Some pecularities of functioning of H⁺‐ATPase from the membranes of the anaerobic bacterium Lactobacillus casei

Abstract

Radiation inactivation analysis gave the target sizes of 176 & 5 kDa and 275 33 kDa for ATPase from anaerobic Lactobacillus casei and aerobic Micrococcus luteus bacteria respectively. The values are close to the known molecular masses of the enzymes. Thus, to function the L. casei ATPase, like the F1-ATPases, requires a complete structure composed of all the enzyme subunits. L. casei ATPase is inhibited by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole owing to modification of an amino acid residue(s) with pK > 8.5. L. casei ATPase consists of six identical subunits and differs from ct3P3y&-type F1-ATPases in a number of catalytic properties. Namely, ATP hydrolysis under the 'unisite' conditions proceeds at a relatively high rate suggesting the absence of cooperative interactions between the catalytic sites. Contrary to mitochondria1 F1-ATPase. L. casei ATPase does not form an inactive complex with ADP. These findings imply essential differences in the operating mechanism for L. casei ATPase and F1 ATPase.Unravelling the molecular mechanism of the H ' -ATPase

Subunit composition of the H+‐ATPase complex from anaerobic bacterium Lactobacillus casei

“…These structural differences can also be due to the fact that the L. casei ATPase complex has no such regulatory systems as a native protein inhibitor [30,311 or SH regulatory system [32]. As was previously shown [14], trypsin treatment of L. casei membranes or the soluble enzyme failed to stimulate ATPase activity. Treatment of these preparations with SH reagent dithiothreitol neither influenced ATPase activity nor stimulated the membrane potential generation as in aerobic bacteria [33] and chloroplasts [32].…”

“…The soluble ATPase was purified by gel filtration on a column packed with Toypearl HW-55F gel (Toyo Soda, Japan) equilibrated with 10 mM Tris/HCl buffer, pH 7.8, 100 mM KCl [14].…”

“…Treatment of these preparations with SH reagent dithiothreitol neither influenced ATPase activity nor stimulated the membrane potential generation as in aerobic bacteria [33] and chloroplasts [32]. Besides, it was shown that L. casei soluble ATPase differs from F,-ATPases in a number of catalytic properties and the most essential among them are unusual parameters of the unisite catalysis [14]. At the substrate concentration comparable to the enzyme concentration, a relatively high rate of catalysis was observed.…”

“…The ability of the ATPase for covalent 4-chloro-7-nitrobenzofuran (NBD-C1)modification [14] indicates a certain similarity between the L. casei ATPase major subunits and the p subunits of F1 ATPases.…”

Avaliação funcional dos inibidores químicos da NAD(P)+ transidrogenase mitocondrial

“…Diante dos resultados favoráveis obtidos em medidas de atividade de NNT e respiração mitocondrial na presença de NBD-Cl, foram feitos testes adicionais com esse composto. Em estudos da literatura, amostras de proteína ou mitocôndrias foram pré-incubadas com NBC-Cl com o intuito de potencializar seu efeito inibitório tanto sobre a NNT quanto sobre a ATP sintase (64,65,(93)(94)(95). No entanto, em nossas condições experimentais, não foi observado aumento da inibição da atividade de NNT por NBD-Cl após 20 minutos de pré-incubação, enquanto ocorreu inibição significativa do controle respiratório mesmo em condições controle (Capítulo I, Figura S1).…”