Changes in Activities of Glucose Metabolising Enzymes in Germ Cells during Spermatogenesis in Rats

The rate of ¹⁴CO₂, liberation from [¹⁴C-1]glucose was identical to that from [¹⁴C-6]glucose in spermatids, but more than the latter in spermatogonia. Rotenone (1 μM) completely inhibited ¹⁴CO₂ release from [¹⁴C-1]glucose in spermatids, but decreased it only 30% in spermatogonia. The activity of glucose-6-phosphate dehydrogenase, but not 6-phosphogluconate dehydrogenase, was markedly lower in spermatocytes and spermatids than in spermatogonia. The activities of the glycolytic enzymes, glucosephosphate isomerase, fructose diphosphatase, glyceraldehyde-3-phosphate dehydrogenase and enolase, differed only slightly in spermatids and spermatogonia. It is concluded that the low glucose-6-phosphate dehydrogenase activity may contribute to the low activity of the pentose cycle in spermatocytes and spermatids.     

Processing and Secretion of Lysosomal Acid α-Glucosidase In Tetrahymena Wild Type and Secretion-Deficient Mutant Cells

ABSTRACT. The proteolytic processing and secretion of a lysosomal enzyme, acid α-glucosidase, was studied by pulse-chase labeling with [³⁵S]methionine in Tetrahymena thermophila CU-399 cells treated with ammonium chloride. This cell secreted a large amount of acid α-glucosidase into the cultured medium during starvation. the secretion was found to be repressed by addition of ammonium chloride (NH₄Cl). Acid α-glucosidase was produced as a precursor form (108 kDa) and then processed to a mature polypeptide (105 kDa) within 60 min. This mature enzyme was secreted into the media within 2-3 h after chase, whereas the precursor form was not secreted by either control cells or NH₄Cl-treated cells. NH₄Cl did not affect the processing of the precursor acid α-glucosidase. Processing profile of this enzyme was apparently indistinguishable from that of the mutant MS-1 defective in lysosomal enzyme secretion. Furthermore, the purified extracellular (CU-399) and intracellular (MS-1) acid a-glucosidases were the same in molecular mass (105 kDa) and enzymatic properties. They contained no mannose 6-phosphate residues in N-linked oligosaccharides. These results suggested that unlike mammalian cells, Tetrahymena acid α-glucosidase may be transferred to lysosomes by a mannose 6-phosphate receptor-independent mechanism, and also that low pH was not essential for the proteolytic processing of precursor polypeptide.     

Malate dehydrogenase and glucose-6-phosphate dehydrogenase, key markers for studying the genetic diversity of Actinobacillus actinomycetemcomitans

Abstract Cell-free extracts of strains belonging to the 5 serotypes of A. actinomycetemcomitans were screened for several enzymes. Enzymes representative of the pentose phosphate pathway/hexose monophosphate shunt and the TCA cycle were present. Of these glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH) were the most readily detected and stable. MDH and G6PDH retained more than 50% of their activities at alkaline pHs (10–11) for up to 6 h and 3 h at 25°C, respectively, while at pH 6.5, 50% of their activities were lost within 2–3 h. The K _m for malate oxidation catalysed by MDH was 5.8×10⁻⁴ M while that for glucose-6-phosphate oxidation was 2.0×10⁻⁴ M. The pH optima for MDH and G6PDH oxidation activities were 10 and 9.5, respectively. Among the 5 designated serotypes of A. actinomycetemcomitans three groups were delineated by multilocus enzyme electrophoresis using MDH and G6PDH.     

Evidence for a pentose phosphate pathway in Helicobacter pylori

Abstract Evidence for the presence of enzymes of the pentose phosphate pathway in Helicobacter pylori was obtained using ³¹P nuclear magnetic resonance spectroscopy. Activities of enzymes which are part of the oxidative and non-oxidative phases of the pathway were observed directly in incubations of bacterial lysates with pathway intermediates. Generation of NADPH and 6-phosphogluconate from NADP⁺ and glucose 6-phosphate indicated the presence of glucose 6-phosphate dehydrogenase and 6-phosphogluconolactonase. Reduction of NADP⁺ with production of ribulose 5-phosphate from 6-phosphogluconate revealed 6-phosphogluconate dehydrogenase activity. Phosphopentose isomerase and transketolase activities were observed in incubations containing ribulose 5-phosphate and xylulose 5-phosphate, respectively. The formation of erythrose 4-phosphate from xylulose 5-phosphate and ribose 5-phosphate suggested the presence of transaldolase. The activities of this enzyme and triosephosphate isomerase were observed directly in incubations of bacterial lysates with dihydroxyacetone phosphate and sedoheptulose 7-phosphate. Glucose-6-phosphate isomerase activity was measured in incubations with fructos 6-phosphate. The presence of these enzymes in H. pylori suggested the existence of a pentose phosphate pathway in the bacterium, possibly as a mechanism to provide NADPH for reductive biosynthesis and ribose 5-phosphate for synthesis of nucleic acids.     

Hydroperoxide inactivation of enzymes within spores of Bacillus megaterium ATCC19213

Abstract Hydroperoxide inactivation of the protoplast enzymes enolase, aldolase and glucose-6-phosphate dehydrogenase in intact spores of Bacillus megaterium ATCC19213 was assessed by first treating the cells with lethal levels of H₂O₂, then germinating them in the presence of chloramphenicol prior to permeabilization and enzyme assays. Glucose-6-phosphate dehydrogenase proved to be more sensitive to H₂O₂than enolase or aldolase, in agreement with findings for isolated enzymes. Average D values (time for 90% inactivation) for spores treated with 0.50% H₂O₂ were 173 min for enolase, 67 min for aldolase and 32 min for glucose-6-phosphate dehydrogenase, compared with a D value of 34 min for spore killing. H₂O₂ killing of spores was found to be conditional in that recoveries of survivors were greater on complex medium than on minimal medium. Overall, it appeared that oxidative inactivation of enzymes may be important for hydroperoxide killing of spores.     

Effects of Boron Deficiency on Rubidium Uptake and Photosynthesis in the Diatom Cylindrotheca fusiformis

Culturing the diatom Cylindrotheca fusiformis under boron-deficient conditions leads to changes in ⁸⁶Rb uptake and photosynthesis prior to any effect on the rate of cell division. The influx rate of ⁸⁶Rb into boron-deficient cells was 79% of the control rate after 5 to 5.5 hours culture. Despite lowered ⁸⁶Rb influx, however, boron-deficient diatoms accumulated more ⁸⁶Rb than did control cells; this was due to the deficient cells' lower efflux rate. After 24 hours culture, boron-deficient cells had accumulated 30% more ⁸⁶Rb than had control cells, while releasing ⁸⁶Rb at only one-half the control rate. Increased photosynthetic rates were another effect of boron deficiency during this early stage of culture. Prior to 20 hours boron-deficient culture, diatoms had photosynthetic rates 37% greater than those of control cells. Corresponding to the increase in photosynthesis, boron-deficient diatoms had 12% more carbohydrate than control cells after 16 hours culture.     

PRODUCTION OF MUCILAGE BY THE ADRIATIC EPIPELIC DIATOM CYLINDROTHECA CLOSTERIUM (BACILLARIOPHYCEAE) UNDER NUTRIENT LIMITATION

The carbon partitioning of the epipelic diatom Cylindrotheca closterium (Ehrenberg) Reiman and Lewin isolated from the Adriatic Sea was studied in the laboratory under varying scenarios of nutrient limitation. Total number of cells, photosynthesis measured at 695 μmol photons·m ^{− 2}·s ^{− 1} irradiance (P_{695- μ mol}), chlorophyll ( a + c ) content, respiration, extracellular polymeric substances (EPS), total particulate carbohydrate (TPC), and dissolved carbohydrate were evaluated under nitrogen and phosphorus deficiencies in culture. The highest total number of cells was found in the control, whereas the nitrogen-limited treatment showed the lowest value. During the transition phase of growth, photosynthesis in the nitrogen-limited treatment was 3-fold lower than in the phosphorus-limited treatment and 4-fold lower than in the control. Differences in respiration rates and chlorophyll ( a + c ) content were even more marked. Dissolved carbohydrate remained the same in all the treatments, whereas during the transition and stationary phase, EPS presented the highest values under phosphorus limitation and the lowest in the control treatment. The production of EPS was closely linked to the periods of carbon assimilation (transition phase) in the nutrient depleted treatments, especially in the phosphorus-limited treatment. These results point out the relevance of the nutrient imbalance (nitrogen or phosphorus) in the production of EPS by the benthic or resuspended diatoms and suggest that these diatoms play an important role in nutrient-unbalanced systems like sediments or marine snow.     

POLYOL ACCUMULATION IN IN VITRO SYSTEMS OF BOMBYX EGGS

The mechanism of polyol accumulation in diapausing Bombyx eggs, conversion of [6-¹⁴C] glucose-6-phosphate into polyols and other neutral sugars was investigated in in vitro reaction systems. When a crude homogenate or a press juice of the eggs was incubated with [6-¹⁴C]glucose-6-P, the labelled trehalose, sorbitol and glycerol accumulated in the reaction mixture. In the press juice incubation system of developing eggs at day 1, ¹⁴C-sorbitol was detected in appreciable amounts, but it decreased rapidly with the development of the embryos. When the press juice was prepared from eggs in diapause, the formation of ¹⁴C-sorbitol was 3–5 times greater in eggs at early stages (day 2 to day 4) than in developing eggs.     

Capacity for hexose respiration in symbiotic Frankia from Alnus incana

Frankia vesicle clusters were prepared from Alnus incana (L.) Moench root nodules containing a local source of Frankia by an improved homogenization-filtration procedure. The capacity of the vesicle clusters to metabolize hexoses was investigated by respirometric and enzymological studies. The vesicle clusters could utilize glucose, glucose-6-phosphate and 6-phosphogluconate provided that appropriate cofactors were added to the preparations. The enzymes hexokinase (EC 2.7.1.1), NADP⁺: glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and NAD⁺;6-phosphogluconate dehydrogenase (EC 1.1.1.44) were found in cell-free extracts of the vesicle clusters and kinetic constants for the enzymes were determined. Hexokinase had a lower K_m for glucose than for fructose. Extracts from both symbiotic and propionate grown Frankia AvcII also showed activity of these hexose-degrading enzymes, indicating that their presence is not necessarily dependent on sugars as carbon source. The NAD⁺- dependent 6-phosphogluconate dehydrogenase was only present in Frankia cells and not in alder root cells, which makes this enzyme a useful Frankia -specific marker in these symbiotic systems.     

Benthic diatom distribution and grazing by Sarotherodon mossambicus in Lake Sibaya, South Africa

SUMMARY. Distributions of benthic diatoms and benthic floc in Lake Sibaya were examined to determine if preferential grazing by juvenile Sarotherodon mossambicus on benthic detrital aggregate in shallow water maximized diatom concentrations in the diet of the fish. Between 0.1 and 12.0 m depth, the range of diatom densities was 1–60 × 10⁴ cells cm⁻² with high but extremely variable densities above 3 m and consistently low densities at greater depths. Light intensity and burial by wave action appear to be primary factors affecting diatom abundance, but the effect of unidentified factors causing local patchiness is also evident. Substrate stabilization by diatoms was observed between 0.3 and 1.8 m depth. Weight of benthic detrital aggregate generally increased with depth such that diatom concentrations were variable above 3 m but were extremely low in benthic detrital aggregate at greater depths. Comparison of diatom concentrations in fish stomachs and benthic detrital aggregate shows that within the shallow waters, fish do not selectively graze those areas richest in diatoms.     

Photosynthesis and carbon metabolism in photoautotrophic cell suspensions of Chenopodium rubrum from different phases of batch growth

Rapidly dividing photoautotrophic cell suspensions from Chenopodium rubrum L. assimilated about 85 μmol CO₂ (mg chlorophyll)⁻¹ h⁻¹. During the late stationary phase of culture growth, CO₂ fixation rate was reduced to about 60 μmol CO₂ (mg chlorophyll)⁻¹ h⁻¹. Actively dividing cells characteristically incorporated a smaller proportion of ¹⁴C into starch than cells from non-dividing stationary phases. In rapidly dividing cells, [¹⁴C]-turnover from free sugars, sugar-phosphates, organic and amino acids was substantially higher compared to non-dividing cells from stationary growth phase. Higher proportions of photosynthetically fixed carbon were channelled into proteins, lipids and structural components in actively dividing cells than in non-dividing cells. In the latter. ¹⁴C was preferentially channeled into starch, and a striking increase in starch accumulation was observed. The transfer of non-dividing, stationary growth-phase cells into fresh culture medium resulted in an increase in the maximum extractable activities of some enzymes involved in the glycolytic and dark respiratory pathways and in the citric acid cycle. In contrast, the maximum extractable activities of the chloroplastic enzymes, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.38) and NADP⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) were highest after the cells had reached the stationary growth phase.     

The capsular (K51) antigen of Escherichia coli 01:K51:H−, an O-acetylated poly-N-acetylglucosamine phosphate

Abstract The capsular K51 antigen of E.coli was isolated from a liquid culture of E.coli 01:K51:H⁻ by Cetavlon precipitation. After purification it was obtained in a yield of about 80 mg/l. The polymer consisted of equimolar amounts of N -acetylglucosamine and phosphate and contained about 1.6 O -acetyl groups per N -acetylglucosamine residue. After de- O -acetylation it was resistant to periodate oxidation. Mild acid hydrolysis yielded N -acetylglucosamine-3-phosphate. With the aid of ¹³C- and ³¹P-NMR spectroscopy it was ascertained that the K51 antigen is a poly- α -1.3- N -acetylglucosamine phosphate, in which most of the hydroxyl groups at C4 and C6 of the N -acetylglucosamine residue are acetylated.     

ACTIVATION OF HEXOSE MONOPHOSPHATE PATHWAY IN BRAIN BY ELECTRICAL STIMULATION IN VITRO

Abstract— The incubation of cerebral cortical slices for 15 min in Krebs-Ringer-tris (pH 7.6) solution at 37°C with [1-¹⁴C]glucose or [6-¹⁴C]glucose as substrates yielded a C-1:C-6 ¹⁴CO₂ ratio of 1.21, whereas this ratio increased to 3.01 after the application of electrical stimulation (ES). Tissue levels of 6-phosphoglu-conate (6PG) and glucose 6-phosphate (G6P), intermediary metabolites of hexose monophosphate (HMP) pathway, were 7 and 180 nmol/g tissue following 15 min incubation, and increased by 33 and 45 per cent respectively following the application of ES. Activities of 6-phosphogluconate dehydrogenase (6PGDH, 6-phospho- d -gluconate: NADP⁺ 2-oxidoreductase, EC 1.1.1.44) and glucose-6-phosphate dehydrogenase (G6PDH, d -glucose-6-phosphate: NADP⁺ 1-oxidoreductase, EC 1.1.1.49), important enzymes in regulating the activity of HMP pathway, in cerebral cortical slices were 689 and 907 pmol/mg protein/min and were increased by 66 and 25 per cent respectively by the application of ES. Synaptosomal G6PDH and 6PGDH activities were maximally activated by the addition of 40 m m -Na⁺ to the reaction mixture, whereas no activation by Na⁺ was observed in microsomal G6PDH and 6PGDH. Amobarbital inhibited more strongly the Embden–Meyerhof (EM) pathway than the HMP pathway, while imipramine had a stronger inhibitory effect on HMP pathway than on EM pathway in the electrically stimulated cerebral tissues.
The present results indicate that the HMP shunt pathway in the cerebral cortex is activated by the application of ES in vitro , possibly at synaptic regions and may play an important metabolic and functional role in the brain.     

DOMOIC ACID PRODUCTION IN NITZSCHIA SP. (BACILLARIOPHYCEAE) ISOLATED FROM A SHRIMP-CULTURE POND IN DO SON, VIETNAM

Domoic acid (DA), a neuroexcitatory amino acid, was detected in batch culture of the newly recognized species Nitzschia navis-varingica Lundholm et Moestrup . The production of DA by this diatom was confirmed by electrospray ionization mass spectrometry. The diatom was collected from a shrimp-culture pond in Do Son, Vietnam. The production of DA (1.7 pg·cell ^{− 1}) is within the levels reported for Pseudo-nitzschia multiseries (Hasle) Hasle. The DA production started during the late exponential growth phase and reached a maximum during the early stationary growth phase. Maximum DA levels in the axenic culture decreased to about half that of the nonaxenic culture (0.9 pg·cell ^{− 1} vs. 1.7 pg·cell ^{− 1}), suggesting that DA production by the new species is influenced by bacteria.     

Consequences of the presence of 24-epibrassinolide, on cultures of a diatom, Asterionella formosa

Addition of the plant hormone 24-epibrassinolide to culture media stimulated the growth of a freshwater diatom, Asterionella formosa. The hormone stimulated activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme from Calvin cycle, by 6-fold. Other key metabolic enzymes, phosphofructokinase and malate dehydrogenase were also stimulated but to a lesser extent. The activity of glucose-6-phosphate dehydrogenase, involved in the oxidative pentose phosphate pathway, also increased in the presence of the hormone but only under non reducing conditions. In cells stimulated by epibrassinolide, activated enzymes were sensitive to oxidized-DTT. GAPDH purified from cells grown in the presence of the hormone was not associated with a small protein of 8.5 kDa shown to be similar to CP12. Consequently the activity of GAPDH was no longer regulated by either oxidizing or reducing conditions. Among enzymes that, like GAPDH, responded positively to reducing agent were fructose-1,6-bisphosphatase (FBPase) and glucose-6-phosphate dehydrogenase (G6PDH). These enzymes were also sensitive to, and were negatively regulated by, oxidized-DTT. The activities in extracts from illuminated cells differed from those from darkened cells: FBPase, G6PDH and GAPDH, that were activated by DTT in darkened cells were no more activated in illuminated cells, but were oxidized by oxidized-DTT. Thus, oxidizing or reducing conditions mimic the conditions in dark and light, respectively. Unlike the other enzymes, phosphofructokinase (PFK) was inhibited by DTT but oxidized-DTT reversed this effect. The enzymes shown to be redox regulated in vitro by reduction/oxidation are very likely candidates for regulation in vivo by thioredoxins.     

In vitro and in vivo regulation of chloroplast glyceraldehydes-3-phosphate dehydrogenase isozymes from Chenopodium rubrum. I. Purification and properties of isozymes

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GPD, EC 1.2.1.13) was purified from leaves of Chenopodium rubrum L. Aggregated (≥ 10⁶) and disaggregated (165 × 10³) molecular weight forms were obtained by gel filtration in the presence of NAD⁺ and NADP⁺, respectively. The disaggregated enzyme was separated into two isozymes by inverse ammonium sulphate gradient solubilization: "NADP-GPD I" was homotetrameric (subunit molecular weight 39 × 10³); "NADP-GPD II" was heterotetrameric (subunit molecular weights 39 × 10³ and 43 × 10³). Isoelectric focusing of the isozymes, both aggregated and disaggregated, revealed two isoelectric forms in each case, at 4.3 and 7.7. Chloroplast GPD was "NADP-suppressed" in crude extracts due to partial oxidation, incubation with dithioerythritol restored full activity.     

Estimating the number of viable animal cells in multiwell culture--a tetrazolium-based assay

A reliable, indirect method (GPD/INT assay) for estimating the number of live animal cells in multiwell culture has been devised. It is based on the glucose-6-phosphate dehydrogenase (Gpdh) and 6-phosphogluconate dehydrogenase activities present in the cytoplasm of viable eukaryotic cells but not in their bathing medium nor in nonviable cells. A single reagent mixture, buffered at pH 7.8 and containing Tris, Triton X-100, glucose-6-phosphate, nicotinamide adenine dinucleotide phosphate (NADP), phenazine methosulfate, and iodonitrotetrazolium violet, is added to the cultures. The Triton X-100 releases the cytoplasmic contents into the medium, facilitating enzyme-catalyzed oxidation of the glucose-6-phosphate and 6-phosphogluconate by NADP. The resulting reduced nicotinamide adenine dinucleotide phosphate, NADPH, reduces tetrazolium violet to its formazan, the color of which reflects the number of living cells that were in the culture. The assay was tested on recombinant Gpdh and the several types of animal and insect cell lines to verify the premise that there is proportionality between the amount of GPdh and number of viable cells in the cultures. The method has been used to quantitate the effects of growth inhibitors on cells in 96-well cultures.     

A STUDY OF Si DEPOSITION SYNCHRONY IN RHIZOSOLENIA (BACILLARIOPHYCEAE) MATS USING A NOVEL 32Si AUTORADIOGRAPHIC METHOD

A ³²Si autoradiographic technique using a liquid photographic emulsion was developed for the study of diatom silica deposition in culture or in natural water samples. The method was used in the Central North Pacific to study silica deposition by diatoms of the genus Rhizosolenia. The species examined form centimeter-sized aggregates commonly referred to as mats. The Rhizosolenia mats examined were composed of a matrix of R. fallax Sundström chains, embedded with chains of larger cells, either R. debyana H. Peragallo or R. acuminata H. Peragallo. The autoradiographs revealed distinct rings of labeled intercalary bands and/or labeled valves. A greater proportion of the frustule of the larger species was labeled during the incubations with ³²Si, implying higher rates of silicification by R. debyana and R. accuminata compared to R. fallax. A quantitative consideration of these differences in species-specific Si production combined with abundance and surface area estimates for each species indicates that cells of the larger species carry out the majority of silica production in Rhizosolenia mats. The large cell size (pervalvar axis 240 to 3000 μm) and elongate frustule morphology of Rhizosolenia cells enabled us to localize the deposition of silica along the pervalvar axis. Positions of labeled bands along this axis indicate progress through the Si deposition cycle, and the results suggest that cell division is phased, with either a bimodal or unimodal age distribution of cells within the cell cycle for all species in a mat. Species-specific doubling times from 25 to 60 h were implied by the mean fractions of frustule that were labeled. ³²Si autoradiography revealed unique species-specific differences in diel patterns of cell division and silica deposition and has potential for studies of Si deposition by other diatom species and assemblages.     

Glucose Metabolism in Neisseria gonorrhoeae

The metabolism of glucose was examined in several clinical isolates of Neisseria gonorrhoeae. Radiorespirometric studies revealed that growing cells metabolized glucose by a combination on the Entner-Doudoroff and pentose phosphate pathways. A portion of the glyceraldehyde-3-phosphate formed via the Entner-Doudoroff pathway was recycled by conversion to glucose-6-phosphate. Subsequent catabolism of this glucose-6-phosphate by either the Entner-Doudoroff or pentose phosphate pathways yielded CO(2) from the original C6 of glucose. Enzyme analyses confirmed the presence of all enzymes of the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways. There was always a high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) relative to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The glucose-6-phosphate dehydrogenase utilized either nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide as electron acceptor. Acetate was the only detectable nongaseous end product of glucose metabolism. Following the disappearance of glucose, acetate was metabolized by the tricarboxylic acid cycle as evidenced by the preferential oxidation of [1-(14)C]acetate over that of [2-(14)C]acetate. When an aerobically grown log-phase culture was subjected to anaerobic conditions, lactate and acetate were formed from glucose. Radiorespirometric studies showed that under these conditions, glucose was dissimilated entirely by the Entner-Doudoroff pathway. Further studies determined that this anaerobic dissimilation of glucose was not growth dependent.