ASSAY AND PROPERTIES OF 4-AMINOBUTYRIC-2-OXOGLUTARIC ACID TRANSAMINASE AND SUCCINIC SEMIALDEHYDE DEHYDROGENASE IN RAT BRAIN TISSUE

Abstract— The activity of 4-aminobutyric-2-oxoglutaric acid transaminase (GABA transaminase) and succinic semialdehyde dehydrogenase was determined in total rat brain homogenate. GABA transaminase activity was measured using a coupled enzyme method which utilizes endogenous succinic semialdehyde dehydrogenase to convert the formed succinic semialdehyde into succinate. The concurrently produced NADH was used as an estimate of GABA transaminase activity. This method could be used since it was shown that the dehydrogenase was about twice as active as the transaminase and because no significant accumulation of the intermediate succinic semialdehyde could be detected. GABA transaminase was inhibited by high ionic strength. In contrast NaCl decreased the apparent K _m and increased V _max for succinic semialdehyde dehydrogenase at high but not al low tissue concentrations. Increasing tissue concentration also resulted in a decrease of the apparent K _m, but did not change the V_max of succinic semialdehyde dehydrogenase and it is suggested that this enzyme can exist in two distinct states of aggregation, one with a high and one with a low affinity for succinic semialdehyde. The high affinity form of the enzyme is thought to prevent succinic semialdehyde from accumulation in the GABA transaminase assay. It is concluded that within certain limits the coupled enzyme method described here can be used for the assay of GABA transaminase activity.     

Use of an axenic medium for differentiation between pathogenic and nonpathogenic Naegleria fowleri isolates.

Growth in an axenic medium composed by Chang (3rd Int. Congr. Parasitol. Munich Abstr. ICPIII 1:187-188, 1974) allowed separation of pathogenic from nonpathogenic Naegleria fowleri strains, since only the former show luxuriant growth in this medium. On the basis of these results, this medium was used in early screening for virulent Naegleria isolates. During an extensive ecological study, data were obtained on 102 Naegleria strains. Twenty of these strains grew luxuriantly in this liquid medium. Seventeen of them were tested by intranasal instillation in mice, and all proved to be highly pathogenic. Strains showing only moderate growth or no growth at all in this axenic medium were found to be nonpathogenic for mice. Moreover, it was found that using this medium in the early stage of Naegleria sampling favors isolation of pathogenic strains in mixtures of Naegleria. During these experiments, further evidence was obtained that thermal polluted waters are the main origin of N. fowleri in the environment.     

Studies on (Na+ +K+) activated ATPase. XLI. Effects of N-ethylmaleimide on overall and partial reactions.

1. Preincubation with N-ethylmaleimide inhibits the overall activity of highly purified (Na+ +K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations of rabbit kidney outer medulla. 2. This inhibition is decreased by addition of ATP or 4-nitrophenylphosphate under non-phosphorylating conditions, and also by addition of ADP or adenylylimidodiphosphate. 3. N-ethylmaleimide treatment leads to inhibition of K+-stimulated 4-nitrophenylphosphatase activity, Na+-stimulated ATPase activity, and phosphorylation by ATP as well as by inorganic phosphate. These inhibitions strictly parallel that of the overal (Na+ +K+)-ATPase reaction. 4. N-ethylmaleimide lowers the number of sites which are phosphorylated by inorganic phosphate, without affecting the dissociation constant of the enzyme-phosphate complex. 5. N-ethylmaleimide does not affect the relative stimulation by ATP of the K+-stimulated 4-nitrophenylphosphatase activity. 6. These effects of N-ethylmaleimide can be explained as a complete loss of active enzyme, either by reaction of N-ethylmaleimide inside the active center, or by alterations in the quaternary structure through reactions outside the active center.     

Is there a plasma-membrane-located anion-sensitive ATPase? II. Further studies on rabbit kidney.

A study has been made to determine whether renal plasma membranes contain an HCO3 stimulated, ouabain insensitive Mg ATPase. Purified mitochondrial, microsomal and brush border membrane fractions have been isolated from rabbit kidney. The microsomal anion-sensitive ATPase activity appears to be entirely of mitochondrial origin on the basis of the effects of inhibitors of mitochondrial Mg ATPase. The brush border membrane fraction is contaminated with mitochondrial fragments and contains an Mg ATPase activity with low anion-sensitivity. Further purification of this fraction causes parallel decreases in anion-sensitivity of the Mg ATPase activity and in cytochrome c oxidase activity. These results indicate that conclusions previously reached by other investigators for a role of anion-sensitive Mg ATPase in the bicarbonate reabsorption of the proximal tubule may no longer be tenable.     

Secretion and reabsorption of uterine luminal fluid in rats

Treatment of ovariectomized rats with oestradiol-17beta and progesterone demonstrated that oestradiol-17beta causes secretion of sodium, potassium and water into the lumen of the uterine horn and that progesterone causes reabsorption of these substances.     

Lack of effect of toxic aluminium concentrations on nitrogen fixation by nodulatedStylosanthes species

Summary Effects of three solution aluminium concentrations (0, 25, and 100M) on nitrogen fixation by well-nodulated plants ofStylosanthes hamata, Stylosanthes humilis andStylosanthes scabra are reported. Plants were inoculated with Rhizobium CB756 and grown for 21 days in an aluminium-free nutrient solution at pH 5.3 before imposition of the aluminium treatments.Nitrogen fixation was measured both by the increase in total nitrogen content of the plants and acetylene reduction in roots of plants harvested at 10 and 20 days after imposition of the aluminium treatments. Solution aluminium concentrations as high as 100M, had no detrimental effect on nitrogen fixation in any species.     

Abscisic acid and gibberellin-like substances in roots and root nodules ofGlycine max

Summary The content of endogenous gibberellin (GA)-like substances of roots and root nodules of SOya, and GA production byRhizobium japonicum cultures, were investigated by a combined thin layer chromatographic (TLC)-dwarf pea epicotyl bioassay technique. GAs were more concentrated in root nodules than in the roots, totalling 1.34 and 0.16 nM GA₃ equivalents g⁻¹ dry wt. respectively. GA production byR. japonicum cultures was demonstrated (1.00 nM GA₃ equivalentsl ⁻¹) and comparison of the GA components of plant and bacterial culture medium extracts, suggested that rhizobial GA production may contribute to the nodule GA content. Cis-trans abscisic acid (ABA) was identified in root and nodule extracts by TLC-gas liquid chromatography (GLC), and amounted to 0.18 and 2.21 nM g⁻¹ dry wt. respectively, whereas 0.30 and 4.63 nM ABA equivalents g⁻¹ dry wt. were detected by a TLC-wheat embryo bioassay technique. ABA was not detected in extracts of bacterial cultures.     

Separation of mannosylretinylphosphate from dolichylmannosylphosphate by chromatography on columns of DEAE-sephacel

A one-step column chromatographic procedure on DEAE-Sephacel allows the separation of mannosylretinylphosphate from dolichylmannosylphosphate with minimal breakdown of the mannosylretinylphosphate. Using this procedure, subcellular fractions of rat liver were shown to be active in synthesizing both mannolipids from GDP-[¹⁴C]mannose in the absence or presence of exogenous retinylphosphate.     

Immunohistochemical evidence for a plasma membrane localization of dopamine-beta-hydroxylase in the mouse neuroblastoma

Dopamine-beta-hydroxylase (D beta H), a glycoprotein enzyme which converts dopamine into noradrenaline, was purified from C1300 mouse neuroblastoma and used to raise antibodies in rabbits. Using an indirect immunofluorescence technique the cellular localization of D beta H in C1300 mouse neuroblastoma was compared with that of the superior cervical ganglion. C1300 neuroblastoma D beta H was found to be predominantly localized in the plasma membrane, in contrast to its intracellular localization in the superior cervical ganglion of A/J mice. At least part of the enzyme was found to be associated with the external side of the plasma membrane.     

Serological detection of H-2K- and H-2D-gene products

Using the fluorescence-activated cell sorter (FACS II), we have analyzed the expression of H-2K- and H-2D-gene products on the membrane of various cellular components of the murine immune system. Using this serological technique we show a basic difference between T and B lymphocytes. Whereas all cellular components analyzed — hydrocortisone-resistant thymocytes, splenic T and B lymphocytes, macrophages and bone-marrow cells — expressed H-2K-subregion-encoded alloantigens at a high density, it seems that the high density expression of H-2D-encoded alloantigens is restricted mainly to B cells and to macrophages. Hydrocortisone-resistant thymocytes, splenic T lymphocytes and bone-marrow cells, on the other hand, showed significant expression of the H-2D alloantigens only at low membrane density. These results, then, provide evidence for the existence of an imbalance in serologically detectable expression of H-2K- and H-2D-region-gene products on the cell membrane of various cells comprising the murine immune system.Abbreviations usedin this paper DTH delayed type hypersensitivity - FCS fetal calf serum - FITC fluorescein isothiocyanate - HrT hydrocortisone-resistant thymocytes - Ig immunoglobulins P. De Baetselier is an EMBO and Euratom postdoctoral fellow