction mechanism, whereas the second may result from interallelic copy correction. These may be mediated by gene conversion processes, as previously described for other multigene families.

Alleles↗
PubMed1991

Cooperative assembly of simian virus 40 T-antigen hexamers on functional halves of the replication origin.

The cofactor ATP stimulates the formation of T-antigen double hexamers on the simian virus 40 core origin of replication (I. A. Mastrangelo, P. V. C. Hough, J. S. Wall, M. Dodson, F. B. Dean, and J. Horwitz, Nature [London] 338:658-662, 1989). We report here the pathway for the assembly of hexamers and double hexamers on the core origin. ATP triggers the cooperative assembly of hexamers on the early and late halves of the origin even when they are completely isolated. Hexamer assembly nucleates at T-antigen recognition pentanucleotides in the early half of the origin. In intact origins, assembly of the first hexamer on the early half of the origin cooperatively stimulates the assembly of a second hexamer on the adjacent late half of the origin. Thus, monomer-monomer and hexamer-hexamer interactions of T antigen, allosterically activated by ATP, constitute two distinct types of cooperative interaction with the origin. Finally, we show that the assembly of T-antigen hexamers on isolated half origins leads to the same array of structural changes that T antigen induces in intact origins. We conclude that the origin is divided into complementary halves that each promote the assembly of functional T-antigen hexamers.

Adenosine Triphosphate↗
PubMed1991

The zinc finger region of simian virus 40 large T antigen is needed for hexamer assembly and origin melting.

Simian virus 40 large T antigen contains a single sequence element with an arrangement of cysteines and histidines that is characteristic of a zinc finger motif. The finger region maps from amino acids 302 through 320 and has the sequence C-302 L K C-305 I K K E Q P S H Y K Y H-317 E K H-320. Previous genetic analysis has shown that the cysteine and histidine sequences and the contiguous S H Y K Y region in the finger are important for DNA replication in vivo. We show here that representative mutations in either of these elements of the finger prevent the assembly of large T antigen into stable hexamers in vitro. These same mutations have a characteristic effect on the interaction of T antigen with the simian virus 40 core origin of replication. The mutant T antigens bind to the central pentanucleotide domain of the core origin but fail to melt the adjacent inverted repeat domain and to untwist the adenine-thymine domain. These defects would prevent the formation of a replication bubble and the initiation of DNA replication. Finger mutations have lesser effects on the helicase function of T antigen and no observable effect on binding of T antigen to the mouse p53 protein. We propose that the zinc finger region contributes to protein-protein interactions essential for the assembly of stable T-antigen hexamers at the origin of replication and that hexamers are needed for subsequent alterations in the structure of origin DNA. We cannot exclude the possibility that the zinc finger region also makes specific contacts with components of origin DNA.

Amino Acid Sequence↗
PubMed1991

Pharmacokinetics of 18F-labeled fluconazole in rabbits with candidal infections studied with positron emission tomography.

[4-18F]Fluconazole was used to measure the pharmacokinetics of fluconazole in normal and infected animals. The biodistribution of fluconazole was determined after administration of the 18F-tracer with/without a pharmacological dose of unlabeled drug by radioactivity measurements on excised tissues. In normal rabbits and rabbits with candidal infection of the thigh, tissue concentrations of drug were determined by serial positron emission tomographic imaging. In rats, coinjection of tracer quantities of [4-18F]fluconazole with a pharmacological dose of unlabeled drug resulted in a relatively uniform distribution of radioactivity in most organs, whereas, when the 18F-tracer was injected alone, spleen, muscle and heart accumulation was decreased and liver accumulation was increased. In rabbits, this effect was less pronounced. Early accumulation of [4-18F]fluconazole was greater in infected muscle. The areas under the 2-hr uptake curves were 4.30 and 6.05 micrograms.hr.ml-1 for normal and infected tissue. A mathematical model was used to summarize the kinetics of fluconazole in normal and infected muscle. The model hypothesizes that fluconazole is compartmentalized in blood and tissue, with rate constants describing the transition between compartments. Direct measurement of the partition coefficient of fluconazole in muscle and predictions of the kinetic model were in close agreement, suggesting that fluconazole enters muscle via a passive transport mechanism. Transport rates of fluconazole, into (Kin) and out of tissue (kout), were increased in infected compared with normal muscle, possibly due to increased capillary permeability (Kin: 0.064 +/- 0.001 vs. 0.0270 +/- 0.0002, kout: 0.063 +/- 0.002 vs. 0.035 +/- 0.001).

Animals↗
PubMed1991

Developmental expression of the embryonic chicken brain DNA polymerase alpha and its binding with monoclonal antibodies against human KB cell DNA polymerase alpha.

Changes in DNA polymerase alpha activity accompanying tissue development have been well established in several systems. In most cases, DNA polymerase alpha activity decreases with development. Here, we report observed changes in DNA polymerase alpha activity throughout embryonic chicken brain (ECB) development. The level of DNA polymerase alpha activity was found to gradually decrease by 60% (2.3 to 0.8 nmol of [3H]dCMP incorporated/mg protein/h) between 9- and 19-day-old ECB. An enzyme-linked immunosorbent assay of DNA polymerase alpha utilizing monoclonal antibody SJK 237-71 (human KB cell DNA pol-alpha binder) also demonstrated a gradual decrease (up to 60%) of antigen over this same range of development. Analysis of DNA polymerase alpha from 11- and 19-day-old ECB by a 10 to 30% glycerol density gradient revealed a high molecular weight peak sedimenting near catalase (11.3 S) with activity at the 11th day being approximately 3-fold greater than activity at the 19th day. A Western immunoblot analysis utilizing monoclonal antibody SJK 237-71 (against human KB cell DNA polymerase alpha) showed a decrease in DNA polymerase alpha from 186 kilodaltons in 9- and 11-day ECB cell-free extracts to 120 kilodaltons in extracts from 13- to 19-day ECB. The conversion of DNA polymerase alpha from a higher to a lower molecular weight form may be a regulatory mechanism in eukaryotic DNA replication.

Animals↗
PubMed1991

A PMA degrading constitutive organomercurial lyase in a broad-spectrum mercury resistant Bacillus pasteurii strain DR2.

A broad-spectrum Hg-resistant strain of B. pasteurii DR2 utilized phenylmercuric acetate (PMA) as sole source of carbon. This bacterial strain contained a constitutive organomercurial lyase which specifically degraded PMA but not other organo-mercurials. This PMA-lyase activity was also stimulated to different extents when this bacterial strain was grown in presence of different organic compounds as sole source of carbon.

Bacillus↗