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Kinetic characterization of plasma membrane atpase from Saccharomyces cerevisiae

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Summary

  1. 1.

    Plasma membrane preparations have been isolated from spheroplasts of Saccharomyces cerevisiae, strain R XII, via lysis and subsequent differential centrifugation. These preparations are almost devoid of mitochondria) contamination.

  2. 2.

    The plasma membrane ATPase is fairly stable when refrigerated, but loses activity at 8 °C and above. Below pH 5.6 the ATPase is irreversibly inactivated. The enzyme also splits GTP and ITP, although to a lesser extent.

  3. 3.

    Mg2+-ions are essential as part of the reactive substrate, MgATP, and furthermore they activate the ATPase. Optimal conditions depend on substrate concentration. When the concentration of free Mg2+ ions exceeds about 0.1 mm, competitive inhibition occurs.

  4. 4.

    In the range of pH 5.6–9.2 two functional groups dissociate. One, with pKb = 8.1 ± 0.1 participated in substrate binding and another one with pKb′ = 8.1 ± 0.1 is involved in substrate splitting.

  5. 5.

    The experiments with group-specific inhibitors suggest that an α-amino group and a sulfhydryl residue are involved in substrate binding and conversion. Furthermore, imidazole, tryptophan and carboxyl residues may be important for the catalytic process.

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Abbreviations

E, EH+ :

free enzyme with various degrees of protonation

S:

substrate

P:

product

Km′:

modified Michaelis constant (in the presence of activator)

\(\tilde K\) m H+, \(\tilde v\) H+ :

apparent constants, which depend on H+ ion concentration

KMg :

dissociation constant of the EMg complex

KMg′:

dissociation constant of the EMgS complex

Ki :

inhibition constant = dissociation constant of the EM92 complex

Tricin:

N-Tris-(hydroxymetyl)-methyl-glycine

MES:

morpholino-ethyl-sulfonic acid

DCCD:

N,N′ dicyclohexyl carbodiimide

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Authors and Affiliations

  1. Institut für Biochemie und Molekularbiologie, Fachbereich Biologie, Freie Universität Berlin, Ehrenbergstraße 26-28, 1000, Berlin 33, Germany

    Jan Ahlers, Erika Ahr & Angelika Seyfarth

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  1. Jan Ahlers
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  2. Erika Ahr
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  3. Angelika Seyfarth
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Ahlers, J., Ahr, E. & Seyfarth, A. Kinetic characterization of plasma membrane atpase from Saccharomyces cerevisiae. Mol Cell Biochem 22, 39–49 (1978). https://doi.org/10.1007/BF00241469

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