R. W. Newburgh

PYRUVATE KINASE ISOZYMES IN NEURONS, GLIA, NEUROBLASTOMA, AND GLIOBLASTOMA

Abstract– The distribution of pyruvate kinase isozymes (EC 2.7.1.40) was examined in cells and tissues from the central and peripheral nervous system of the rat. Most tissues contain significant quantities of both the K4 (fetal type) and M4 (skeletal muscle type) isozymes plus tetrameric hybrids comprised of various combination of the type M and type K subunits. Retina, for example, contains a five‐mem‐bered hybrid set weighted toward K4, while sciatic nerve and spinal cord have patterns very similar to that of adult brain, consisting predominantly of M4 with small amounts of K4 and K‐M hybrids. This adult pattern is achieved by a gradual shift from a hybrid set dominated by K4 in fetal life, to the pattern at birth at which time the two most prominent bands were M4 and K2M2, and finally to the adult pattern by about 28 days after birth.

INHIBITION OF 5′‐NUCLEOTIDASE IN RAT BRAIN BY METHYLXANTHINES

—Rat brain 5′‐nucleotidase (EC 3.1.3.5) is inhibited by methylxanthines such as theophylline. Inhibition of the 5′‐nucleotidase by theophylline appears more efficient than the inhibition of cAMP phosphodiesterase by this drug. A similar inhibition is observed with caffeine, theobromine, 7′‐methyl‐xanthine and 1‐methylxanthine.

Nutrition and metabolism of methyl donors and related compounds in the blowfly, Phormia regina (Meigen).

Abstract In addition to carnitine and 2,2-dimethylaminoethanol, γ-butyrobetaine and O -acetylcarnitine are shown to be effective replacements for choline in the diet of P. regina , an organism which shows a choline requirement when grown on a chemically defined diet under sterile conditions. dl -Carnitine is shown to be approximately equivalent to choline in the diet on a molar basis and slightly more effective than γ-butyrobetaine. The choline inhibitor 2-amino-2-methylpropanol is shown to be effective in P. regina , and its effects can be reversed by those compounds which are able to replace choline in the diet as well as by choline itself. The order of effectiveness is choline > 2,2-dimethylaminoethanol > γ-butyrobetaine > carnitine. Phospholipides containing serine, ethanolamine, and choline are shown to be normal constituents of the third instar larvae. Formate although apparently not being used in the biosynthesis of methyl groups for choline synthesis is shown to be metabolized to CO 2 both by the whole organism and by larval homogenates. The biosynthesis of choline from ethanolamine is shown either not to occur or to be unimportant in P. regina .

The isolation of a methylcholine containing phospholipid from Phormiaregina larvae

Abstract Although carnitine, β-hydroxy-γ-butyrobetaine, occurs in many animal systems, its function and intermediary metabolism remains obscure (Fraenkel 1957). The results of previous experiments in our laboratory indicated that on a chemically defined diet, carnitine and several related compounds, including γ-butyrobetaine, etc., replaced the choline requirement. (Hodgson, Cheldelin and Newburgh 1956, 1960). Since choline is a major constitent in Phormia regina phospholipids it seemed reasonable to postulate that when larvae are reared on a diet devoid of choline but containing carnitine, a phospholipid in which choline is replaced by carnitine might be observed. This communication reports the formation of a new “lecithin” when blowflies are reared in the presence of carnitine.

Enzyme studies of various stages of the blowfly Phormia regina (meig.)

Abstract Investigations on carbohydrate dissimilation in the blowfly Phormia regina have been conducted. Extensive citric acid cycle activity was found to be localized in adult mitochondria, but could not be demonstrated in larvae. Cytochrome oxidase activity was greater in the adult than in larvae. Glycolysis and the pentose cycle system were present in soluble fractions from both the adult and larvae, and the pentose cycle was also indicated in the egg. The presence of three TPN-specific enzymes was demonstrated: isocitric, G-6-P, and 6-PGA dehydrogenases.

Carnitine and derivatives in embryonic chick tissue

Abstract 1. 1. Carnitine, acetylcarnitine and long-chain acylcarnitine concentrations were determined for heart, brain and liver of chick embryos during various stages of development. The total carnitine concentration was approximately the same in all three organs and showed only small variations during development. Acetylcarnitine was not detected in any organ until the 17th day of incubation and represented about 20% of the total carnitine in each organ on the day of hatch. Concentrations of long-chain acylcarnitine generally represented from 5 to 10% of the total carnitine in each case. 2. 2. The levels of carnitine acetyltransferase follow the increase in acetylcarnitine concentrations with development in the heart and liver. The enzyme activity in the yolk sac is localized in the yolk-sac membrane. The possibility that this yolk-sac enzyme may function in the transfer of fatty acyl groups from the yolk into the embryo is discussed. 3. 3. Perchloric acid extracts of chick embryonic tissues contained a substance which interferes with the assays for carnitine and acetylcarnitine. The interference was attributed to an inhibitor of the carnitine acetyltransferase reaction used in the assays. By careful adjustment of the concentration of tissue extract in the assay mixture, carnitine and acetylcarnitine were accurately determined in the presence of the inhibitor.

Properties of cyclic nucleotide phosphodiesterase in the central nervous system of Manduca sexta.

The properties of cyclic nucleotide phosphodiesterase were studied in soluble and particulate fractions from the central nervous system of Manduca sexta (Lepidoptera: Sphingidae). It was determined that: (1) The highest levels of phosphodiesterase occur in nervous tissue. (2) The total and specific enzyme activities of larval and adult brains are greater than those of the remaining ganglia. (3) Specific central nervous sy stem phosphodiesterase activities of the adult are lower than those of the larva, but both protein and total phosphodiesterase contents are considerably greater in the adult central nervous system. (4) Mg2+ is not absolutely required for either cyclic AMP-phosphodiesterase or cyclic GMP-phosphodiesterase activity. (5) Phosphodiesterase is inhibited by a variety of physiological and non-physiological compounds, nucleoside triphosphates being particularly effective; Some potent inhibitors of mammalian phosphodiesterase are comparatively ineffective toward Manduca sexta phosphodiesterase. (6) Kinetic analyses of soluble and particulate phosphodiesterase revealed non-linear double-reciprocal plots for the hydrolysis of both cyclic AMP and cyclic GMP, with Michaelis constants of approximately 10 mu M and 20 mu M; (7) The hydrolysis of both cyclic nucleotides appears in part to be the function of a single enzyme or related enzymes in the insect central nervous system. It follows that the intracellular level of one cyclic nucleotide may influence the concentration of the other by inhibiting its DEGRADATION.

Oxalate oxidation by an obligately parasitic fungus Tilletia contraversa.

Abstract Enzyme preparations from teliospores of Tilletia contraversa oxidize oxalic acid to carbon dioxide and probably hydrogen peroxide. The oxidation is maximum near pH 2.6 and is 50% inhibited by 2.4 × 10 −4 M fluoride and by 1.6 × 10 −2 M cyanide. The oxidation is stimulated by riboflavine and flavine mononucleotide, but not by flavine adenine dinucleotide.

A DNA-like RNA fraction in the posterior silkgland of the wax moth, Galleria mellonella (L.).

Abstract Extraction of the 28-S rRNA from the posterior silkgland of Galleria in an acetate-sodium dodecyl sulfate solution at 45° results in the conversion of the rRNA to a 18-S component. When silkgland RNA is labeled with [3H]uridine for 2 h, a highly radioactive RNA specie is found that separates by sucrose gradient centrifugation at the same place as the 28-S rRNA. This radioactive RNA is not converted by heat-treatment to the 18-S component. It is no longer radioactive when the incubation in the presence of [3H]uridine is carried out for 8 h, indicating a rapid turnover. Methylation experiments suggested that the major component of this RNA is not likely a 30-S rRNA precursor. Furthermore, characterizations of this RNA by base composition analysis and RNA-DNA hybridization tests strongly indicated that this is “DNA-like” RNA. These results suggest that there is probably an unstable mRNA in the posterior silkgland of Galleria. This possibility was discussed in comparison with the so-called stable mRNA for the silk fibroin of Bombyx.

Cyclic nucleotide-stimulable protein kinases in the central nervous system of Manduca sexta

Cyclic nucleotide-stimulable protein kinase (EC 1.7.1.37) has been studied in crude extracts from the central nervous system of the tobacco hornworm Manduca sexta (Lepidoptera: Sphingidae). The insect kinase was fulfhydryl-sensitive and required Mg-2+ for optimal activity. Polyacrylamide gel electrophoresis of supernatants demonstrated the presence of multiple kinases in the larval nerve cord. At low concentrations, cyclic AMP was a much more potent activator of soluble and particulate activities than was cyclic GMP. The specific activity of coluble kinase and the magnitude of its activations by cyclic AMP were greater in the adult than in the larval central nervous system. The exogenous protein substrate specificity of the insect enzyme was similar to that of rat brain kinase with the sole exception that protamine was more readily phosphorylated than histone by nerve cord kinase. It was observed that cyclic AMP lowered the Km of Manduca sexta kinase for ATP, a phenomenon which is apparently nervous tissue=specific in mammals. An effective inhibitor of cyclic AMP-dependent protein kinase was prepared from the larval central nervous system.