Effect of adenosine on the adenine nucleotide content and metabolism of hepatocytes.

ADENOSINE (0.5 MM) added to hepatocyte suspensions increased the intracellular concentration of ATP and total adenine nucleotides within 60 min up to three-fold. 2. Adenosine at 0.5 mM inhibited gluconeogenesis from lactate by about 50%. At higher adenosine concentrations the inhibition was less. There was no strict parallelism between the time-course of the increase of the adenine nucleotide content and the time-course of the inhibition of gluconeogenesis from lactate. 3. Adenosine abolished the accelerating effects of oleate and dibutyryl cyclic AMP on gluconeogenesis from lactate. 4. Gluconeogenesis was no significant effect of adenosine with fructose, dihydroxyacetone or glycerol. With asparagine, adenosine caused anacceleration of glucose formation. 5. Adenosine incorporation into adenine nucleotides accounted for about 20% of the adenosine removal. 6. Inosine, hypoxanthine or adenine compared with adenosine gave relatively slight increases of adenine nucleotides. 7. Urea synthesis from NH4Cl under optimum conditions i.e. in the presence of ornithine, lactate and oleate, was also inhibited by adenosine. The inhibition increased with the adenosine concentration and was 65% at 4 mM-adenosine. Again there was no correlation between the degree of inhibition of urea synthesis and the increase in the adenine nucleotide content. 8. The basal O2 consumption, the increased O2 consumption on the addition of oleate and the rate of formation of ketone bodies were not affected by the addition of adenosine. The [beta-hydroxybutyrate]/[acetoacetate] ratio was increased by adenosine, provided that lactate was present. 9. The increase of the adenine nucleotide content of the hepatocytes on the addition of adenosine may be explained on the assumption that adenosine kinase is not regulated by feedback but by substrate supply.     

Acceleration of gluconeogenesis from lactate by lysine (Short Communication)

l-Lysine (2mm) causes an increase (mean 60%) in the rate of gluconeogenesis from lactate in isolated liver cells. The effect is of a catalytic nature. No other amino acid has the same effect, though ornithine is slightly active. The effect is additional to the stimulatory effects of oleate and of dibutyryl cyclic AMP.     

MACROMOLECULAR ABSORPTION : Mechanism of Horseradish Peroxidase Uptake and Transport in Adult and Neonatal Rat Intestine

The immature small intestine of neonatal mammals is permeable to gamma globulins as a source of passive immunity. Allegedly, macromolecular absorption ceases when the epithelial cell membrane matures. However, some evidence exists that adult animals retain a limited capacity to transport antigenic and biologically active quantities of large molecules. In this study, the mechanism of absorption of the tracer protein, horseradish peroxidase (HRP), was tested in neonatal and adult rat gut sacs. Transport into serosal fluid was quantitated by enzymatic assay and monitored morphologically by histochemical techniques. A greater transport of HRP was noted in the adult jejunum compared to adult ileum and neonatal intestine. Morphologically, the uptake mechanism in adult intestine was similar to the endocytosis previously reported in neonatal animals Like other endocytotic processes, HRP uptake in adult rats is an energy-dependent process as determined by metabolic inhibitors and temperature-controlled studies. An understanding of the mechanism whereby macromolecules are bound to intestinal membranes and engulfed by them is necessary before the action of physiologic macromolecules such as enterotoxins can be appreciated.     

Digitonin perfusion of rat liver. A new approach in the study of intra-acinar and intracellular compartmentation in the liver.

Perfusing a rat liver with digitonin in the concentration range 2-20 mg/ml results in complete decolorization of the organ within 45-250 s. Decolorization progresses with time in the direction of flow, and it is therefore possible, by collecting the eluate, to obtain material from specific intracellular compartments of hepatocytes in different zones in the microcirculatory unit of the liver. The results demonstrate that cytoplasmic marker enzymes from periportal or perivenous hepatocytes can be collected with as little contamination from the other compartment as is obtained in micro-dissection studies. Furthermore, a fraction enriched in mitochondrial marker enzymes can be achieved with only 10-20% contamination by cytoplasmic material.     

Commerson's dolphin (Cephalorhynchus commersonii): A discussion of the first live birth within a marine zoological park

Six adult Commerson's dolphins (Cephalorhynchus commersonii) have been housed at Sea World, San Diego, since 1983. Details of their husbandry at Sea World are briefly presented. Pregnancy was determined in one of the females by means of radioimmunoassay, following observations of copulation. Food consumption of the pregnant female decreased beginning 5 days prior to parturition and ceased entirely 6 hours prior to delivery. A single male calf, 55–65 cm in length and weighing 4.5–5.5 kg, was delivered after 35 minutes of labor. Details of the delivery, calf's appearance, suckling behavior, and early respiratory pattern are presented. Similarities between the birth of the Commerson's dolphin calf and previous births of bottlenosed dolphin (Tursiops truncatus) calves are discussed. The successful propagation of new species, such as Commerson's dolphins, permits documentation of the event with greater detail and accuracy than is possible in the wild. Details of growth and reproduction thus gained may provide information applicable to the natural history and proper management of the species in question.     

Lactate-stimulated ethanol oxidation in isolated hepatocytes

1. Hepatocytes isolated from starved rats and incubated without other substrates oxidized ethanol at a rate of 0.8-0.9mumol/min per g wet wt. of cells. Addition of 10mm-lactate increased this rate 2-fold. 2. Quinolinate (5mm) or tryptophan (1mm) decreased the rate of gluconeogenesis with 10mm-lactate and 8mm-ethanol from 0.39 to 0.04-0.08mumol/min per g wet wt. of cells, but rates of ethanol oxidation were not decreased. From these results it appears that acceleration of ethanol oxidation by lactate is not dependent upon the stimulation of gluconeogenesis and the consequent increased demand for ATP. 3. As another test of the relationship between ethanol oxidation and gluconeogenesis, the initial lactate concentration was varied from 0.5mm to 10mm and pyruvate was added to give an initial [lactate]/[pyruvate] ratio of 10. This substrate combination gave a large stimulation of ethanol oxidation (from 0.8 to 2.6mumol/min per g wet wt. of cells) at low lactate concentrations (0.5-2.0mm), but rates remained nearly constant (2.6-3.0mumol/min per g wet wt. of cells) at higher lactate concentrations (2.0-10mm). 4. In contrast, owing to the presence of ethanol, the rate of glucose synthesis was only slightly increased (from 0.08 to 0.12mumol/min per g wet wt. of cells) between 0.5mm- and 2.0mm-lactate and continued to increase (from 0.12 to 0.65mumol/min per g wet wt. of cells) with lactate concentrations between 2 and 10mm. 5. In the presence of ethanol, O(2) uptake increased with increasing substrate concentration over the entire range. 6. Changes in concentrations of glutamate and 2-oxoglutarate closely paralleled changes in the rate of ethanol oxidation. 7. In isolated hepatocytes, rates of ethanol oxidation are lower than those in vivo apparently because of depletion of malate-aspartate shuttle intermediates during cell preparation. Rates are returned to those observed in vivo by substrates that increase the intracellular concentration of shuttle metabolites.     

Differential inhibition of ketogenesis by malonyl-CoA in mitochondria from fed and starved rats.

Rates of ketogenesis in mitochondria from fed or starved rats were identical at optimal substrate concentrations, but responded differently to inhibition by malonyl-CoA. Kinetic data suggest that the K1 for malonyl-CoA is greater in the starved animal. These results indicate that, for the regulation of ketogenesis in the starved state, the lower sensitivity of carnitine palmitoyltransferase to inhibition by malonyl-CoA may be more important than the concentration of malonyl-CoA.     

Modifications in the enzymatic assay for inorganic phosphate.

A recent publication from this laboratory (1) described an enzymatic assay for inorganic phosphate (P_i) which eliminates the need for standards and, through mild reaction conditions, avoids the hydrolysis of labile organic phosphates. Those features are advantageous, particularly for P_i measurements in biological samples. However, subsequent inquiries from other laboratories and our own experience indicated that the assay, as described (1), does not perform well. Specifically, it was found that the assay range was 10-fold narrower than that reported, completion times were 3- to 5-fold longer, and the reaction with P_i standards was only 90–95% complete.Because of these deficiencies we have systematically evaluated every aspect of the assay and have found that the difficulties are eliminated and the assay is improved and simplified by the following changes; (i) Triethanolamine is used in place of Tris as the assay buffer; (ii) triose phosphate isomerase is eliminated and the levels of other enzymes are adjusted to obtain optimum reaction conditions; (iii) ammonium sulfate is removed from the analytical enzymes. The modified procedure described below is more convenient, is linear up to P_i concentrations of 0.1 μmol/ml in the assay, gives complete reaction of P_i standards and quantitative recovery of P_i added to biological extracts, and comes to stable endpoints in 30 min or less (depending on the amount of P_i in the assay).     

Hemin-dependent growth stimulation and cytochrome synthesis in Corynebacterium pyogenes.

Growth of Corynebacterium pyogenes, an important pathogen in animals, was greatly increased on addition of hemin to a medium of tryptose plus mineral. The synthesis of a type b cytochrome in this organism appeared to depend on the presence of hemin in the growth medium.     

Mutational analysis of a nucleosidase involved in quorum-sensing autoinducer-2 biosynthesis

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is important in a number of cellular functions such as polyamine biosynthesis, methionine salvaging, biological methylation, and quorum sensing. The nucleosidase is found in many microbes but not in mammalian systems, thus making MTAN a broad-spectrum antimicrobial drug target. Substrate binding and catalytic residues were identified from the crystal structure of MTAN complexed with 5'-methylthiotubercidin [Lee, J. E., Cornell, K. A., Riscoe, M. K. and Howell, P. L. (2003) J. Biol. Chem. 278 (10) 8761-8770]. The roles of active site residues Met9, Glu12, Ile50, Ser76, Val102, Phe105, Tyr107, Phe151, Met173, Glu174, Arg193, Ser196, Asp197, and Phe207 have been investigated by site-directed mutagenesis and steady-state kinetics. Mutagenesis of residues Glu12, Glu174, and Asp197 completely abolished activity. The location of Asp197 and Glu12 in the active site is consistent with their having a direct role in enzyme catalysis. Glu174 is suggested to be involved in catalysis by stabilizing the transition state positive charge at the O3', C2', and C3' atoms and by polarizing the 3'-hydroxyl to aid in the flow of electrons to the electron withdrawing purine base. This represents the first indication of the importance of the 3'-hydroxyl in the stabilization of the transition state. Furthermore, mutation of Arg193 to alanine shows that the nucleophilic water is able to direct its attack without assistance from the enzyme. This mutagenesis study has allowed a reevaluation of the catalytic mechanism.