The topology of phosphogluconate dehydrogenases in rat liver microsomes.
Rat liver microsomes are known to contain a 6-phosphogluconate dehydrogenase which differs from the 6-phosphogluconate dehydrogenase in the soluble fraction. Microsomes which were washed once bind the soluble phosphogluconate dehydrogenase more tightly than they do glucose-6-phosphate dehydrogenase. Microsomes washed three times in 0.15 M Tris-HCl, pH 8.0, contain only the microsomal 6-phosphogluconate dehydrogenase. Two observations show that this dehydrogenase is located in the cisternae. First, this dehydrogenase is inactive in intact, three times washed microsomes. Second, proteolytic inactivation of 6-phosphogluconate dehydrogenase like that of the cisternal enzyme glucose-6-phosphatase requires disruption of the membrane. Under the conditions used, detergent did not affect the proteolytic inactivation of NADPH-cytochrome c reductase, an enzyme located on the external surface. The excellent correspondence between the activations of hexose phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in microsomes at various stages of disruption of the microsomal membrane produced by detergent supports the earlier contention that these two dehydrogenases are reducing NADP in the same region of the microsomes. A similar experiment which shows an exact correspondence between the activations of 6-phosphogluconate dehydrogenase and mannose-6-phosphatase with increasing concentrations of detergent indicates that the activation of the dehydrogenase can be explained solely by the penetration of the substrates to the active dehydrogenase within the microsomes and strongly suggests that the dehydrogenase is catalytically active in the cisternae.
Bublitz
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Department of Biochemistry, Biophysics, and Genetics, University of Colorado Health Science Center, Denver 80262.
Lawler
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CA
Steavenson
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D000818:Animals; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000494:pharmacology; D010734:Phosphogluconate Dehydrogenase / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D051381:Rats; D013050:Spectrometry, Fluorescence
D010734:Phosphogluconate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; C023553:mannose-6-phosphatase; D010744:Phosphoric Monoester Hydrolases; D010447:Peptide Hydrolases