Algae acid phosphatase affected by ten metals and enzymatic parameters determination in presence of mercury and copper

Acid phosphatases play important roles in algae metabolism such availability/recycling of inorganic phosphate and autophagic digestive processes. Chemicals released into the environment from agricultural activities and through industrial and urban wastes, may impair algae enzyme activity. The aim of this work was to evaluate the in vitro activation/inhibition effect of ten metals, commonly present as contaminants in soil and water, on the acid phospahatase extracted from the green algae Pseudokirchneriella subcapitata. Results demonstrated that Hg, Al, Mo, Pb, Se, and Cd inhibited the enzyme activity in 56.3, 54.5, 30.6, 25.5, 23.1 and 11.5 per cent respectively. This corresponds to the maximum percentage of effect attained at the metal concentration tested (0.02-2.0 mM). On the other hand, Cu, Zn, Ni and Cr exhibited an increment on phosphatase activity equal to 95.5, 87.6, 87.6, 77.6 and 42.8 per cent, respectively. Kinetic parameters values were calculated for the metals that showed hisghest effects. Thus, Ki ( inhibition constant) and Kd (dissociation constant) values equal to 0.0400 and 0.0016 mM were determined for Hg and Cu, respectively. A non-competitive inhibition mechanism was attributed to the former. Results improved the understanding of the basic events of the impact of metals at biochemical lebels in primary producers organisms.

Citotoxicidade do promotor de tumor e sua ação mitogênica sobre os linfócitos humanos / Cytotoxicity studies of okadaic acid on human lymphocytes and its mitogenic effect

Proteínas fosfatases são moléculas sinalizadoras que agem juntamente com as proteínas quinases para regular uma variedade de processos celulares fundamentais, bem como, crescimento celular, mitogênese, metabolismo, transcrição de gene, ciclo celular e resposta ao estresse e imune. O ácido okadáico é um potente e específico inibidor de proteína serina/treonina fosfatase (PP1 e PP2A). O objetivo deste estudo foi avaliar o efeito citotóxico do ácido okadáico na viabilidade de linfócitos humanos e sua ação mitogênica. Nos estudos de citotoxicidade avaliamos o efeito do ácido okadáico através dos seguintes parâmetros: redução do MTT (integridade mitocondrial), conteúdo total de proteínas (número de células) e atividade fosfatásica (metabolismo celular). O valor para redução do MTT e atividade fosfatase foram: 50nM e 100nM, respectivamente; não foi encontrado valor de IC50 para o conteúdo de proteína. A atividade fosfatásica não foi afetada pelo ácido okadáico (100nM) quando este composto foi adicionado no extrato celular. A proliferação de linfócitos foi estimulada em 25 porcento quando as células foram tratadas com o ácido okadáico durante o plaqueamento e na ausência da fitohemaglutinina (mitogêno). O estímulo máximo foi até 30 minutos. O ácido okadáico não foi citotóxico para os linfócitos. A ação mitogênica deste composto foi confirmada pelo conteúdo de proteína.

Purification and characterization of a low-molecular-weight bovine kidney acid phosphatase

A relative low molecular mass bovine kidney acid phosphatase was purified 1,640-fold to homogeneity, with 7 percent recovery. The purified enzyme (specific activity 100 mumol min-1 mg-1) was electrophoretically homogeneous with a relative molecular massa of 17.8 kDa, as determined by SDS-polyacrylamide gel electrophoresis. A broad pH optimum of 4.0-5.5 and a maximal enzyme activity at 60 degrees Celsius were determined for the p-nitrophenyl phosphate hydrolysis. Apparent Km values of 0.14 mM, 0.4 mM, 0.3 mM and 7.9 mM were obtained, at 37 degrees Celsius and pH 5.0, for the best substrates p-nitrophenyl phosphate, beta-naphtyl-phosphate, flavin mononucleotide and tyrosine-phosphate, respectively. The enzyme activity was enhanced by guanosine but inhibited by ZnCl2 and CuSO4, p-cloromercuribenzoate and ammonium molybdate. Vanadate (Ki 0.47 muM), pyridoxal 5'-phosphate (Ki 2.2 muM), inorganic phosphate (Ki 0.77 mM) are competitive inhibitors. Both glycerol and methanol increased significantly the acide phosphatase activity, acting as good phosphate acceptors in the transphosphorylation reaction.

Kinetic properties of poly (2'-0-methycytidylic acid)- directed reverse transcriptase reaction

Poly (2'-O-methylcytidylic acid) is recognized as a template in reactions catalyzed by RNA-dependent DNA polymerases in the presence of Mn2+ as divalent cation. We report that kinetic data obtained for dGTP and template under optimal experimental conditions in the reaction catalyzed by reverse transcriptase showed some similarities between the poly (2'-O-methylcytidylic acid)Mn2+ and polyribocytidylic acid/Mg2+ systems. The reaction was inhibited by the action of N-ethylmaleimide and novobiocin, and to a lesser extent by ethidium bromide and tetramethyl ethidium bromide

O reverso da transcriçäo / The reverse of the transcription

O objetivo da presente revisäo é mostrar alguns aspectos relacionados com o reverso da transcriçäo, incluindo descriçäo do retrovírus, ciclo de vida do vírus, mecanismo do reverso da transcriçäo, propriedades cinéticas e imunológicas da transcritase reversa, e inibidores da enzima viral. Estäo também incluídas algumas referências sobre o retrovírus AIDS. O reverso da transcriçäo parece näo ser restrito apenas aos vírus de RNA. Alguns vírus de DNA, como o vírus da hepatite B, e o vírus do mosaico da couve-flor, e outros elementos genéticos, como pseudogenes e elementos de transposiçäo, parecem utilizar o reverso da transcriçäo para suas existências

Poly (2'-0-methyladenylic acid) as template of reverse transcriptase

Poly (2'-O-methyladenylic acid), poly (Am), is recognized as template in the reaction catalyzed by avian myeloblastosis virus, AMV, reverse transcriptase better in the presence of Mn than Mg as divalent cation. An apparent KM value of 2.5 x 10 M in relation to TTP and an inhibition of the reaction at poly (Am concentration higher than 5 microng/ml have been observed. Ethidium bromide, EtBr, tetramethyl ethidium bromide, TMEtBr, dideoxy TTP, and novobiocin are inhibitors of the poly (Am)-directed reverse transcriptase reaction in the presence of Mn

Threonyl-tRNA synthetase from bovine liver: purification and kinetic properties in the ATP-pyrophosphate exchange reaction

Treonil-RNA sintetase (E.C. 6.1.1.3) foi purificada quase à homogeneidade de fígado bovino cerca de 500 vezes com um rendimento de 48%. Duas bandas de pesos moleculares 90.000 e 82.000, respectivamente, foram obtidas por eletroforese em gel em presença de dodecil sulfato de sódio. A enzima tem um ponto isoelétrico de 5,2 por eletroforese em gel de poliacrilamida contendo anfoline. Utilizando-se a reaçäo de intercâmbio ATP-PPi foram determinadas as condiçöes ótimas de ensaio e os valores aparentes de Km. através da mesma reaçäo foram também observados efeitos de cátions divalentes e diaminas em substituiçäo ao Mg2+ e efeitos de reagentes sulfidrílicos