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1.
Biochemistry ; 35(34): 11019-25, 1996 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-8780503

RESUMO

The sarcoplasmic reticulum ATPase segment (Thr316-Leu356) connecting the extramembranous phosphorylation domain to the preceding transmembrane helix M4 (which is an integral component of the Ca2+ binding domain) retains a high degree of sequence homology with other cation transport ATPases. Single, non conservative mutations of homologous residues in this segment produces enzyme inhibition (Zhang et al., 1995). We have now produced single and multiple mutations of non-homologous residues in this segment of the Ca2+ ATPase to match the corresponding residues of the Na+, K+ ATPase. We find that the main characteristics of the ATPase mechanism (i.e., Ca2+ dependent phosphoenzyme formation and thapsigargin sensitivity) are retained even when the entire 41-amino acid (Thr316-Leu356) segment of the Ca2+ ATPase is rendered identical to the corresponding segment of the Na+, K+ ATPase by sequential mutations of the 14 non-homologous amino acids. However, the phosphoenzyme turnover (likely rate limited by the "Ca2.E1-P-->Ca.E2-P transition") is progressively reduced if four or more Ca2+ ATPase residues are mutated to the corresponding residues of the Na+, K+ ATPase. The time course of enzyme inactivation by EGTA (likely rate limited by the "E1 to E2 transition") is also prolonged. Our findings suggest that an analogous peptide segment provides a functional linkage for energy transduction between phosphorylation and cation binding domains in various cation transport ATPase. However, its kinetic influence on rate-limiting conformational transitions is dependent on matching specific structures in each ATPase.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Retículo Sarcoplasmático/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Transporte Biológico/fisiologia , Cálcio/farmacologia , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/genética , Linhagem Celular , Galinhas , Transferência de Energia , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos/metabolismo , Fosfopeptídeos , Fosforilação , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Terpenos/farmacologia , Tapsigargina , Transfecção
2.
Rev Esp Fisiol ; 38(3): 327-32, 1982 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-6218543

RESUMO

A method to optimize enzymatic assays by using pyruvate kinase and lactate dehydrogenase enzymes is presented and applied to mitochondrial ATPase as an example. Optimum amounts of auxiliary enzymes, to obtain either a 99% of the initial rate in a given time (t99) or a given lag period (L), are calculated from their apparent Michaelis constants (Kapp) in the medium used and their prices per enzymatic international unit.


Assuntos
L-Lactato Desidrogenase , ATPases Translocadoras de Prótons/análise , Piruvato Quinase , Animais , Bovinos , Análise Custo-Benefício , Cinética , Matemática , Métodos , Mitocôndrias/enzimologia , Fatores Acopladores da Fosforilação Oxidativa/análise
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