RESUMO
The in-situ localisation of a desulpho-glucosinolate precursor has been studied by microautoradiography of cryo-sections from immature seeds and pods of the high-glucosinolate Brassica napus L. cv. Argentine collected 23 days after pollination. After feeding with the tritium-labelled glucosinolate precursor [4,5-3H](beta-D-glucopyranosyl)-4-pentenethiohydroxamic acid, embryo radicles, cotyledons and pod-wall were frozen in liquid nitrogen. Cryotome sections were freeze-dried and coated with nuclear emulsion autoradiographic film. A distinct pattern of radioactivity derived from the glucosinolate precursor was found in specific cells in both radicle and cotyledons. In contrast, the labelling in pod walls was not cell specific, but general at the inner side of the pod wall. The results show that the glucosinolate/desulphoglucosinolate was localised in specific cells, in a pattern resembling that of myrosin cells known to contain myrosinase (EC 3.2.3.1). In addition [4,5-3H](beta-D-glucopyranosyl)-4-pentenethiohydroxamic acid was fed to immature seeds and pods of B. napus and a quantitative incorporation into 2-hydroxy-3-butenylglucosinolate and 3-butenyl-glucosinolate was observed. When [4,5-3H](beta-D-glucopyranosyl)-4-pentenethiohydroxamic acid was fed to 4-day-old seedlings the label was taken up by all tissues. We propose a model in which glucosinolate/desulphoglucosinolates are transported to myrosin cells to participate in the myrosinase-glucosinolate multifunctional defence system.
Assuntos
Brassica napus/metabolismo , Glucosinolatos/metabolismo , Glicosídeo Hidrolases/metabolismo , Adaptação Fisiológica , Ácido Ascórbico/farmacologia , Autorradiografia , Brassica napus/citologia , Brassica napus/embriologia , Brassica napus/crescimento & desenvolvimento , Radioisótopos de Carbono , Estruturas Vegetais/citologia , Estruturas Vegetais/embriologia , Estruturas Vegetais/crescimento & desenvolvimentoRESUMO
Human T-cell lymphotropic virus 1 (HTLV-1) is a type C human retrovirus, which is the causative agent of Adult T-cell Leukemia and other diseases. The reverse transcriptase of HTLV-1 (E.C. 2.7.7.49) is synthesized as part of a Gag--Pro--Pol precursor protein, and the mature Gag, Pro, and Pol proteins, including the reverse transcriptase, are created by proteolytic processing catalyzed by the viral protease. The location of the proteolytic cleavage site, which creates the N-terminus of mature HTLV-1 reverse transcriptase, has not been previously identified. By using sequence comparisons of several retroviral polymerases, as well as information about the location of the ribosomal frameshift, we tentatively identified this N-terminal processing site. PCR amplification was used to construct a clone, which spans a region of the pro--pol junction of HTLV-1, to produce a recombinant Pro--Pol protein spanning the locations of those cleavage sites proposed by others as well as the one identified by our sequence alignment. Cleavage of the recombinant Pro--Pol protein by HTLV-1 protease generated a 5.5-kDa fragment. Analysis of this fragment by capillary LC-MS and MS/MS revealed the N-terminal cleavage site to be between Leu(147)--Pro(148) of the pro ORF. This is the first physical identification of the authentic amino acid sequence of the reverse transcriptase of HTLV-1. The data reported here provides a basis for further investigation of the function and structural aspects of protein-nucleic interaction.
Assuntos
Vírus Linfotrópico T Tipo 1 Humano/enzimologia , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Primers do DNA/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Técnicas In Vitro , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , DNA Polimerase Dirigida por RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TripsinaRESUMO
We have investigated the ability of pyridoxal-5'-phosphate to inhibit a recombinant deletion mutant of human immunodeficiency virus type 1(HIV-1) reverse transcriptase (RT) which is missing the last 23 amino acids of the C-terminus. This mutant reverse transcriptase is characterized by normal polymerase activity as compared with full-length enzyme; however, it has no RNase H activity. Inhibition studies with pyridoxal-5'-phosphate showed several differences as compared with inhibition of full-length enzyme: (1) Inhibition of mutant reverse transcriptase was independent of divalent cation, (2) Either substrate alone could protect mutant reverse transcriptase from inactivation by pyridoxal-5'-phosphate, and (3) stoichiometry of pyridoxal-5'-phosphate binding to mutant reverse transcriptase was 2 mol/mol under the same conditions in which 1 mol/mol bound to full-length enzyme. Furthermore, in the presence of either substrate alone, the stoichiometry of pyridoxal-5'-phosphate binding to the mutant was reduced to 1 mol/mol. These results indicate that the second binding site for pyridoxal-5'-phosphate seen in the mutant reverse transcriptase is at or near the primer-template binding site of the enzyme. They also suggest that the RNase H domain of HIV RT plays a functional role in substrate binding at the polymerase domain.
Assuntos
HIV-1/enzimologia , Inibidores da Síntese de Ácido Nucleico , Fosfato de Piridoxal/farmacologia , Inibidores da Transcriptase Reversa , Ribonuclease H/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , HIV-1/genética , Cinética , Magnésio/metabolismo , Mutação , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Ribonuclease H/genéticaRESUMO
Human immunodeficiency virus Type I reverse transcriptase is active as either the homodimer (p66/p66) or the heterodimer (p66/p51). Purified recombinant p66 and p51 expressed in yeast were reconstituted in the presence of 60 mM sodium pyrophosphate to enhance dimer formation. Comparison of the processivity of these two active reconstituted forms shows that the heterodimer is more processive than the homodimer with a cycle almost twice as long as judged by assays utilizing poly (U,G) as a challenger to primer-template. Binding assays demonstrated that the heterodimer has a higher affinity for primer-template than the homodimer and that the p51 subunit has an affinity equal to that of the heterodimer. These results suggest that the p51 subunit functions to increase processivity in the heterodimer.
Assuntos
HIV-1/enzimologia , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Clonagem Molecular , Genes Virais , HIV-1/genética , Cinética , Substâncias Macromoleculares , DNA Polimerase Dirigida por RNA/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Moldes GenéticosRESUMO
The reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) is comprised of two subunits of approximately 66kD and 51kD. We have defined the carboxyl terminus of the 51kD molecule using the 66kD RT and HIV-1 protease (PR) expressed in yeast. Precise constructs encoding the 66kD and 51kD molecules were expressed individually, in yeast, at high levels. The purified recombinant subunits were shown to associate into heterodimers that retained both RT and RNase H activities. Only the 66kD molecule could associate into homodimers. Such homodimers retained approximately 80% of the RT activity of the heterodimers. Our data demonstrates that the 51/66kD heterodimer, analogous to that found in vivo, can be reconstituted in vitro and is more efficient in both RT and RNase H activity than the homodimer.
Assuntos
HIV-1/genética , DNA Polimerase Dirigida por RNA/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Cromatografia em Gel , Clonagem Molecular , DNA Viral/genética , HIV-1/enzimologia , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , DNA Polimerase Dirigida por RNA/isolamento & purificação , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
We have isolated a highly enriched preparation of the multienzyme complex which synthesizes deoxyribonucleoside triphosphates (dNTPs) from bacteriophage T4-infected bacteria. By a combination of SDS polyacrylamide gel electrophoresis and assays for specific enzyme activities, we have been able to identify in our final preparation ten different gene products which were previously identified as constituents of this complex, based upon studies with crude preparations. The complex dissociates at high concentrations of NaCl and MgCl2 but is stable under ionic conditions thought to exist in vivo. The purified complex catalyzes the efficient five-step conversion of dCTP to dTTP. Experiments with several T4 mutants have demonstrated that gene products encoded by cd, regA, nrdA, and nrdB are necessary to retain physical integrity of the complex throughout the preparative procedure, while gp44, gp55, and gppseT are not required. We conclude from this evidence that the T4 early gene products which function in dNTP biosynthesis are, in fact, physically linked as a multienzyme complex, and that regA contributes to the integrity of this complex. However, the dNTP-synthesizing complex as we isolate it contains no detectable DNA polymerase, nor have other known replication proteins been detected.