The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5'-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic e...

Proplastids from developing castor bean (Ricinus communis) endosperm have a pyruvate kinase activity which is extremely unstable on isolation from the organelle. It can be stabilized by 20 mm 2-mercaptoethanol in 20% ethylene glycol. In contrast the soluble pyruvate kinase is stable at 60 C for 10 minutes. The two activities have different pH optima. The soluble and the proplastid activities ar...

Pyruvate kinase multimers have been investigated by charge detection mass spectrometry (CDMS). In CDMS, the m/z and z are simultaneously measured for each ion, so the mass is determined directly. The measurements were made using a modified cone trap that incorporates an image charge detector with a cryogenically cooled preamplifier. With non-denaturing solution conditions, the tetrameric form o...

The percentage of total pyruvate dehydrogenase in the active nonphosphorylated form was measured in intact rat liver mitochondria after rapid separation of the mitochondria through silicone oil and extraction with glycerol buffer at 10”. The contents of ATP, ADP, Mg2+, and Ca2+ in the mitochondrial matrix of separated mitochondria were determined in parallel experiments. Mitochondria were incub...

Purified pig heart pyruvate dehydrogenase complex is denuded of its intrinsic pyruvate dehydrogenase kinase activity by sedimentation from dilute solution (60munits/ml). Kinase activity is restored by a supernatant fraction prepared by high-speed centrifugation of rat heart mitochondrial extracts; the factor responsible is referred to as kinase/activator. Kinase/activator was also assayed by it...

The allosteric inhibition of M1-type pyruvate kinase from rabbit skeletal muscle by phenylalanine is reciprocally dependent on Mg2+ and phosphoenolpyruvate concentrations. At pH 8, phenylalanine acts as a competitive inhibitor with respect to Mg2+ and phosphoenolpyruvate, and vice versa. Phenylalanine introduces sigmoidicity into the dependence of the reaction velocity on [Mg2+]. In vitro kinet...

Mutants have been isolated in S. cerevisiae with the phenotype of growth on pyruvate but not on glucose, or growth on rich medium with pyruvate but inhibition by glucose. Screening of mutagenized cultures was either without an enrichment step, or after enrichment using the antibiotic netropsin (Young et al. 1976) or inositol starvation (Henry, Donahue and Culbertson 1975). One class of mutants ...

Prenatal testing for pyruvate kinase deficiency is often requested by parents who already have an affected child. However, before the development of molecular biologic techniques there were no suitable diagnostic methods. We present here two cases in which the diagnosis was established, one using amniotic fluid cells, the other cord blood. Two different approaches were used. The first, using a ...