Altered brain phosphocreatine and ATP regulation when mitochondrial creatine kinase is absent.

In cerebral gray matter, ATP concentration is closely maintained despite rapid, large increases in turnover and low substrate reserves. As seen in vivo by (31)P nuclear magnetic resonance (NMR) spectroscopy, brain ATP is stable early in seizures, a state of high energy demand, and in mild hypoxia, a state of substrate deficiency. Like other tissues with high and variable ATP turnover, cerebral gray matter has high phosphocreatine (PCr) concentration and both cytosolic and mitochondrial creatine kinase (UbMi-CK) isoenzymes. To understand the physiology of brain creatine kinases, we used (31)P NMR to study PCr and ATP regulation during seizures and hypoxia in mice with targeted deletion of the UbMi-CK gene. The baseline CK reaction rate constant (k) was higher in mutants than wild-types. During seizures, PCr and ATP decreased in mutants but not in wild-types. The k-value for the CK catalyzed reaction rate increased in wild-types but not in the mutants. Hypoxic mutants and wild-types showed similar PCr losses and stable ATP. During recovery from hypoxia, brain PCr and ATP concentrations returned to baseline in wild-types but were 20% higher than baseline in the mutants. We propose that UbMi-CK couples ATP turnover to the CK catalyzed reaction rate and regulates ATP concentration when synthesis is increased.

Brain creatine kinase and creatine transporter proteins in normal and creatine-treated rabbit pups.

Systemic creatine (Cr) supplementation increases brain phosphocreatine (PCr) and prevents hypoxic seizures in 15-day-old rabbits. Between 5 and 30 days of age during normal development, rabbit gray matter mitochondrial creatine kinase (Mi-CK) increases 400% while cytosolic CK (BB-CK) increases 60%. In white matter, both isoenzymes show smaller, similar increases (40%) during this period. The Cr transporter protein decreases 60% between 5 and 15 days in both regions. In vivo CK rate constants measured by (31)P nuclear magnetic resonance increase 30% between 10 and 20 days, and then fall 50% between 20 and 30 days in predominantly gray matter slices. Similar maturational changes are seen in predominantly white matter slices. Injecting Cr at 15 days does not significantly change BB-CK or Mi-CK isoenzymes or the in vivo CK reaction rate constants. Thus, the largest change in the CK system associated with suppression of hypoxic seizures in Cr-treated rabbits is increased PCr in gray and white matter.

Flavin substrate specificity of the vitamin B2-aldehyde-forming enzyme from Schizophyllum commune.

Vitamin B2-aldehyde-forming enzyme from Schizophyllum commune catalyzes oxidation of the 5'-hydroxymethyl of riboflavin to the formyl group. We have monitored enzyme activity by spectrophotometrically measuring the reduction of 2,6-dichlorophenol-indolphenol as electron acceptor to assess 35 riboflavin analogs as potential substrates or competitive inhibitors with the purpose of delimiting structural requirements of the substrate binding site. Analogs with side chains of two- to six-carbon length modified by deletion of secondary hydroxyls or by changes in their epimeric configuration are not oxidized. The omega-hydroxyalkyl-flavins (n = 2-6) are competitive inhibitors (Ki = 7-16 microM) of riboflavin oxidation, as are some analogs with L-secondary hydroxyls in the side chain. Analogs with bulky substituents on the isoalloxazine ring are also not substrates. The enzyme does not significantly bind flavins with an 8 alpha-N-imidazole; diethylamino, methylethylamino, dimethylamino, ethylamino, or ethoxy groups at position 8; methyl at 6; and beta-hydroxyethylamino at position 2. Also the replacement of N with CH in 1-deazariboflavin disallows substrate reaction. Analogs with fluoro, chloro, methyl, amino, or methylamino at position 8; chloro at 7; methyl or carboxylmethyl at 3; thio at 2, and C replacing N at positions 3 or 5 are substrates with relative Vmax values ranging from 27 to 110% that of riboflavin. The Km values for the analogs oxidized are all found to be in the micromolar range (22-176 microM). Overall specificity of the enzyme for riboflavin is found to be rather narrow and sterically limited, which suggests that the vitamin is the natural substrate.

Hysteretic interaction of NADH and Mg2+ with mammalian NADH:CoQ reductase from beef heart.

Preincubation of submitochondrial particles (SMP) from beef heart in a reaction mixture containing low concentrations of Mg2+ induces a time lag in the NADH:oxidase activity. Preconditioning of the SMP by NADH, but not by NAD+, prevents the Mg2+-related time lag. The data obtained show that there exists a tight binding site for Mg2+ regulating the rate of electron transfer from NADH to the natural acceptor. The ability of Mg2+ to form a catalytically inactive complex with the enzyme is regulated by NADH.