ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 168, 744-753 (1973) Relation of Phosph:olipase D Activity to the Decay of Succinate Dehydrogenase and of Covalently Bound Flavin in Yeast Cells Undergoing Glucose Repression’ SHLOMO GROSSMAN; JOHN COBLEY; P. K. HOGUE, EDNA B. KEARNEY, AND THOMAS P. SINGER Department of Biochemistry and Biophysics, University of California, San Francisco, California Q.#Y, and Molecular Biology Division, Veterans Administration Hospital, San Francisco, California 94121 Received July 13, 1973 The disappearance of succinate dehydrogenase activity and of protein-bound histidyl flavin were studied in aerobic yeast cells incubated with high glucose con- centrations. The decay of succinate dehydrogenase activity, covalently bound flavin, and of respiration is prevented by cycloheximide but not by chloramphenicol. Dur- ing t,his decay there is a large increase in mitochondrial phospholipase D activity; the appearance of this enzyme is also prevented by cycloheximide. It seems pos- sible, therefore, that the formation of phospholipase D may be important in trigger- ing the disappearance of covalently bound flavin, succinate dehydrogenase, and of other mitochondrial enzymes during glucose repression of aerobic yeast cells. During the past few years enzymes containing covalently bound FAD have been shown to be linked to the peptide chain via the SLY-CHZ group of the flavin (1). In succinate dehydrogenase the bond is to an imidazole nitrogen, in monoamine oxidase, and the flavocytochrome C652 of Chromatium to a cysteine sulfur via a thioether and a thiohemiacetal linkage, respectively. Current studies in this and other laboratories indicate the existence of at, least six other enzymes containing covalently bound flavin, in each of which the 8a! position is involved. Despite these advances in the elucidation of the struc- ture of this interesting class of flavoenzymes nothing is known about the mechanism by which the flavin is inserted into the covalent linkage, the intermediate enzymes 1 Supported, in part, from NSF Grant No. GB 30078. 2 On leave of absence from Bar-Ilan University, Ramat-Gan, Israel. 3 Fellow of the European Molecular Biology Organization. involved in the biosynthesis of covalently bound flavin or about their catabolic conversion to free flavin. Two years ago a program was initiated in this Laboratory, aimed at the elucida- tion of the mechanism of the biosynthesis and metabolic degradation of covalently bound flavins. Succinate dehydrogenase was used as a model system in view of the availability of relatively large quantities of synthetic histidyl-&-riboflavin (2) and the ease of its analytical determination (3). Exploratory experiments showed that histidyl riboflavin is uniquely stable meta- bolically. Thus incubation with broken aerobic yeast cells, liver homogenates, isolated fractions of liver cells, with or without the presence of a variety of cofac- tors, failed to reveal any evidence of reduc- tive cleavage to riboflavin or hydrolysis to 8-hydroxyriboflavin. It seemed logical, therefore, to turn to aerobic yeast under- going glucose repression, since it is known that the incubation of aerobic yeast with high glucose concentrations leads to dis- 744 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.