Metabolism of threo-beta-fluoroaspartate by H4 cells. Inhibition of adenylosuccinate lyase by fluoro analogs of its substrates

DL-threo-beta-Fluoroaspartate is a substrate for the two enzymes in de novo purine biosynthesis that use aspartate, namely 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) synthetase and adenylosuccinate synthetase. With both enzymes, Vmax with threo-beta-fluoroaspartate is about 50% of that observed with aspartate. The products of the two enzyme reactions, threo-beta-fluoro-SAICAR and threo-beta-fluoroadenylosuccinate, are inhibitors of adenylosuccinate lyase purified from rat skeletal muscle. In 20 mM phosphate buffer, pH 7.4, the KI values for threo-beta-fluoro-SAICAR are 5 and 3 microM and for threo-beta-fluoroadenylosuccinate are 3 and 1 microM, in the SAICAR and adenylosuccinate cleavage reactions, respectively. In 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, the KI values for threo-beta-fluoro-SAICAR are approximately 0.14 and 0.03 microM and for threo-beta-fluoroadenylosuccinate are approximately 0.05 and 0.015 microM, in the same two reactions, respectively. These KI values are one-half to one-hundredth of the Km values for SAICAR and adenylosuccinate, the two substrates of adenylosuccinate lyase. After an 8-h incubation with 45 microM threo-beta-fluoroaspartate, H4 cells contain 200-300 microM threo-beta-fluoro-SAICAR and 60-90 microM threo-beta-fluoroadenylosuccinate. These concentrations of fluoro analogs are sufficient to substantially inhibit adenylosuccinate lyase and hence the de novo synthesis of purines in H4 cells.     

Citrate and the conversion of carbohydrate into fat. The regulation of fatty acid synthesis by rat liver extracts

1. The rate of fatty acid synthesis by particle-free extracts prepared from rat liver is increased greatly if the enzyme system is first activated with citrate. 2. The extent of the activation depends on the citrate concentration and on the time of activation in an interdependent manner. 3. Citrate activation is strongly dependent on temperature. 4. Tricarballylate can replace citrate as an activator, but its presence in the assay inhibits fatty acid synthesis. 5. Mg(2+) ions can replace citrate in the activation but not in the complete reaction system. 6. ATP prevents the activating effect of citrate and Mg(2+) ions. 7. The rate of fatty acid synthesis is increased by palmitoyl-dl-carnitine. This type of activation, additional to that caused by citrate, is rapid and does not depend on prior incubation. 8. Inhibition of fatty acid synthesis by palmitoyl-CoA can be prevented by palmitoyl-dl-carnitine or by increasing the concentration of protein.     

Lactogenesis in the rat. Changes in metabolic parameters at parturition

1. Tissue concentrations of nucleic acids, protein, fat, water, metabolites and lactose, and the activities of seven enzymes concerned in milk biosynthesis, were measured in the rat mammary gland at closely spaced times before, at and after parturition. 2. Changes are seen in the tissue concentrations of most substances, and several changes are initiated at least during the day preceding parturition. 3. Lactose, which is absent 1 day before parturition, is found in amounts of 12μmoles/g. fresh wt. of tissue at parturition. 4. From the tissue activities before parturition of three enzymes on the biosynthetic pathway of lactose, and, from the small changes observed in their activities at parturition itself, it is concluded that the factors responsible for the appearance of lactose at parturition remain to be demonstrated.     

Comparative aspects of some bacterial dehydrogenases and transhydrogenases

Ragland, T. E. (Brandeis University, Waltham, Mass.), T. Kawasaki, and J. M. Lowenstein. Comparative aspects of some bacterial dehydrogenases and transhydrogenases. J. Bacteriol. 91:236-244. 1966.-Twenty-eight diverse bacterial species were surveyed for the activities and coenzyme specificities of four enzymes: isocitrate dehydrogenase (ICDH), glucose-6-phosphate dehydrogenase (G-6-PDH), 6-phosphogluconate dehydrogenase (6-PGDH), and reduced nicotinamide adenine dinucleotide phosphate-nicotinamide adenine dinucleotide (NAD) transhydrogenase (TH). Most of the species that exhibited a nicotinamide adenine dinucleotide phosphate (NADP)-linked ICDH also showed significant TH activity, but there were several which did not. Only one of the organisms tested, Xanthomonas pruni, had an ICDH active with both NAD and NADP; it was devoid of TH activity. Acetobacter suboxydans, which lacks ICDH altogether, also had no TH. Some of the species examined had G-6-PDH or 6-PGDH (or both) of dual coenzyme specificity, but there was no apparent relation between these findings and the presence or absence of TH. The TH reaction was assayed by use of analogues of NAD as acceptors. The bacteria could be divided into two groups on the basis of TH specificity, one group reacting at a much faster rate with the 3-acetylpyridine analogue of NAD than with the thionicotinamide analogue, whereas the converse was true for the other group. A few organisms showed no marked specificity for either analogue. This division of specificity can be related to the currently accepted taxonomic classification of the organisms, although a few apparent anomalies were found.     

Halophilic Archaea cultured from ancient halite,Death Valley,California

Halophilic Archaea cultured from ancient fluid inclusions in a 90‐m‐long (0‐ to 100 000‐year‐old) salt core from Death Valley, California, demonstrate survival of bacterial cells in subsurface halite for up to 34 000 years. Five enrichment cultures, representing three genera of halophilic Archaea (Halorubrum, Natronomonas and Haloterrigena), were obtained from five surface‐sterilized halite crystals exclusively in one section of the core (13.0–17.8 m; 22 000–34 000 years old) containing perennial saline lake deposits. Prokaryote cells were observed microscopically in situ within fluid inclusions from every layer that produced culturable cells. Another 876 crystals analysed from depths of 8.1–86.7 m (10 000–100 000 years old) failed to yield live halophilic Archaea. Considering the number of halite crystals tested (culturing success of 0.6%), microbial survival in fluid inclusions in halite is rare and related to the paleoenvironment, which controls the distribution and abundance of trapped microorganisms. Two cultures from two crystals at 17.8 m that yielded identical 16S rRNA sequences (genus: Haloterrigena) demonstrate intra‐laboratory reproducibility. Inter‐laboratory reproducibility is shown by two halophilic Archaea (genus: Natronomonas), with 99.3% similarity of 16S rRNA sequences, cultured from the same core interval, but at separate laboratories.