Ronald W. Brosemer

The Na+,K+-ATPase: a plausible trigger for voltage-independent release of cytoplasmic neurotransmitters.

Abstract: A comparison was made between the releasability of eight neurotransmitters from eight regions of mouse brain in response to either 60 μM‐K+ or 20 μM‐ouabain, a specific inhibitor of the Na+, K+‐ATPase. With few exceptions, all transmitters were released by either or both agents from each brain region examined. Potassium was superior in releasing the biogenic amines and acetylcholine, while the putative amino acid transmitters were generally releasable by both agents. Measurements of tissue depolarization using [3H]‐tetraphenylphosphonium uptake indicated that 60 μM‐K+ is capable of depolarizing brain tissue above the threshold necessary for initiating an action potential, but 20μM‐ouabain is not. The pattern of release by ouabain coupled with its failure to depolarize brain tissue at 20μM suggests that inhibition of the Na+,K+‐ATPase is capable of releasing cytoplasmic neurotransmitters in a voltage‐independent manner.

Pathway of proline oxidation in insect flight muscle.

Summary 1 Enzymes catalyzing the two-step oxidation of L -proline to L -glutamate in insect flight muscle have been measured. The pathway is similar to that in mammalian liver and in microorganisms. 2 Proline oxidase, which catalyzes the oxidation of L -proline to Δ1-pyrroline-5-carboxylate, is tightly bound to the particulate fraction. The requirement for cytochrome c and the effects of various electron transport inhibitors indicate that the enzyme system consists of a flavoprotein linked to the cytochrome chain. 3 Δ1-Pyrroline-5-carboxylate dehydrogenase (Δ1-pyrroline-S-carboxylate: DPN+ oxidoreductase) catalyzes the DPN+-dependent oxidation of the proline oxidase product to glutamate. This enzyme requires Mg2+ ions and is not as tightly bound to particles as is proline oxidase. 4 The possible role of these two enzymes in insect flight metabolism is discussed.

Insect extramitochondrial glycerophosphate dehydrogenase I. Crystallization and physical properties of the enzyme from honeybee (Apis mellifera) thoraces

Extramitochondrial glycerophosphate dehydrogenase (L-glycerol-3-phosphate: DPN+ oxidoreductase, EC 1.1.1.8) was purified and crystallized from honeybee (Apis mellifera) thoraces. The isolation procedure is quite simple, since it consists merely of a series of (NH4)2SO4 precipitations. The crystalline enzyme is homogeneous according to the following criteria: recrystallization to constant specific activity, electrophoresis on cellulose acetate strips, absence of 3 other glycolytic enzyme activities, chromatography on Sephadex G-200, and immunodiffusion on cellulose acetate strips. Sedimentation velocity studies indicate that the native protein readily associates at moderate protein concentrations. The minimum molecular weight as determined by Sephadex G-200 chromatography is around 67 000. Absorbance and fluorescence spectra show that neither DPNH nor adenosine diphosphate ribose is bound to the enzyme. The bee enzyme differs from the crystalline rabbit-muscle enzyme in electrophoretic, ultracentrifugal, and immunological properties.

Effects of inhibitors of Na+,K+-ATPase on the membrane potentials and neurotransmitter efflux in rat brain slices.

The potassium potential EK, of rat brain slices was estimated by determining the uptake of 86Rb+. The ERb was the same for slices prepared from five rostral brain regions, the average value being 66.4 mV. The ERb values in the presence of 20 microM ouabain were only slightly lower than the resting values; increasing concentrations of ouabain above 20 microM resulted in a graded depolarization in all five brain regions. High concentrations (1 mM) of two other inhibitors of Na+,K+-ATPase, dihydro-ouabain and strophanthidin, produced no more depolarization than did 20 microM ouabain. Competitive binding studies indicated that the differential effects were due to the relative binding to brain slices. Erythrosin B, an inhibitor of Na+,K+-ATPase, had no measurable effect on ERb. Intermediate concentrations of the Na+/H+ ionophore monensin slightly hyperpolarized striatal slices, whereas the same monensin concentrations plus 20 microM ouabain, 1 mM strophanthidin or 70 microM erythrosin B resulted in marked depolarization. Measurement of the membrane potential via uptake of methyltriphenylphosphonium cation indicated that ERb was indeed a valid estimation of the membrane potential. EK was measured directly by monitoring 42K+ uptake in striatal slices and was found to be essentially identical to ERb. Uptake of 22Na+ was consistent with the values for ERb or EK. Several conditions that resulted in little or no measurable depolarization of striatal slices did induce efflux of exogenously loaded GABA and dopamine; these conditions included 20 microM ouabain, 1 mM dihydro-ouabain or strophanthidin, and 70 microM erythrosin B. Neurotransmitter efflux in the absence of general cell depolarization was not accompanied by altered rates of respiration or decreased ATP levels.

Characterization of DNA from three bee species

Abstract DNA was isolated from three species of bees: the common honey-bee ( Apis mellifera ), the Indian honey-bee ( Apis cerana ), and the leaf-cutting bee ( Megachile rotundata ). The guanine-cytosine distribution of the two honey-bee DNAs is relatively broad and skewed toward lower values; leaf-cutting bee DNA shows a narrow distribution. No distinct satellite bands are present in any of the species. Renaturation kinetics indicate that the genome sizes of the two honey-bee species are identical to one another and somewhat smaller than that of the leaf-cutting bee. About 89 per cent of the genome of all three species renatures slowly; this fraction presumably comprises the single-copy (unique) sequence portion of the genome. Sufficient DNA was isolated from the dwarf honey-bee ( Apis florea ) to determine a guanine-cytosine distribution. Like the other honey-bee species, the distribution is relatively broad.

Insect extramitochondrial glycerophosphate dehydrogenase II. Enzymic properties and amino acid composition of the enzyme from honeybee (Apis mellifera) thoraces

Several enzymic properties of crystalline honeybee (Apis mellifera) thoracic glycerophosphate dehydrogenase (L-glycerol-3-phosphate:DPN+ oxidoreductase, EC 1.1.1.8) were determined. The apparent Michaelis constant for dihydroxyacetone phosphate is 0.33 mM; there is no increase in activity with substrate concn. above 0.5 mM. The bee enzyme has a broad pH optimum around pH 6.6, while the rabbits-muscle enzyme has a sharp optimum at pH 7.7. The bee enzyme is stable for 15 min at 21° from pH 4.8 to 9.9. The temperature coefficient, Q10, for the bee enzyme is 1.7 in the range from 21° 36°. The enzyme is stable for 5 min at 55° and completely inactivated at 61°. The bee enzyme shows the same relative reactivity with 2 DPN+ analogues as does the rabbit enzyme. The bee enzyme is inhibited by a low concentration of p-mercuribenzoate (PCMB), is less sensitive to N-ethylmaleimide, and is not inhibited by 1 mM iodoacetate. Glutathione does not activate the enzyme. The amino acid composition of the bee enzyme is quite different from the previously reported composition of the rabbits-muscle enzyme. The minimum molecular weight of the bee enzyme based on 1 tryptophan residue is 32 700. Since the molecular weight determined on a Sephadex G-200 column is around 67 000, the amino acid composition indicates a mol. wt. of 65 400.

Crystallization of glycerol 3-phosphate dehydrogenase, triosephosphate dehydrogenase, arginine kinase, and cytochrome c from a single extract of honeybees

Abstract The isolation of homogeneous preparations of four proteins in high yields from a single extract of honeybee thoraces is described. The four proteins are: glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), triosephosphate dehydrogenase (EC 1.2.1.12), arginine kinase (EC 2.7.3.3), and cytochrome c. This is the first reported isolation of arginine kinase from an insect source.

Changes in glycerolphosphate dehydrogenase activity during development of the grasshopper Schistocerca vaga

Abstract 1. 1. Extramitochondrial glycerolphosphate dehydrogenase ( l -glycerol-3-phosphate:DPN+ oxidoreductase, EC 1.1.1.8) was assayed in various tissues of the grasshopper Schistocerca vaga during the period from 3 days before to 8 days after the last molt. 2. 2. The specific activity, on protein basis, increase 8-fold in longitudinal dorsal flight muscles and 6-fold in the remaining pterothoracic flight muscles. 3. 3. The specific activity increases about 2-fold in two leg muscles (the flexor and extensor) and in fat body. 4. 4. The specific activity increases 6-fold in both mono- and bifunctional thoracic muscles. The former, the tergosternal muscles, are essentially only flight muscles. The latter, the thoracic coxal muscles, are active not only during adult flight but also during walking of the nymphs and adults. 5. 5. The relationship between tissue function and enzymitic development is discussed.

Glyceraldehydephosphate dehydrogenase: Crystallization from honeybees; Quantitative immunochemical and electrophoretic comparisons of the enzyme in other insects

Glyceraldehydephosphate dehydrogenase (EC 1.2.1.12) was crystallized from honeybee (Apis mellifera) thoraces. The yellow colour of the crystals is due to diphosphopyridine nucleotide bound to the enzyme. The sedimentation coefficient of the honeybee enzyme is the same as that reported for the enzyme from other species. A small amount of the dehydrogenase was also crystallized from bumblebee (Bombus nevadensis) thoraces. Rabbit antibodies directed against the pure honeybee enzyme were prepared, and the cross-reaction with extracts of other insects quantitatively measured with the micro-complement fixation technique. The order of decreasing cross-reaction is: honeybee, leafcutting bee, bumblebee (three species)—lobster—mining bee, flesh fly. The order: honeybee, leafcutting bee, bumblebee, was also observed in two-dimensional immunodiffusion and in a precipitin test. The relative cross-reaction of this enzyme, thus, varies considerably from the classical taxonomy of the species tested. The anomalous immunochemical properties of the glyceraldehyde-P dehydrogenases may be due to the relatively conservative changes in primary structure of this protein during evolution. Electrophoretic patterns of glyceraldehyde-P dehydrogenase activity reveal one band in extracts of honeybees and bumblebees, three bands in extracts of leafcutting bees and flesh flies, and up to five bands in extracts of mining bees.

Cellular localization of α-ketoglutarate: Glyoxylate carboligase in rat tissues

Abstract α-Ketoglutarate : glyoxylate carboligase activity has been reported by other laboratories to be present in mitochondria and in the cytosol of mammalian tissues; the mitochondrial activity is associated with the α-ketoglutarate decarboxylase moiety of the α-ketoglutarate dehydrogenase complex. The cellular distribution of the carboligase has been re-examined here using marker enzymes of known localization in order to monitor the composition of subcellular fractions prepared by differential centrifugation. Carboligase activity paralleled the activity of the mitochondrial matrix enzyme citrate synthase in subcellular fractions prepared from rat liver, heart and brain as well as from rabbit liver. Whole rat liver mitochondria upon lysis released both carboligase and citrate synthase. The activity patterns of several other extramitochondrial marker enzymes differed significantly from that of carboligase in rat liver. In addition, the distribution pattern of carboligase was similar to that of α-ketoglutarate decarboxylase and of α-ketoglutarate dehydrogenase complex. The data indicate that α-ketoglutarate : gloxylate carboligase activity is located exclusively within the mitochondria of the rat and rabbit tissues investigated. There is no evidence for a cytosolic form of the enzyme. Thus the report from another laboratory that the molecular etiology of the human genetic disorder hyperoxaluria type I is a deficiency of cytosolic carboligase must be questioned.