Studies on peroxisomes. VI. Relationship between the peroxisomal core and urate oxidase.

The peroxisomal core from the liver of rats was purified 450-fold as a marker of urate oxidase [EC 1.7.3.3.] activity. This preparation has a high specific activity of urate oxidase but not of other peroxisomal enzymes: D-amino acid oxidase [EC 1.4.3.3.], L-alpha-hydroxy acid oxidase [EC 1.1.3.15], or catalase [EC 1.11.1.6]. No activity of marker enzymes for other subcellular particles; cytochrome c oxidase [EC1.9.3.1] (mitochondria), acid phosphatase [EC 3.1.3.2] (lysosomes), or glucose-6-phosphatase [EC 3.1.3.9] (microsomes), was detected in this preparation. The core obtained showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the position of the band was found to correspond to a molecular weight 35,000. When the peroxisomal core was subjected to treatment at various pH's with 0.1 M carbonate buffer, urate oxidase was almost completely solubulized at pH 11.0, although approximately 35% of the core protein still remained in the pellet After solubilization of the core at pH 11.0, the specific activity of urate oxidase in the supernatant increased about 1.6 times; the density of the insoluble protein remaining in the pellet was identical with the that of the original core on sucrose density gradient centrifugation.     

Sphingolipid Long-Chain Base Synthesis in Plants (Characterization of Serine Palmitoyltransferase Activity in Squash Fruit Microsomes)

The activity of serine palmitoyltransferase (palmitoyl-coenzyme A [CoA]:L-serine [Ser]-C-palmitoyltransferase [decarboxylating], EC 2.3.1.50), the enzyme catalyzing the first step in the synthesis of the long-chain base required for sphingolipid assembly, has been characterized in a plant system. Enzyme activity in a microsomal membrane fraction from summer squash fruit (Cucurbita pepo L. cv Early Prolific Straightneck) was assayed by monitoring the incorporation of L-[3H]Ser into the chloroform-soluble product, 3-ketosphinganine. Addition of NADPH to the assay system resulted in the conversion of 3-ketosphinganine to sphinganine. The apparent Km for Ser was approximately 1.8 mM. The enzyme exhibited a strong preference for palmitoyl-CoA, with optimal activity at a substrate concentration of 200 [mu]M. Pyridoxal 5[prime]-phosphate was required as a coenzyme. The pH optimum was 7.6, and the temperature optimum was 36 to 40[deg]C. Enzyme activity was greatest in the microsomal fraction obtained by differential centrifugation and was localized to the endoplasmic reticulum using marker enzymes. Two known mechanism-based inhibitors of the mammalian enzyme, L-cycloserine and [beta]-chloro-L-alanine, were effective inhibitors of enzyme activity in squash microsomes. Changes in enzyme activity with size (age) of squash fruit were observed. The results from this study suggest that the properties and catalytic mechanism of Ser palmitoyltransferase from squash are similar to those of the animal, fungal, and bacterial enzyme in most respects. The specific activity of the enzyme in squash microsomes ranged from 0.57 to 0.84 nmol min-1 mg-1 of protein, values 2- to 20-fold higher than those previously reported for preparations from animal tissues.     

Purification and properties of a new cathepsin from rat liver.

1) A lysosomal protease, a new cathepsin that inactivates glucose-6-phosphate dehydrogenase [EC 1.1.1.49] and some other enzymes and differs from cathepsin B [EC 3.4.22.1] was purified about 2,200-fold from crude extracts of rat liver by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, and DEAE Sephadex and CM-Sephadex column chromatographies. 2) The new cathepsin was markedly activated by the thiol-reagent, 2-mercaptoethanol and inhibited by monoiodoacetate. 3) The molecular weight of the new cathepsin was found by Sephadex G-75 column chromatography to be 22,000, which is smaller than that of cathepsin B. 4) The optimum pH of the enzyme for inactivation of glucose-6-phosphate dehydrogenase was pH 5.0--5.5. The enzyme was unstable in alkali and on heat treatment. 5) The rates of inactivation of glucose-6-phosphate dehydrogenase, apo-ornithine aminotransferase [EC 2.6.1.13], apo-tyrosine aminotransferase [EC 2.6.1.5], apo-cystathionase [EC 4.4.1.1], glucokinase [EC 2.7.1.2], glyceraldehyde-3-phosphate dehydrogenase [EC 1.2.1.12], and malate dehydrogenase [EC 1.1.1.37] by the new cathepsin were higher than those by cathepsin B. However aldolase [EC 4.1.2.13] was inactivated more rapidly by cathepsin B than by the new cathepsin. Lactate dehydrogenase [EC 1.1.1.27], glutamate dehydrogenase [EC 1.4.1.2] and alcohol dehydrogenase [EC 1.1.1.1] were not inactivated by either cathepsin. Unlike cathepsin B, the new cathepsin scarcely hydrolyzes N-substituted derivatives of arginine.     

Cholate-independent retinyl ester hydrolysis. Stimulation by Apo-cellular retinol-binding protein

Apo-cellular retinol-binding protein (apoCRBP) activated the hydrolysis of endogenous retinyl esters in rat liver microsomes by a cholate independent retinyl ester hydrolase. A Michaelis-Menten relationship was observed between the apoCRBP concentration and the rate of retinol formation, with half-maximum stimulation at 2.6 +/- 0.6 microM (mean +/- S.D., n = 5). Two other retinol-binding proteins, bovine serum albumin and beta-lactoglobulin, acceptors for the rapid and spontaneous hydration of retinol from membranes, had no effect up to 90 microM. These data suggest activation of the hydrolase by apoCRBP directly, rather than by facilitating removal of retinol from membranes. The hydrolase responding was the cholate-independent/cholate-inhibited retinyl ester hydrolase as shown by: 60% inhibition of the apoCRBP effect by 3 mM cholate; apoCRBP enhancement of retinyl ester hydrolysis in liver microsomes that had no detectable cholate-enhanced activity; inhibition of cholate-dependent, but not apoCRBP-stimulated retinyl ester hydrolysis by rabbit anti-rat cholesteryl esterase. Compared to the rate (mean +/- S.D. of [n] different preparations) supported by 5 microM apoCRBP in liver microsomes of 6.7 +/- 3.7 pmol/min/mg protein [10], microsomes from rat lung, kidney, and testes had endogenous retinyl ester hydrolysis rates of 1.8 +/- 0.3 [5], 0.5 +/- 0.2 [3], and 0.3 +/- 0.2 [5] pmol/min/mg protein, respectively. N-Ethylmaleimide and N-tosyl-L-phenylalanine chloromethyl ketone were potent inhibitors of apoCRBP-stimulated hydrolysis with IC50 values of 0.25 and 0.15 mM, respectively, but phenylmethylsulfonyl fluoride and diisopropyl-fluorophosphate were less effective with IC50 values of 1 mM, indicating the importance of imidazole and sulfhydryl groups to the activity. These data provide evidence of a physiological role for the cholate-independent hydrolase in retinoid metabolism and suggest that apoCRBP is a signal for retinyl ester mobilization.     

Carbohydrate metabolism during early fruit development of sweet melon (Cucumis melo)

In sink tissues of cucurbits, including sweet melon fruits, the galactosyl-sucrose oligosaccharides, stachyose and raffinose, together with sucrose, are the major translocated carbohydrates. In the present study we investigated the carbohydrate metabolism of young melon ( Cucumis melo L. cv. C-8) fruit during the period of initial fruit set and development, from 3 days prior to anthesis until 20 days after anthesis (DAA), prior to the onset of sucrose accumulation. The enzymes assayed could be classified into two categories according to developmental patterns. Two of the enzymes, alkaline α -galactosidase I [EC 3.2.1.22], which hydrolyzes both raffinose and stachyose, and acid invertase [EC 3.2.1.26] either increased or remained stable during the first 10 DAA. The remaining measured enzymes (the stachyose-specific alkaline α -galactosidase form II, acid α -galactosidase, alkaline invertase, sucrose synthase [EC 2.4.1.13], galactokinase [EC 2.7.1.6], UDP-Gal PPase [EC 2.7.7.10], UDP-Glc-4 epimerase [EC 5.1.3.2], UDP-Glc PPase [EC 2.7.7.9], phosphoglucomutase [EC 5.4.2.2] and phosphoglucoisomerase [EC 5.3.1.9]) all showed a similar developmental pattern of steady decrease in activity following anthesis. We also compared the saccharide metabolism of pollinated and non-pollinated ovaries during the initial days following anthesis. In the absence of pollination, ovary growth dramatically decreased by the first DAA and was accompanied by a sharp decrease in the activity of UDP-Glc PPase. Other enzymes in the pathway, including the enzymes of stachyose and raffinose hydrolysis, did not decrease in activity until 2 or 4 DAA, after ovary growth was affected. These results provide information to assess the possible regulating enzymes in cucurbit ovary development and fruit set.     

Biochemical studies on sulfate-reducing bacteria. XIV. Enzyme levels of adenylylsulfate reductase, inorganic pyrophosphatase, sulfite reductase, hydrogenase, and adenosine triphosphatase in cells grown on sulfate, sulfite, and thiosulfate.

Sulfate-reducing bacteria, Desulfovibrio vulgaris, strain Miyazaki, were grown on either sulfate, sulfite, or thiosulfate as the terminal electron acceptor. Better growth was observed on sulfite and less growth on thiosulfate than on sulfate. Enzyme levels of adenylylsulfate (APS) reductase [EC 1.8.99.2], reductant-activated inorganic pyrophosphatase [EC 3.6.1.1], sulfite reductase [EC 1.8.99.1] (desulfoviridin), hydrogenase [EC 1.12.2.1], and Mg2+-activated ATPase [EC 3.6.1.3] were compared in crude extracts of these cells at various stages of growth. 1) The specific activity of APS reductase in sulfite-grown cells was only one-fourth that in sulfate-grown cells throughout growth. Thiosulfate-grown cells had an activity intermediate between those of sulfate- and sulfite-grown cells. 2) Cells grown on sulfite had lower specific activity of reductant-activated inorganic pyrophosphatase than cells grown on sulfate or thiosulfate. 3) The specific activity of sulfite reductase (desulfoviridin) was highest in sulfite-grown cells. The sulfite medium gave the enzyme in high yield as well as with high specific activity. 4) The specific activities of hydrogenase and Mg2+-ATPase were not significantly altered by electron acceptors in the growth medium.     

Effects of adenosine triphosphate and magnesium chloride on affinity elution of aminoacyl-transfer ribonucleic acid synthetases from phosphocellulose with transfer ribonucleic acids.

Aminoacyl-tRNA synthetases of bakers' yeast (Saccharomyces cerevisiae) were adsorbed to a phosphocellulose (P-cellulose) column, and those specific for tyrosine [EC 6.1.1.1], threonine [EC 6.1.1.3], valine [EC 6.1.1.9], and isoleucine [EC 6.1.1.5] were eluted with several specific tRNAs. Elutions of these synthetases were affected by ATP and/or MgCl2. The effects of ATP and MgCl2 differ with synthetases. Elutions of tyrosyl- and valyl-tRNA synthetases with their cognate tRNAs were more specific in the presence of MgCl2. Isoleucyl-tRNA synthetase was eluted with its cognate tRNA in the presence of both ATP and MgCl2. On the other hand, threonyl-tRNA synthetase was eluted in the absence of ATP and MgCl2 with unfractionated tRNA but not with some non-cognate tRNAs. This suggests that elution of threonyl-tRNA synthetase is highly specific. The present data on the effects of ATP or MgCl2 or both on this affinity elution will be useful for simple and rapid purification of the synthetases.     

A novel scintillation proximity assay for fatty acid amide hydrolase compatible with inhibitor screening

A binding assay for human fatty acid amide hydrolase (FAAH) using the scintillation proximity assay (SPA) technology is described. This SPA uses the specific interactions of [3H]R(+)-methanandamide (MAEA) and FAAH expressing microsomes to evaluate the displacement activity of FAAH inhibitors. We observed that a competitive nonhydrolyzed FAAH inhibitor, [3H]MAEA, bound specifically to the FAAH microsomes. Coincubation with an FAAH inhibitor, URB-597, competitively displaced the [3H]MAEA on the FAAH microsomes. The released radiolabel was then detected through an interaction with the SPA beads. The assay is specific for FAAH given that microsomes prepared from cells expressing the inactive FAAH-S241A mutant or vector alone had no significant ability to bind [3H]MAEA. Furthermore, the binding of [3H]MAEA to FAAH microsomes was abolished by selective FAAH inhibitors in a dose-dependent manner, with IC50 values comparable to those seen in a functional assay. This novel SPA has been validated and demonstrated to be simple, sensitive, and amenable to high-throughput screening.     

Radiorespirometric studies of carbohydrate metabolism by washed spermatozoa of various species.

1. The patterns of 14CO2 evolution from specifically labeled glucose substrates by washed bull, ram, boar, rabbit, dog, rooster and turkey spermatozoa were similar and indicated the Embden-Meyerhof and Kreb's cycle pathways as the major route of energy metabolism. 2. Honey bee spermatozoa metabolized glucose-3,4-[14C], glucose-[U-14C] or fructose-[U-14C], but not glucose-1-[14C], glucose-2-[14C]or glucose-6-[14C], indicating the presence of the glycolytic pathway, but the absence of respiration via the Kreb's cycle. 3. The rate of glycolysis exceeded the rate of respiration in the spermatozoa of all the species studied. 4. A preferential utilization of glucose-1-[14C] over glucose-6-[14C] was evident in some sperm samples, but no consistent indication of pentose cycle metabolism was observed, due to considerable variability between samples within each group. 5. Fructose metabolism was greater than glucose metabolism in the rooster, less in the dog, boar and turkey, and similar in the spermatozoa from the other species examined. 6. Only ram and bull spermatozoa metabolized acetate-1-[14C] to any extent.     

Identification of serine esterases in tissue homogenates

Serine esterases react with [3H]diisopropylphosphofluoridate ([3H]DFP) to produce radioactive adducts that can be resolved by denaturing slab gel electrophoresis. To identify an esterase or its catalytic subunit, a potential substrate was included in the reaction mixture with the expectation that it would suppress the enzyme's reaction with [3H]DFP. The nature of the enzyme could be inferred from the character of the substrates that suppress labeling. The validity of this analytical method was tested with two serine proteases, trypsin and alpha-chymotrypsin, and two serine esterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), and several of their natural or model substrates or inhibitors. Application of the method to complex biological systems was tested with chicken embryo brain microsomes. Trypsin labeling with [3H]DFP was suppressed by alpha-N-benzoyl-l-arginine ethyl ester (BAEE) and poly-l-lysine but not by benzoyl-l-tyrosine ethyl ester (BTEE). [3H]DFP labeling of chymotrypsin was suppressed by both BAEE and BTEE. Labeling of AChE and BuChE was suppressed by their natural and some related substrates and inhibitors. [3H]DFP reacted with brain microsomes to produce nine distinct radioactive bands. When the relevant substrates and inhibitors of AChE were included in the reaction mixtures, labeling of only the 95-kDa band was suppressed, implicating it as AChE. Labeling of the 85- and 79-kDa bands was inhibited by butyrylcholine, suggesting that these proteins have BuChE activity.     

Substrate properties of stable fly (Diptera: Muscidae) developmental sites associated with round bale hay feeding sites in eastern Nebraska

Residues at sites where stationary feeders were used to provide hay as supplemental forage for cattle during the winter are developmental substrates for immature stable flies, Stomoxys calcitrans (L.), in the central United States. Spatial patterns in physical (substrate depth, temperature, water content), chemical (pH, electrical conductivity [EC(lab)], total nitrogen [N] and carbon [C], ammoniacal nitrogen [NH(4)-N], extractable phosphorus [P]), and biological (microbial respiration rate) substrate properties for two feeding sites were estimated and the correlations between these properties and adult emergence were characterized. Hay feeding sites had a circular footprint with residues extending ≈7 m from the feeder. With the exception of extractable P and total N, all substrate properties exhibited spatial patterns centered on the feeder location. Adult stable fly emergence densities were significantly correlated with substrate microbial respiration rate, NH(4)-N concentration, EC(lab), total C concentration, pH, and moisture content. Logistic regression indicated that EC best predicted the probability of stable flies emerging from a substrate and that the other properties did not provide additional information. A better understanding of the physical, chemical, and biological conditions needed for stable fly larval development may help in identifying previously unrecognized developmental habitats and management of this pest. Targeted implementation of management practices such as sanitation and chemical treatments can be applied to smaller areas reducing labor and improving cost effectiveness.     

Circadian rhythms in digestive enzymes in the small intestine of rats. I. Patterns of the rhythms in various regions of the small intestine.

The activities of the digestive enzymes, maltase [EC 3.2.1.20], sucrase [EC 3.2.1.26], trehalase [EC 3.2.1.28], Leucine aminopeptidase [EC 3.4.11.1], and alkaline phosphatase [EC 3.1.3.1] were measured in various regions of the small intestine of rats. The activities of all these enzymes were much higher in the jejunum than in the ileum, and in the distal regions of the ileum no sucrase, trehalase or alkaline phosphatase activity was detected. In the jejunum, the activities of all the enzymes tested exhibited clear circadian variations with the highest activity at 0000-0400 h and the lowest at 1200 h when the rats were fed ad libitum. In the ileum, maltase and sucrase also exhibited circadian variations, but the amplitude of the rhythm was smaller than that in the jejenum. Trehalase and alkaline phosphatase did not show any circadian variation in the ileum. Leucine aminopeptidase showed a circadian variation in the ileum with the same amplitude as in the jejunum. The phase of the circadian variations shifted about half a day when the rats were fed in the daytime, but the amplitude of the rhythm did not change.     

Effects of phthalate esters on the respiration of rat liver mitochondria.

The effects of phthalate esters on the oxidation of succinate, glutamate, beta-hydroxybutyrate and NADH by rat liver mitochondria were examined and it was found that di-n-butyl phthalate (DBP) strongly inhibited the succinate oxidation by intact and sonicated rat mitochondria, but did not inhibit the State 4 respiration with NAD-linked substrates such as glutamate and beta-hydroxybutyrate. However, oxygen uptake accelerated by the presence of ADP and substrate (State 3) was inhibited and the rate of oxygen uptake decreased to that without ADP (State 4). It was concluded that phthalate esters were electron and energy transport inhibitors but not uncouplers. Phthalate esters also inhibited NADH oxidation by sonicated mitochondria. The degree of inhibition depended on the carbon number of alkyl groups of phthalate esters, and DBP was the most potent inhibitor of respiration. The activity of purified beef liver glutamate dehydrogenase [EC 1.4.1.3] was slightly inhibited by phthalate esters.     

Stimulatory effect of n-alkanes on CTP: Cholinephosphate cytidyltransferase activity in rat liver microsomal membranes in vitro.

To assess the effect of alteration of membrane structure on the enzymic activities related to phospholipid synthesis in microsomal membrane, the effects of several organic solvents have been studied in an in vitro system, in which the cytoplasmic extract prepared from rat liver incorporated [14C]choline or [14C]CDP-choline into phosphatidycholine (lecithin). The optimum conditions for the incorporation were determined. Among several organic solvents examined, n-alkanes such as n-hexane, n-octane, and n-tetradecane stimulated the incorporation. It was shown that n-alkanes stimulated one of three enzymic steps of lecithin biosynthesis from choline; that is, the formulation of CDP-choline catalyzed by CTP: cholinephosphate cytidyltransferase [EC 2.7.7.15], an enzyme on the microsomal membrane. It was further shown that the same enzyme was also stimulated by preincubation of microsomes in the absence of substrate. It is suggested that alteration of the lipid environment of the microsomal membrane induced by n-alkanes caused activation of this enzymic step.     

Creatine kinase of heart mitochondria. Control of oxidative phosphorylation by the extramitochondrial concentrations of creatine and phosphocreatine

Defining how extramitochondrial high-energy phosphate acceptors influence the rates of heart oxidative phosphorylation is essential for understanding the control of myocardial respiration. When the production of phosphocreatine is coupled to electron transport via mitochondrial creatine kinase, the net reaction can be expressed by the balanced equation: creatine + Pi----phosphocreatine + H2O. This suggests that rates of oxygen consumption could be regulated by changes in [creatine], [Pi], or [phosphocreatine], alone or in combination. The effects of altering these metabolites upon mitochondrial rates of respiration were examined in vitro. Rat heart mitochondria were incubated in succinate-containing oxygraph medium (pH 7.2, 37 degrees C) supplemented with five combinations of creatine (1.0-20 mM), phosphocreatine (0-25 mM), and Pi (0.25-5.0 mM). In all cases, the mitochondrial creatine kinase reaction was initiated by additions of 0.5 mM ATP. To emphasize the duality of control, the results are presented as three-dimensional stereoscopic projections. Under physiological conditions, with 5.0 mM creatine, increases in Pi or decreases in phosphocreatine had little influence upon mitochondrial respiration. When phosphocreatine was held constant (15 mM), changes in [creatine] modestly stimulated respiratory rates, whereas Pi again showed little effect. With 1.0 mM Pi, respiration clearly became dependent upon changes in [creatine] and [phosphocreatine]. Initially, respiratory rates increased as a function of [creatine]. However, at [phosphocreatine] values below 10 mM, product "deinhibition" was observed, and respiratory rates rapidly increased to 80% State 3. With 2.0 mM Pi or higher, respiration could be regulated from State 4 to 100% State 3. Overall, the data show how increasing [creatine] and decreasing [phosphocreatine] influence the rates of oxidative phosphorylation when mediated by mitochondrial creatine kinase. Thus, these changes may become secondary cytoplasmic signals regulating heart oxygen consumption.     

γ-Aminobutyric Acid and Glycine Modulate Each Other''s Release Through Heterocarriers Sited on the Releasing Axon Terminals of Rat CNS

The ability of gamma-aminobutyric acid (GABA) and glycine (Gly) to modulate each other's release was studied in synaptosomes from rat spinal cord, cerebellum, cerebral cortex, or hippocampus, prelabeled with [3H]GABA or [3H]Gly and exposed in superfusion to Gly or to GABA, respectively. GABA increased the spontaneous outflow of [3H]Gly (EC50, 20.8 microM) from spinal cord synaptosomes. Neither muscimol nor (-)-baclofen, up to 300 microM, mimicked the effect of GABA, which was not antagonized by either bicuculline or picrotoxin. However, the effect of GABA was counteracted by the GABA uptake inhibitors nipecotic acid and N-(4,4-diphenyl-3-butenyl)nipecotic acid. Moreover, the GABA-induced [3H]Gly release was Na+ dependent and disappeared when the medium contained 23 mM Na+. The effect of GABA was Ca2+ independent and tetrodotoxin insensitive. Conversely, Gly enhanced the outflow of [3H]GABA from rat spinal cord synaptosomes (EC50, 100.9 microM). This effect was insensitive to both strychnine and 7-chlorokynurenic acid, antagonists at Gly receptors, but it was strongly Na+ dependent. Also, the Gly-evoked [3H]GABA release was Ca2+ independent and tetrodotoxin insensitive. GABA increased the outflow of [3H]Gly (EC50, 11.1 microM) from cerebellar synaptosomes; the effect was not mimicked by either muscimol or (-)-baclofen nor was it prevented by bicuculline or picrotoxin. The GABA effect was, however, blocked by GABA uptake inhibitors and was Na+ dependent. Gly increased [3H]GABA release from cerebellar synaptosomes (EC50, 110.7 microM) in a strychnine- and 7-chlorokynurenic acid-insensitive manner. This effect was Na+ dependent. The effects of GABA on [3H]Gly release seen in spinal cord and cerebellum could be reproduced also with cerebrocortical synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and characterization of 5-azido-UDP-glucuronic acid. A new photoaffinity probe for UDP-glucuronic acid-utilizing proteins.

A new active site-directed photoaffinity analogue, [beta-32P]5-azido-UDP-glucuronic acid (UDP-GlcA), was enzymatically synthesized from [beta-32P]5-N3UDP-Glc using UDP-glucose dehydrogenase. The product was characterized by its mobility on ion exchange and two thin-layer chromatographic systems, by its UV absorbance at 288 nm, and the loss of this absorbance after UV irradiation of the compound. Photoincorporation of [beta-32P]5-N3UDP-GlcA into bovine liver UDP-Glc dehydrogenase (EC 1.1.1.22) was saturable with an apparent Kd of 12.5 microM, and was inhibited by the known active-site effectors UDP-GlcA, UDP-Glc, and UDP-xylose. When human liver microsomes with known UDP-glucuronosyltransferase (EC 2.4.1.17) activities were photolabeled with [beta-32P]5-N3UDP-GlcA, major photolabeled bands of 35-37 and 50-54 kDa were detected. When rat liver microsomes from phenobarbital-injected rats were photolabeled with [beta-32P]5-N3UDP-GlcA, there was a marked increase in photoincorporation of a 51-kDa protein as compared with control animals. Evidence is presented which suggests that the photolabeled 51-54-kDa proteins in the liver microsomes from both tissues are UDP-glucuronosyltransferase and that [beta-32P]5-N3UDP-GlcA represents a new alternative approach in the study of UDP-glucuronosyltransferase and other UDP-GlcA-utilizing enzymes.     

Purification and properties of the intact form of NADH-cytochrome b5 reductase from rabbit liver microsomes.

NADH-cytochrome b5 reductase [EC 1.6.2.2] has been solubilized with Triton X-100 and purified to homogeneity from rabbit liver microsomes. The purified enzyme is essentially free of the detergent and phospholipids and exists in aqueous media as an oligomeric aggregate of about 13 S. Its monomeric molecular weight is about 33,000 and 1 mole of FAD is associated with 1 mole of the monomeric unit. The enzyme catalyzes the reductions by NADH of ferricyanide and 2,6-dichlorophenol indophenol at an activity ratio of 1 : 0.09. Although the intact form of cytochrome b5 is a poorer electron acceptor than its hydrophilic fragment for the purified flavoprotein, electron transfer from the reductase to the intact cytochrome can be markedly stimulated by detergents or phospholipids, which also cause profound enhancement of the NADH-cytochrome c reductase activity reconstituted from the reducatse and cytochrome b5. Upon digestion with trypsin [EC 3.4.21.4], the ability of the reductase to form an active NADH-cytochrome c reductase system with the intact form of cytochrome b5 and Triton X-100 is rapidly lost. This loss of the reconstitution capability can be prevented by preincubation of the reductase with phosphatidylcholine liposomes. Trypsin digestion also results in the cleavage of the reductase molecule to a protein having a molecular weight of about 25,000 and a smaller fragment. The purified flavoprotein can bind to liver microsomes, liver mitochondria, sonicated human erythrocyte ghosts, and phosphatidylcholine liposomes. The reductase solubilized directly from liver microsomes by lysosomal digestion however, is devoid of membrane-binding capacity. It is concluded that the intact form of NADH-cytochrome b5 reductase is an amphipathic protein and its hydrophobic moiety, which is removable by lysosomal digestion, is responsible for the tight binding of the reductase to microsomes and for its normal functioning in the membrane.     

Gaba shunt in developing soybean seeds is associated with hypoxia

In the present study we investigated the proposal that the γ-aminobutyrate (Gaba) shunt in developing soybean (Glycine max [L.] Merr.) seeds is associated with hypoxia. The ontogeny and pH profile of enzymes associated with glutamate metabolism (glutamate decarboxylase [EC 4.1.1.15]. Gaba transaminase [EC 2.6.1.19], succinic semialdehyde dehydrogenase [EC 1.2.1.16], glutamate dehydrogenase [EC 1.4.1.2], glutamate:oxaloacetate transaminase [EC 2.6.1.1], glutamate:pyruvate transaminase [EC 2.6.1.2] and 2-oxoglutarate dehydrogenase complex [EC 1.2.4.2]) and hypoxia (alcohol dehydrogenase [ADH, EC 1.1.1.1] and pyruvate decarboxylase [PDC, EC 4.1.1.1]) were determined in cotyledons, nucellus and seed-coat tissues. Gaba-shunt enzymes were ubiquitous in the developing seed. Activities of enzymes catalyzing glutamate-C entry into the Krebs cycle via 2-oxoglutarate were generally greater than those of Gaba-shunt enzymes. In cotyledons, the activity of ADH increased throughout seed development (up to 72 days after anthesis [DAA]), whereas PDC was static during early development, then increased. In contrast, the activities of ADH and PDC in maternal tissues (nucellus and seed coat) were initially high, then declined dramatically after 37 DAA. The adenylate energy charge (AEC) = ([ATP]+0.5 [ADP])/ ([ATP] + [ADP] + [AMP]) of soybean seeds from fruits (37 DAA) frozen in situ was low (0.67±0.01) compared to the AEC of adjacent pod tissue (0.82 ± 0.04) and cotyledons exposed to air (0.84 ± 0.01). A 60-min time-course study showed that the rate of [U-¹⁴C]-glutamate catabolism by an intact excised cotyledon at 37 DAA was markedly lower at 8 and 0% O₂ than at 21%; the pool size of [¹⁴C]-Gaba was unaffected. The data indicated that: (1) Gaba-shunt activity is not a response to limited glutamate deamination/transamination: (2) the soybean seed is hypoxic; and (3) the relative partitioning of glutamate-C through glutamate decarboxylase is increased by hypoxia.     

Turnover of bis-diphosphoinositol tetrakisphosphate in a smooth muscle cell line is regulated by beta2-adrenergic receptors through a cAMP-mediated, A-kinase-independent mechanism.

Bis-diphosphoinositol tetrakisphosphate ([PP]2-InsP4 or 'InsP8') is a 'high-energy' inositol phosphate; we report that its metabolism is receptor-regulated in DDT1 MF-2 smooth muscle cells. This conclusion arose by pursuing the mechanism by which F- decreased cellular levels of [PP]2-InsP4 up to 70%. A similar effect was induced by elevating cyclic nucleotide levels, either with IBMX or by application of either Bt2cAMP (EC50 = 14.7 microM), Bt2cGMP (EC50 = 7.9 microM) or isoproterenol (EC50 = 0.4 nM). Isoproterenol (1 microM) decreased [PP]2-InsP4 levels 25% by 5 min, and 71% by 60 min. This novel, agonist-mediated regulation of [PP]2-InsP4 turnover was very specific; isoproterenol did not decrease the cellular levels of either inositol pentakisphosphate, inositol hexakisphosphate or other diphosphorylated inositol polyphosphates. Bradykinin, which activated phospholipase C, did not affect [PP]2-InsP4 levels. Regulation of [PP]2-InsP4 turnover by both isoproterenol and cell-permeant cyclic nucleotides was unaffected by inhibitors of protein kinases A and G. The effectiveness of the kinase inhibitors was confirmed by their ability to block phosphorylation of the cAMP response element-binding protein. Our results indicate a new signaling action of cAMP, and furnish an important focus for future research into the roles of diphosphorylated inositol phosphates in signal transduction.