Luminal secretion of myo-inositol by the rat epididymis perfused in vitro

A technique for perfusing the lumen of rat epididymal tubules maintained in vitro showed that [3H]inulin was largely excluded from the lumen of unravelled tubules from the cauda and tubules from the corpus if the connective tissue capsule was removed. The preparation transported [3H]inositol from the bath fluid for 3 h against a concentration gradient in both regions with activity rising (16-29% of bath fluid values) in the cauda and reaching a plateau (18%) in the corpus epididymidis. HPLC showed that radioactivity was solely associated with inositol and its movement to the lumen was reduced by raising inositol in the bath fluid from 50 microM (plasma levels) to 10 mM, but not affected by reducing the glucose concentration in the bath fluid or introducing physiological concentrations of inositol (30 mM) into the lumen. Secretion into the caudal lumen of unlabelled inositol measured by g.l.c. was maintained for 3 h at concentrations (300 microM) greater than those in the bath fluid and was not reduced when glucose or inositol were removed from the bath. In contrast, glucose was only detectable in the lumen when it was present in the bathing medium, reaching 1% of this concentration. Radioactivity appeared in the epididymal lumen reaching a plateau (19% of bath fluid values) in the corpus and cauda when [3H]glucose was added to the bath fluid, but no radiolabelled inositol was found in the lumen. We conclude that epididymal tissue is a major source of secreted inositol.     

Sulfated glycoprotein-1 (saposin precursor) in the reproductive tract of the male rat

Sulfated glycoprotein-1 is one of the major protein secretion products of rat Sertoli cells in culture. This 70,000 Mr protein shares substantial sequence similarity with human prosaposin, the precursor of lysosomal saposins. Saposins are known to enhance the activity of lipid modifying enzymes presumably by solubilizing the lipids. We report here the immunolocalization of sulfated glycoprotein-1 in the cells and fluid of the male reproductive tract. The protein is present in secondary lysosomes of Sertoli cells and also in the luminal fluid of seminiferous tubules and epididymis. The highest concentrations of the protein are in seminiferous tubule fluid and rete testis fluid, while relatively low amounts are found in cauda epididymal fluid and serum. Sulfated glycoprotein-1 is believed to be involved in degradation of lipids in residual bodies and may also assist in modification of membrane lipids during sperm maturation.     

Formation,d'inositol par les chloroplastes isolés de pois

It was possible to synthesize d-inositol-1-phosphate from glucose-6-phosphate with extracts of chloroplasts isolated from pea-leaves. (U-¹⁴C)Inositol produced by alkaline phosphatase hydrolysis was studied under various conditions. The enzyme d-glucose-6-phosphate cyclase was isolated from the chloroplast preparation. The results showed the role of chloroplasts in the synthesis of their endogenous inositol.     

Enzymes of myo-inositol and inositol lipid metabolism in rats with streptozotocin-induced diabetes.

Diabetes, with only mild ketosis, was induced in male rats by a single injection of streptozotocin. After 12 weeks the specific activities of enzymes concerned with the metabolism of inositol and of inositol lipids were measured in various tissues. Inositol 1-phosphate synthase (EC 5.5.1.4) was most active in testis and the activity was significantly less in diabetic rats than in controls on a similar diet. Inositol oxygenase (EC 1.13.99.1), which converts myo-inositol into glucuronic acid, was also less active in kidney from diabetic animals. CDP-diacylglycerol-inositol phosphatidyltransferase (EC 2.7.8.11) and phosphatidylinositol 4-phosphate kinase (EC 2.7.1.68) showed decreased specific activities in brain and sciatic nerve of diabetic rats. By contrast the diabetic state did not affect the specific activities of phosphatidylinositol kinase (EC 2.7.1.67) or phosphatidylinositol 4,5-bisphosphate phosphatase (EC 3.1.3.36) in these tissues. The results are discussed in relation to diabetic neuropathy.     

Clusterin (SGP-2) in epididymal luminal fluid and its association with epididymal spermatozoa in androgen-deprived rats.

Clusterin is a heterodimeric glycoprotein synthesized and secreted by rat Sertoli cells and epididymal epithelium. The goal of this study was to determine the presence of clusterin in the luminal fluid of the cauda epididymides and its association with the membranes of developing spermatozoa in the presence and absence of androgen. We have previously demonstrated by two-dimensional (2-D) Western blot probing for clusterin that in epididymal fluid the amounts of clusterin were: caput greater than corpus greater than cauda. Luminal fluid from cauda epididymides was collected from control and orchiectomized rats (6 and 12 days) and orchiectomized animals that received testosterone implants. Equal volumes of fluid were analyzed by 2-D Western blot probing for clusterin. Following orchiectomy, there was an increase in clusterin in the luminal fluid after 6 days and maximal amount after 12 days compared with control cauda fluid. Orchiectomized animals which received testosterone treatment showed levels of clusterin comparable to that of controls. Serum clusterin was detected in fluid of orchiectomized animals with and without testosterone. Western blots of cauda sperm membrane extracts of control animals and orchiectomized animals treated with testosterone had a very low level of epididymal clusterin, whereas extracts collected from orchiectomized animals revealed high levels of clusterin. We suggest that, in the normal animal, clusterin is secreted into the lumen of the proximal epididymis where it binds to the sperm membrane. In the distal epididymis, clusterin dissociates from sperm and is processed (proteolysis/endocytosis). We hypothesize that, in the absence of androgen, the processing and regulation of clusterin is disrupted.     

Patterns of resistance to 2-deoxy-D-glucose in pig kidney cells

Variants resistant to 2-deoxy-D-glucose have been isolated from a clonal line of pig kidney cells by serial cultivation in the presence of inhibitor. Hexokinase activity may be affected directly in this system, since the oxidation of glucose to 6-phosphogluconate by extracts from sensitive and resistant cells is blocked by the addition of 2-deoxy-glucose to the reaction mixture. This blockage was removed by the addition of glucose-6-phosphate to the system, but not by ATP. Resistant cells were found to accumulate significantly less 2-deoxyglucose-6-phosphate than sensitive cells. The rate of phosphorylation of 2-deoxyglucose, however, was higher in extracts from the resistant line. Alkaline phosphatase does not account for the reduced level of 2-deoxyglucose-6-phosphate since this enzyme is not detectable in sensitive or resistant pig kidney cells. Increased acid phosphatase activity was observed in resistant cells, but extracts with high acid phosphatase activity proved incapable of hydrolyzing either 2-deoxyglucose-6-phosphate or glucose-6-phosphate. In comparative growth studies, cells resistant to 2-deoxyglucose proliferated more extensively than sensitive cells in a low glucose nutrient. They removed glucose more effectively from this medium, and were less stimulated by the addition of intermediates from the tricarboxylic acid cycle. The evidence suggests that resistance to 2-deoxyglucose in the cells under study may be based on the ability of the resistant cells to proliferate at concentrations of glucose too low to support the growth of sensitive cells.     

Gossypol-induced modifications in the microenvironment of rat epididymal spermatozoa

Gossypol acetic acid (20, 25 or 30 mg/kg/day orally for 5 weeks) decreased epididymal weight in adult Sprague-Dawley rats but the epididymal concentrations of proteins, lactate dehydrogenase and acid phosphatase were unchanged. The concentrations of carnitine, inositol and potassium in epididymal fluid were decreased in a dose-related manner. These modifications were not due to disturbances of Leydig and Sertoli cell functions which were normal. We suggest that the reduction in epididymal secretion results from a decrease in the number of spermatozoa rather than from a direct action of gossypol on the epididymal epithelium.     

Control of inositol biosynthesis in Saccharomyces cerevisiae: properties of a repressible enzyme system in extracts of wild-type (Ino+) cells.

Inositol biosynthesis was studied in soluble, cell extracts of a wild-type (Ino) strain of Saccharomyces cerevisiae. Two reactions were detected: (i) conversion of D-glucose-6-phosphate to a phosphorylated form of inositol, presumably inositol-1-phosphate (IP synthethase, EC5.5.1.4), and (ii) conversion of phosphorylated inositol to inositol (IP phosphatase, EC3.1.3.25). The in vitro rate of conversion of glucose-6-phosphate to inositol was proportional to incubaion time and enzyme concentration. The pH optimum was 7.0. The synthesis of inositol required oxidized nicotinamide adenine dinucleotide (NAD) and was stimulated byNH4C1 and MgC12. NADP substituted poorly for NAD, and NADH inhibitedthe reaction. Phosphorylated inositol accumulated in the absence of MgC12, suggesting that inositol-phosphate is an intermediate in the pathway and that Mg ions stimulate the dephosphorylation of inositol-phosphate. IP synthetase was inhibited approximately 20% in the presence of inositol in the reaction mixture at concentrations exceeding 1 mM. The enzyme was repressed approximately 50-fold when inositol was present in the growth medium at concentrations exceeding 50 muM. IP synthetase reached the fully repressed level approximately 10 h after the addition of inositol to logarithmic cultures grown in the absence of inositol. The specific activity of the enzyme increased with time in logarithmically growing cultures lacking inositol andapproached the fully depressed level as the cells entered stationary phase.     

Characterization of the expression and regulation of genes necessary for myo-inositol biosynthesis and transport in the seminiferous epithelium

In many mammals, the concentration of myo-inositol in the fluid of the seminiferous tubules is dramatically higher than levels found in serum. Two enzymes involved in myo-inositol synthesis: myo-inositol-1-phosphate synthase (ISYNA1) and myo-inositol monophosphatase-1 (IMPA1), are known to have high activity in the testes. ISYNA1 is an isomerase that catalyzes the conversion of glucose-6-phoshate to myo-inositol-1-phosphate. IMPA1 then hydrolyzes the phosphate group to produce myo-inositol. Although no physiological role for the high concentration of myo-inositol has yet to be elucidated, it has been suggested that it could be involved in osmoregulation. Previous research on these enzymes in the testis has focused on enzyme activity. The objective of this study was to evaluate the expression of these genes and the myo-inositol transporter, Slc5a3, within the testis. Using Northern blot analyses, we found that all three genes, Impa1, Isyna1, and Slc5a3 are expressed in Sertoli cells. Isyna1 is highly expressed in two types of germ cells, pachytene spermatocytes and round spermatids. IMPA1 was expressed in round spermatids. Slc5a3 expression is upregulated when Sertoli cells are treated with 0.1 mM dibutyryl cAMP. When Sertoli cells were cultured in a hypertonic medium, there was an increase in the expression of Isyna1 and Slc5a3. We postulate that this upregulation is a result of the capability of the Sertoli cell to sense and then react to a change in osmolarity by increasing the transport and production of the osmolyte myo-inositol.     

Mannitol production in fungi during glucose catabolism.

The levels of phosphofructokinase (EC 2.7.1.11) and mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) have been determined in a number of Mucor and Penicillium species. Mannitol-1-phosphate dehydrogenase was found in only one species of mucor, Mucor rouxii, and this with a specific activity much lower than that found in Penicillium species. All of the fungi tested in the Ascomycetes class exhibited mannitol-1-phosphate dehydrogenase activity. Interference from both mannitol-1-phosphate dehydrogenase and NADH oxidase (EC 1.6.99.5) caused some difficulty initially in detecting phosphofructokinase in Penicillium species; the Penicillium phosphofructokinase is very unstable. Penicillium notatum accumulates mannitol intracellularly; detection of mannitol-1-phosphate dehydrogenase and mannitol-1-phosphatase (EC 3.1.3.22) activity in cell-free extracts indicates that the mannitol is formed from glucose via fructose-6-phosphate and mannitol-1-phosphate; no direct reduction of fructose to mannitol could be detected. The mannitol-1-phosphate dehydrogenase was specific for mannitol-1-phosphate and fructose-6-phosphate; NADP+(H) could not replace NAD+(H). The phosphatase (EC3.1.3.22) exhibited a distinct preference for mannitol-1-phosphate as substrate; all other substrates tested exhibited less than 25% of the activity observed with mannitol-1-phosphate.     

Mechanism and contribution of the pentose phosphate cycle to glucose metabolism in epididymal fat tissue

1. Glucose 6-phosphate, fructose 6-phosphate and altroheptulose 7-phosphate are the major products formed non-oxidatively from ribose 5-phosphate by rat epididymal fat pad enzymes. 2. Arabinose 5-phosphate was detected among the reaction products and significant activity of the new enzyme of the L-type pentose pathway, D-glycero D-ido octulose 1,8-bisphosphate: D-altroheptulose 7-phosphotransferase was found. 3. The glucose moieties of glucose 1-phosphate, glucose 6-phosphate and glucose 1,6-bisphosphate were degraded and showed that epididymal fat pad enzymes relocate 14C from [2-14C]glucose into C-1, C-2, and C-3 of each hexose-phosphate. 4. The 14C-distribution patterns in the hexose-phosphates revealed that these intermediates were not in isotopic equilibrium and the rate of the transaldolase exchange reaction was relatively small. 5. The 14C-distribution data suggest that glucose 1-phosphate, rather than glucose 6-phosphate, is the first intermediate in the path of glycogen synthesis from glucose in this tissue. 6. The data provide the first proof of the mechanism of the pentose pathway in adipose tissue.     

Presence of two trehalose-6-phosphate synthase enzymes in Candida utilis

Abstract Total trehalose-6-phosphate synthase activity decreased in cell extracts from Candida utilis under conditions inducing activation of the regulatory trehalase by protein kinase catalysed phosphorylation. The synthase activity was reactivated by treatment with alkaline phosphatase revealing the presence of an enzyme whose activity is inactivated by reversible phosphorylation. The occurrence in the trehalose-6-phosphate synthase complex of a second synthase enzyme whose activity is not controlled by phosphorylation and dephosphorylation was demonstrated following gel filtration of cell extracts. The activity of the isolated enzymes was differently modified in vitro by the presence of alkaline phosphatase, ATP, glucose or protein kinase.     

Histochemical localization and quantification of alpha-glucosidase in the epididymis of men and laboratory animals

The seminal marker of epididymal function alpha-1, 4-glucosidase was localized histochemically in the cytoplasm of the efferent duct epithelium and the brush border of the entire length of the human epididymis. Quantification using the specific inhibitor castanospermine revealed strongest activity in the corpus and cauda regions. Selective inhibition of the brush border enzyme activities by maltotriose identified these as the neutral isoenzymes. Despite detection of alpha-glucosidase in the renal tubules of all the animals studied, the enzyme was not detectable in epididymides of hamsters or mice. In rabbits and monkeys, it was absent from the entire brush border but present weakly in the cytoplasm of the proximal epididymides. An enzyme distribution pattern similar to that in the human epididymis was found in rats, except for the absence of histochemical staining at pH 6.5 from the initial segment and distal cauda epididymidis. Experiments in which endogenous testosterone was depleted in rats demonstrated the dependence of epididymal alpha-glucosidase on androgen, albeit with a low sensitivity. This study suggests the rat to be a suitable model for the investigation of the role of epididymal alpha-glucosidase in fertility.     

NADP(H) Phosphatase Activities of Archaeal Inositol Monophosphatase and Eubacterial 3′-Phosphoadenosine 5′-Phosphate Phosphatase

NADP(H) phosphatase has not been identified in eubacteria and eukaryotes. In archaea, MJ0917 of hyperthermophilic Methanococcus jannaschii is a fusion protein comprising NAD kinase and an inositol monophosphatase homologue that exhibits high NADP(H) phosphatase activity (S. Kawai, C. Fukuda, T. Mukai, and K. Murata, J. Biol. Chem. 280:39200-39207, 2005). In this study, we showed that the other archaeal inositol monophosphatases, MJ0109 of M. jannaschii and AF2372 of hyperthermophilic Archaeoglobus fulgidus, exhibit NADP(H) phosphatase activity in addition to the already-known inositol monophosphatase and fructose-1,6-bisphosphatase activities. Kinetic values for NADP⁺ and NADPH of MJ0109 and AF2372 were comparable to those for inositol monophosphate and fructose-1,6-bisphosphate. This implies that the physiological role of the two enzymes is that of an NADP(H) phosphatase. Further, the two enzymes showed inositol polyphosphate 1-phosphatase activity but not 3′-phosphoadenosine 5′-phosphate phosphatase activity. The inositol polyphosphate 1-phosphatase activity of archaeal inositol monophosphatase was considered to be compatible with the similar tertiary structures of inositol monophosphatase, fructose-1,6-bisphosphatase, inositol polyphosphate 1-phosphatase, and 3′-phosphoadenosine 5′-phosphate phosphatase. Based on this fact, we found that 3′-phosphoadenosine 5′-phosphate phosphatase (CysQ) of Escherichia coli exhibited NADP(H) phosphatase and fructose-1,6-bisphosphatase activities, although inositol monophosphatase (SuhB) and fructose-1,6-bisphosphatase (Fbp) of E. coli did not exhibit any NADP(H) phosphatase activity. However, the kinetic values of CysQ and the known phenotype of the cysQ mutant indicated that CysQ functions physiologically as 3′-phosphoadenosine 5′-phosphate phosphatase rather than as NADP(H) phosphatase.     

Characterization of a phosphatidylinositol 4-phosphate-specific phosphomonoesterase in rat liver nuclear envelopes

Incubation of rat liver nuclear envelopes with [gamma-32P]ATP resulted in the synthesis of phosphatidylinositol-[4-32P]phosphate (PIP). Degradation of endogenously labeled PIP was observed upon the dilution of the labeled ATP with an excess of unlabeled ATP. This degradation was most rapid in the presence of EDTA, and was inhibited by MgCl2 and CaCl2. To further characterize the degradative activity, phosphatidylinositol[4-32P]phosphate and phosphatidylinositol [4,5-32P]bisphosphate (PIP2) were synthesized and isolated from erythrocyte plasma membranes. The 32P-labeled phospholipids were then resuspended in 0.4% Tween 80, a detergent that did not inhibit degradation of endogenously labeled PIP, and mixed with nuclear envelopes. [32P]PIP and [32P]PIP2 were degraded at rates of 2.25 and 0.04 nmol min-1 mg nuclear envelope protein-1, respectively. Only 32P was released from phosphatidyl[2-3H]inositol-[4-32P]phosphate, indicating that hydrolysis of PIP was due to a phosphomonoesterase activity (EC 3.1.3.36) in nuclear envelopes. Similarly, anion-exchange chromatographic analysis of the water-soluble products released from [32P]PIP indicated that inorganic phosphate was the sole 32P-labeled product. Hydrolysis of PIP was most rapid at neutral pH, and was not affected by inhibitors of acid phosphatase or alkaline phosphatase. Hydrolysis of PIP was also not inhibited by nonspecific phosphatase substrates, such as glycerophosphate, p-nitrophenylphosphate, AMP, or glucose 6-phosphate. Hydrolysis was stimulated by putrescine, and was inhibited by inositol 2-phosphate, spermidine, spermine, and neomycin.     

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.     

Localization of ornithine decarboxylase in rat testicular cells and epididymal spermatozoa

We have employed a monospecific, polyclonal antibody to ornithine decarboxylase (ODC) for the immunocytochemical localization of ODC in freshly isolated testicular cells, epididymal spermatozoa, and cultured Sertoli cells. Antigenically detectable material was present in the cytoplasm of all cell types tested and was highly concentrated in the acrosomal vesicle of round spermatids and in the acrosome region of epididymal spermatozoa. The specific enzymatic activity of ODC, as measured biochemically, was much higher in the interstitial cells than in the other testicular cell types, and no ODC activity was detected in the epididymal spermatozoa or in the Sertoli cells after 5 days in culture. These studies showed that, while all testicular cell types studied contained ODC-like immunoreactive molecules, only testicular germ cells and interstitial cells exhibited detectable ODC activity.     

Myxospore coat synthesis in Myxococcus xanthus: enzymes associated with uridine 5''-diphosphate-N-acetylgalactosamine formation during myxospore development.

Activities of the enzymes glutamine synthetase (EC 6.3.1.2.), glucosamine 6-phosphate acetyltransferase (EC 2.3.1.4.), uridine 5'-diphosphate (UDP)-N-acetylglucosamine pyrophosphorylase (EC 2.7.23.), UDP-N-acetylglucosamine 4-epimerase (EC 5.1.3.7.), fructose 1,6-diphosphate phosphatase (EC 3.13.11.), L-glutamine-fructose 6-phosphate transamidase (EC 5.3.1.19.), alkaline phosphatase (EC 3.1.3.1.), and malic dehydrogenase (EC 1.1.1.37) were assayed in partially purified extracts prepared at different stages of myxospore formation and germination in liquid cultures of Myxococcus xanthus. The specific activities of the first six of these enzymes increased 4.5- to 7.5-fold after 2 h of induction with 0.5 M glycerol or 0.2 M dimethyl sulfoxide. The increase in specific activities of these six enzymes was not observed in a mutant unable to be induced with glycerol. During the first 2 to 4 h of induction and during the first hour of germination, the level of these enzymes decreased to the level characteristic of vegetative cells. It is suggested that the six enzymes are responsible for the increased conversion of fructose 1,6-diphosphate to UDP-N-acetylgalactosamine, the major precursor of the myxospore coat.     

Spent media from immature seminiferous tubules and Sertoli cells inhibit adult rat Leydig cell aromatase activity

Adult rat Leydig cell aromatase activity is stimulated 2.5 fold by LH or dbcAMP. Spent media prepared from seminiferous tubules or Sertoli cells of immature rats depress both the basal and the LH stimulated estradiol syntheses (25 and 20% decreases, respectively). These inhibitory effects are further enhanced when FSH is added to the culture medium of seminiferous tubules or Sertoli cells. Rat serum as well as culture media from other cell lines are ineffective while seminiferous tubule media from other immature animals (mouse, guinea-pig, calf) inhibit the aromatase activity. This Sertoli cell factor is a heat stable protein (molecular weight greater than 10 kDa), different from the LHRH-like Sertoli cell compound, which acts on the aromatase activity at a step beyond the adenylate cyclase.     

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.