Patricia Lund

Purification and properties of malate synthetase

Abstract Procedures are described for the purification of malate synthetase from bakers yeast and from glycollate-grown Pseudomonas ovalis Chester. The properties of the enzymes are closely similar: both re optimally active at pH 8.5 but that from yeast shows greater activity below this pH than that from Ps. ovalis . The K m for glyoxylate was 9.3· 10−5 M (yeast) and 6.3· −5 M ( Pseudomonas ). The K m for acetyl-S-CoA was −5 M . The enymes require Mg ++ for activity ( K m =5·10 −4 M ) and are competitively inhibited by the C 2 -acids oxalate ( K i =1.9·10 −5 M ) fluoroacetate ( K i =2.46·10−4 M ) and glycollate ( K i =3.08·10 −4 M ). The purified enzymes are specific for glyxoxylate, and do not cytalyse the cleavage of acetyl-S-CoA in the presence of oxaloacetate, pyruvate, α-ketoglutarate, glyoxal, glycolaldehyde, formaldehyde, or acetaldehyde. In the presence of glyoxylate, there was no reaction with S,N-diacetylβ-mercaptoethylamine, S-acetyl pantetheine, propionyl coenzyme A or butyryl coenzyme A, but fluoroacetyl A was split at approximately one quarter of the rate observed with similar concentrations of acetyl-S-CoA. Malyl-S-CoA was not split by the enzymes, either alone or in the presence of glyoxylate. The equilibrium of the reaction strongly favours malate formation: no reversal of the reaction was detected. Procedures are described for the preparation of [ 35 S]coenzyme A, which was used to test for the possible formation of an acyl enzyme as an intermediate in the malate synthetase reaction. Then non-enzymic isotopic exchange of 35 S from CoA 35 SH with acetyl-S-Coenzyme A observed was not affected by the presence of malate synthetase. The properties of malate synthetase are compared with those of the citrate-forming condensing enzyme.

Allosteric properties of phosphate-activated glutaminase of human liver mitochondria

The kinetics of human liver phosphate-activated glutaminase were studied in mitochondria isolated from surgical biopsies. The pH profile and activation by phosphate closely resembled rat liver glutaminase and differed clearly from human or rat kidney mitochondrial glutaminases. The activity responses to glutamine or phosphate were allosteric, showing positive cooperativity, as in the rat liver enzyme. Exogenous 1 mM NH4Cl shifted the glutamine concentration at half-maximal velocity, [Gln]0.5, to lower values without changing Vmax or sigmoidicity. Hill plots showed a parallel shift to the left with NH4Cl and the apparent number of binding sites, nH, was 2-3. 25 mM KHCO3 gave the same effects as NH4Cl on [Gln]0.5, Vmax, sigmoidicity and nH. The combination of the two activators was less than additive. Glutamate did not inhibit. We postulate that liver glutaminase is allosteric in its kinetics because it plays a key role in urea synthesis by regulating provision of glutamate for synthesis of N-acetylglutamate, the obligatory co-factor of carbamoylphosphate synthetase.

The effect of paromomycin and [psi] on the suppression of mitochondrial mutations in Saccharomyces cerevisiae

SummaryParomomycin has been found to suppress certain nonsense mutations located in several mitochondrial genes in yeast. In the mosaic genes, paromomycin preferentially suppresses those mutations located in the introns. There is a strong correlation between this phenotypic suppression by paromomycin and the genetic suppression due to various informational mitoribosomal suppressors. No effect of the cytoplasmic element [psi] on mitoribosomal protein synthesis was observed. This work provides strong evidence for the translation of mRNA maturase proteins from open reading frames of the mitochondrial introns.

Biosynthetic capacity of hepatocytes after storage at 4°C

Capacity to synthesize glucose, urea, and ketone bodies is well maintained in hepatocytes after storage for at least 24 h at 4°C. Substrates and albumin are the only requirements.

Amino acid profiles during development of the fetal rat

Umbilical blood venous-arterial differences for amino acids across the rat fetus show uptake of disproportionately large amounts of glutamine, alanine and lysine relative to their contribution to total body protein. Free amino acid concentrations in fetal liver tend to increase during development, but show a rapid adjustment to near-adult values within 24 h after birth.

Maintenance of energy-linked functions in rat liver mitochondria

EGTA and EDTA are compared with respect to their ability to preserve ATP-requiring reactions in rat liver mitochondria. The presence of EGTA sustains the high ATP requirement of citrulline synthesis. EDTA does not, even with excess Mg2+. Carboxylation of pyruvate, which has a lower ATP demand, is not influenced by the type of chelating agent. In mitochondria stored for 24 h at 4 degrees C, EGTA is more effective than EDTA in preventing loss of these energy-linked functions.

Chelating agents and rat liver mitochondria

The chelating agents (EGTA and EDTA) and inorganic phosphate (Pi) are the most variable components of experiments involving isolated liver mitochondria. In the absence of EGTA or EDTA, swelling induced by Pi leads to rapid loss of endogenous adenine nucleotides to adenosine. Chelating agents prevent swelling and loss of adenine nucleotides. Concentrations below about 0.1 mM are ineffective. The protective effects depend on the continuous presence of the chelating agent; they are lost on washing EGTA-containing suspensions with chelating-agent-free medium. We question the accepted view that chelating agents stabilize mitochondria by binding Ca2+ to prevent activation of phospholipase.

Free Radicals Appear to Affect Survival of Hepatocytes at 4°C

1) Rat hepatocytes, stored in a simple salts medium for 24 h at 4°C, retain more than 80% of their capacity to synthesize glucose from lactate.2) The combination of NH4Cl with oleate is cytotoxic during storage and during subsequent incubation of hepatocytes from 48 h starved rats, but not to hepatocytes from fed rats.3) Protection against cytotoxicity is afforded by albumin and by a number of other compounds, notably polyols and glycerol.4) These compounds appear to exert their effects by scavenging free radicals and, in the case of polyols and glycerol, by supplying reducing equivalents to maintain the redox state of the cell in the face of increased flux through glutathione peroxidase.