A series of experiments are described that were carried out with the goal of determining how the membrane-bound ATPase from H. saccharovorum is related to V- and F-type ATPases. They reflect three approaches: the use of inhibitors; structural studies; and immunological relatedness

Hochstein, L. I.

English

NASA Technical Reports Server

N92-13627
ON THE CHIMERICAL NATURE OF THE MEMBRANE-BOUND ATPASE
FROM
HALOBACTERIUM SACCHAROVORUM
L. I. Hochstein*
Planetary Biology Branch, Ames Research Center
There is increasing evidence that proton translocating ATPases evolved from a
progenitor that had the structural characteristics of vacuolar ATPases (V-ATPases) but,
unlike this class of enzymes, was capable of synthesizing ATP. Interestingly, the
Archaebacteria, a diverse collection of organisms that diverged from the Prokaryotes
at an early stage in the evolution of life, possess ATPases more closely related to V-
ATPases than to FoF1-ATPases(F-ATPases).
However, an antiserum against the "13" subunit from S. acidocaldadus reacts with the
13subunit from an F-ATPase as well as subunit II from Halobacterium saccharovorum
ATPase (subunit II is probably the functional equivalent to the 13 subunit). This
communication describes a series of experiments carried out with the goal of
determining how the membrane-bound ATPase from H. saccharovorum is related to V-
and F-type ATPases, and reflect three approaches: the use of inhibitors; structural
studies; and immunological relatedness.
Nitrate is extremely useful for distinguishing between V- and F-ATPases since only the
former are inhibited by this reagent. The enzyme from H. saccharovorum is also
inhibited by nitrate and at concentrations that inhibit V-ATPases and ATPases from
other Archaebacteria. N-ethylmaleimide, which inhibits V- and F-ATPases, also
inhibits the ATPase from H. sac_harovorum. However, whereas V-type enzymes are
inhibited by I_M concentrations of this reagent, F-type and the halobacterial enzyme
are inhibited by mM concentrations of NEM. 4-chloro-7-nitrobenzofurazan, a
nucleotide analog, inhibits V-, F-, and the halobacterial ATPases. In the case of the V-
ATPases inhibition is accompanied by the binding of the inhibitor to the largest of the
subunits, which is the catalytic subunit. 4-chloro-7-nitrobenzofurazan binds to the 13-
subunit of F-ATPases, which is the catalytic subunit. 4-chloro-7-nitrobenzofurazan
binds to subunit II of the halobacterial enzyme, which also contains the
dicylcohexylcarbodiimide-binding site. In spite of this apparent similarity, the
halobacterial enzyme is unrelated to F-type ATPases based on a comparison of the
isoelectric points, the amino acid compositions, the molecular mass, and the
proteolytic products of the two largest subunits from each enzyme. Furthermore,
antisera prepared against the largest subunit from the V-ATPase from _9..I,E.0.._..g.Z_
crassa reacts with subunit I from the halobacterial enzyme. On the basis of these
observations, the halobacterial enzyme may represent a chimera. Subunit II appears
to be immunologically and functionally related to the 13subunit from F-ATPases. On
the other hand, subunit I may be structurally related to the largest of the largest
subunits from V-ATPases but possibly functionally related to the (z-subunit from F-
ATPases (which has a regulatory function). Whether this reflects the nature of the
progenitor enzyme or mixture of structure and function which arose after the evolution
of the proton ATPases into V- and F-type enzymes is not clear, but it is another
example of the evolutionary richness of the extremely halophilic bacteria.
63
https://ntrs.nasa.gov/search.jsp?R=19920004409 2020-03-17T13:48:31+00:00Z


On the chimerical nature of the membrane-bound ATPase from halobacterium saccharovorum

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