Measurement of positional isotope exchange rates in enzyme-catalyzed reactions by fast atom bombardment mass spectrometry: application to argininosuccinate synthetase

Fast atom bombardment mass spectrometry (FAB-MS) has been used to measure positional isotope exchange rates in enzyme-catalyzed reactions. The technique has been applied to the reactions catalyzed by acetyl-CoA synthetase and argininosuccinate synthetase. The FAB technique is also able to quantitatively determine the oxygen-18 or oxygen-17 content of nucleotides on as little as 10 nmol of material with no prior derivatization. Acetyl-CoA synthetase has been shown by FAB-MS to catalyze the positional exchange of an oxygen-18 of ATP from the beta-nonbridge position to the alpha beta-bridge position in the presence of acetate. These results are consistent with acetyl adenylate as a reactive intermediate in this reaction. Argininosuccinate synthetase was shown not to catalyze a positional isotope exchange reaction designed to test for the formation of citrulline adenylate as a reactive intermediate. Argininosuccinate synthetase was also found not to catalyze the transfer of oxygen-18 from [ureido-18O]citrulline to the alpha-phosphorus of ATP in the absence of added aspartate. This experiment was designed to test for the transient formation of carbodiimide as a reactive intermediate. These results suggest that either argininosuccinate synthetase does not catalyze the formation of citrulline adenylate or the enzyme is able to completely suppress the rotation of the phosphoryl groups of PPi.     

Cyclic AMP suppresses expression of v-rasH oncogene linked to the mouse mammary tumor virus promoter

Clone 433.3 of NIH 3T3 cells is a stable carrier of the MMTV LTR:v-rasH chimeric DNA. Only in the presence of dexamethasone (a synthetic glucocorticoid), 433.3 cells exhibit an induced level of p21 transforming protein and phenotypic transformation. N6,O2'-dibutyryl cAMP (DBcAMP) antagonized the effect of dexamethasone in a time - and concentration - dependent manner. DBcAMP (5 X 10(-4)M) added 18 hr prior to the addition of dexamethasone (10(-7)M) almost completely blocked the hormone effect: cells contained levels of p21 20% of that in the cells treated with dexamethasone alone, and formed flat, contact inhibited monolayers. On the basis of these results together with our previous data on mammary carcinomas in vivo, we postulate that cAMP may be an intracellular suppressor acting at a regulatory locus of both cellular and viral ras genes.     

Possible regulation of pyruvate carboxylase fromThiobacillus novellus by hydroxypyruvate

Aspartate, glutamate, or dicarboxylic acids did not inhibit the activity of a highly purified but not homogeneous preparation of pyruvate carboxylase fromThiobacillus novellus. The only effective inhibitors were end-products of the reaction and to a lesser degree hydroxypyruvate. The latter has not been shown previously to regulate the enzyme's activity. Lineweaver-Burk plots revealed that it was uncompetitive with respect to acetyl CoA with a K_ii of 3.6 mM, and noncompetitive with respect to bicarbonate, magnesium ATP, and pyruvate with respective K_ii values of 7.1, 5.5, and 6.47 mM. The corresponding K_is values were 7.02, 5.4, and 4.25 mM. A mathematical model is presented that supports the findings.     

Comparison of uptake kinetics in freshly isolated suspensions and short-term primary cultures of rat hepatocytes

The apparent kinetics of uptake of various model substrates were examined for hepatocytes in suspension and primary culture up to 72 h. The ability of hepatocytes to take up taurocholate and ouabain was decreased in culture. Vmax for uptake of both substrates diminished rapidly with increasing time in culture. An increase in Km was observed in cultures 6 h after plating, but there was no further change with prolongation of culture time. The decrease of uptake of taurocholate and ouabain during culture may be due to the reduction in the number of transport carriers plus a decrease of affinity of the carrier to substrates. The nonsaturable component of cadmium uptake was much reduced in cultured cells compared with the suspensions. The saturable process was lower in 6 h culture but increased to a level comparable with the fresh cells at longer culture time. No significant change was found in the Km between suspensions and cultures. Uptake of alpha-aminoisobutyric acid was greater in culture while that of 3-O-methyl-D-glucose was relatively stable but about one-half that found in cell suspension. Thus, uptake of two substrates, taurocholate and ouabain, is clearly compromised with increasing time in primary culture, while uptake of the other substrates does not reflect such a dramatic decrease. It is therefore apparent that the cell preparation of choice in uptake studies depends on the substrate and the nature of the experiments.