Pyrophosphate-induced acidification of trans cisternal elements of rat liver Golgi apparatus.
Trans cisternal elements of the Golgi apparatus from rat liver, identified by thiamin pyrophosphatase cytochemistry, were isolated by preparative free-flow electrophoresis and were found to undergo acidification as measured by a spectral shift in the absorbance of acridine orange. Acidification was supported not only by adenosine triphosphate (ATP) but nearly to the same degree by inorganic pyrophosphate (PPi). The proton gradients generated by either ATP or PPi were collapsed by addition of a neutral H+/K+ exchanger, nigericin, or the protonophore, carbonyl cyanide m-chlorophenylhydrazone, both at 1.5 microM. Both ATP hydrolysis and ATP-driven proton translocation as well as pyrophosphate hydrolysis and pyrophosphate-driven acidification were stimulated by chloride ions. However, ATP-dependent activities were optimum at pH 6.6, whereas pyrophosphate-dependent activities were optimum at pH 7.6. The Mg2+ optima also were different, being 0.5 mM with ATP and 5 mM with pyrophosphate. With both ATPase and especially pyrophosphatase activity, both by cytochemistry and analysis of free-flow electrophoresis fractions, hydrolysis was more evenly distributed across the Golgi apparatus stack than was either ATP- or PPi-induced inward transport of protons. Proton transport colocalized more closely with thiamin pyrophosphatase activity than did either pyrophosphatase or ATPase activity. ATP- and pyrophosphatase-dependent acidification were maximal in different electrophoretic fractions consistent with the operation of two distinct proton translocation activities, one driven by ATP and one driven by pyrophosphate.
Brightman
A O
AO
Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, IN 47907.
Navas
P
P
Minnifield
N M
NM
Morré
D J
DJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002712:Chlorides / Q000502:physiology; D011756:Diphosphates / Q000494:pharmacology*; D004586:Electrophoresis; D006056:Golgi Apparatus / Q000737:chemistry / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D008297:Male; D008854:Microscopy, Electron; D011755:Pyrophosphatases / Q000378:metabolism; D051381:Rats
D002712:Chlorides; D011756:Diphosphates; D000251:Adenosine Triphosphatases; D011755:Pyrophosphatases