Evidence indicating that pig renal phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane

Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2 mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase (a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate, 20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.     

Chemical modification of spinach plastocyanin using 4-chloro-3,5-dinitrobenzoic acid: characterization of four singly-modified forms

Chemical modification of plastocyanin was carried out using 4-chloro-3,5-dinitrobenzoic acid, which has the effect of replacing positive charges on amino groups with negatively charged carboxyl groups. Four singly-modified forms were obtained which were separated using anion exchange FPLC. The four forms were modified at the N-terminal valine and at lysines 54, 71 and 77. The rates of reaction with mammalian cytochrome c were increased for all four modified plastocyanins. In contrast, the rates of reaction with cytochrome f were inhibited for the forms modified at residues 1, 54 and 77, whereas no effect was observed for the form modified at residue 71. Modification had no effect on either the midpoint redox potential or the reaction with K3Fe(CN)6. These results are consistent with a model in which charged residues on plastocyanin located at or near the binding site for cytochrome f recognize the positively-charged binding site on cytochrome f. In contrast, charged residues located at points on plastocyanin distant from the cytochrome f binding site recognize the net negative charge on the cytochrome f molecule. Based on these considerations, Glu-68 may be within the interaction sphere of cytochrome f, suggesting that cytochrome f may donate electrons to plastocyanin at either Tyr-83 or His-87.     

The effects of maturation on self-induced dynamic body sway frequencies of girls performing acrobatics or classical dance

We investigated the effects of maturation on the dynamic body sways of healthy girls. Prepubertal and postpubertal girls practising professional physical activities requiring a good ability to maintain equilibrium (acrobats and dancers) were asked to stand on a free seesaw platform and the results compared to those for untrained age-matched girls. This platform (stabilometer) allows self-induced body sways. Stabilograms were obtained by a double integration of the angular acceleration from the recordings of the platform sways made with an accelerometer. Fast Fourier transform processing of stabilograms allowed spectral frequency analysis. The total spectrum energy and the energies of three frequency bands (0–0.5 Hz, 0.5–2 Hz, 2–20 Hz) were determined. ANOVA showed that, for all groups of different equilibrium activity and independent of visual input, prepubertal girls had higher energy values than postpubertal girls in the 0- to 0.5-Hz band whereas the opposite was true for 0.5- to 2-Hz band. Ballet dancers were more dependent than acrobats on visual inputs for the regulation of their postural control but were less dependent than untrained girls at both ages. Maturation seemed to shift body sways towards higher frequencies and the utilization of the cues of postural control was different according to the type of equilibrium activity practised by the subjects. Accepted: 7 February 1997     

Chemo-enzymatic synthesis of the Galili epitope Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc on a homogeneously soluble PEG polymer by a multi-enzyme system.

The alpha-Gal trisaccharide Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric support (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of beta-1,4-galactosyltransferase (EC 2.4.1.38), alpha-1,3-galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free alpha-Gal trisaccharide.     

Biosorption of Cd, Zn and Cu by Sargassum sp. waste biomass

Biosorption is becoming an important component in the integrated approach to the treatment of aqueous effluents. The economics of biomass technical applications are improved by using waste biomass instead of purposely-produced biomass. Biomass derived from an alginate extraction industry - Sargassum sp. - was examined for its ability to function as a biosorbent for metals such as cadmium, zinc and copper. For use in column applications, biomass should be immobilized. To the algae reinforcement, the biomass was embedded in polyethleneimine (PEI), followed by glutaraldehyde crosslinking. Equilibrium Zn and Cu isotherms were analysed using the immobilization ratio that showed the best Cd performance. Either Freundlich or Langmuir models can describe the passive biosorption equilibrium of cadmium, zinc and cooper. The preference for this series of metals by the biomass was found to be Cd > Zn > Cu, with maximum uptake values of 157.8, 118.5 and 77.4 mg/g dry weight biomass for Cd, Zn and Cu. respectively. The metal binding capacity by non-living biomass is an important quality for industrial use.     

Lung microsomal p-nitrophenol hydroxylase -- characterization and reconstitution of its activity

1. Formation of catechols from benzene and nitrobenzene have been implicated in the carcinogenic activity of these chemicals. In liver, p-nitrophenol, an intermediate of p-nitrobenzene is enzymatically converted to 4-nitrocatechol. 2. For the first time in this study, the presence of a highly active enzyme catalyzing the formation of 4-nitrocatechol from p-nitrophenol was detected in lung microsomes. The average specific activity of lung p-nitrophenol hydroxylase was found to be 0.494 nmol 4-nitrocatechol formed mg prot-1 min-1. 3. The optimum conditions for sheep lung microsomal p-nitrophenol hydroxylase were established. The maximal activity was noted at pH 6.8. The rate of p-nitrophenol hydroxylation was linear up to 2 mg prot/ml of incubation mixture. The maximal rate of 4-nitrocatechol formation was observed with 0.25 mM p-nitrophenol. 4. The Lineweaver-Burk and Eadie-Hofstee plots were found to be curve-linear. Two different Km values were calculated as 11.6 and 71.4 microM from the Lineweaver-Burk plot and as 10.7 and 74.5 microM from the Eadie-Hofstee plot. This suggested that there were either two forms of enzyme or two different enzymes participating in ortho hydroxylation of p-nitrophenol in lung microsomes. 5. Lung microsomal p-nitrophenol hydroxylase activity of sheep was reconstituted in the presence of purified lung microsomal cytochrome P-450, NADPH dependent cytochrome P-450 reductase and synthetic lipid, phosphatidylcholine dilauroyl.