Identification of tyrosine-phosphorylated proteins of the mitochondrial oxidative phosphorylation machinery.

The role of some serine/threonine kinases in the regulation of mitochondrial physiology is now well established, but little is known about mitochondrial tyrosine kinases. We showed that tyrosine phosphorylation of rat brain mitochondrial proteins was increased by in vitro addition of ATP and H2O2, and also during in situ ATP production at state 3, and maximal reactive oxygen species production. The Src kinase inhibitor PP2 decreased tyrosine phosphorylation and respiratory rates at state 3. We found that the 39-kDa subunit of complex I was tyrosine phosphorylated, and we identified putative tyrosine-phosphorylated subunits for the other complexes. We also have strong evidence that the FoF1-ATP synthase alpha chain is probably tyrosine-phosphorylated, but demonstrated that the beta chain is not. The tyrosine phosphatase PTP 1B was found in brain but not in muscle, heart or liver mitochondria. Our results suggest that tyrosine kinases and phosphatases are involved in the regulation of oxidative phosphorylation.

Phosphoserine aminotransferase, the second step-catalyzing enzyme for serine biosynthesis.

As a step toward analyzing the serine biosynthetic pathway in mammals, we have studied the properties of phosphoserine aminotransferase, the second step-catalyzing enzyme. The K(m) values for 3-phosphohydroxypyruvate and L-phosphoserine are 5 and 35 microM, respectively, and those for glutamate and alpha-ketoglutarate are 1.2 and 0.8 mM, respectively. The product inhibition studies strengthened the support for a ping-pong mechanism and allowed evaluation of Ki values for the four substrates. The equilibrium constant evaluated from the kinetic parameters is approximately 40. Additionally, some physical properties relative to the bound coenzyme and the secondary structure were investigated. The results are consistent with a structural relationship between the Escherichia coli enzyme and the mammalian enzyme. The mammalian enzyme has specific kinetic parameters, the determination of which is a prerequisite to analyzing the serine biosynthetic pathway in mammals.

Interaction of phosphonates related to glutathione with the rat kidney gamma-glutamylcysteine synthetase.

Various phosphonic and sulfonic glutamate analogues as well as phosphonopeptides related to glutathione were studied for their interaction with rat kidney gamma-glutamylcysteine synthetase activity. We found, in all cases, that the presence of a phosphonic group increases the affinity for the enzyme. Among the tripeptides tested, the phosphonic analogue of ophthalmic acid (gamma Glu-Abu-Gly-P) is the most potent inhibitor. The glutamate and cysteine sites of the enzyme seem to be involved in the binding of this compound, since either substrate protects against inhibition. The types of inhibition with respect to the different substrates show dissimilar behaviors of the tripeptides, in spite of their structural analogy. Investigations relative to the role of the divalent ion Mg2+ provided evidence that the actual inhibitors are Mg2+-tripeptide complexes for the phosphonic compounds, whereas chelation with a metal ion is not required for inhibition by glutathione.