Particulate Enzyme Research Articles

Metabolic Fate of Adenosine and Purine Metabolism Enzymes in Young Plants of Peach Tree

Extracts of young plants of peach tree, Prunus persica L. Batsch, exhibited the ability to recycle adenosine into adenine nucleotides either by phosphorylation into AMP by a very active adenosine kinase (EC 2.7.1.20) or by the successive intervention of adenosine nucleosidase (EC 3.2.2.7) and adenine phosphoribosyltransferase (EC 2.4.2.7). This latter sequence appeared to be the preferential salvage pathway of exogenous [U-14C]adenosine in situ. In situ radiolabelling gave evidence of interconversion of adenine nucleotides into guanine nucleotides. Characterization of AMP aminohydrolase (EC 3.5.4.6) and guanylate kinase (EC 2.7.4.8) activities in extracts supported this observation. Peach tree AMP aminohydrolase, a particulate allosteric enzyme, revealed to be activated by K+ ions and free ATP while guanylate kinase, also a particulate enzyme, required Mg-ATP2- complex as substrate. Extracts of peach tree exhibited 5' nucleosidase and inosine nucleosidase activity (EC 3.2.2.2), likely involved in purine nucleotide catabolism occurring in situ. A metabolic scheme of salvage, interconversion and catabolic pathways of adenosine derivatives in young plants of peach tree is established and discussed.

Solubilization and characterization of diacylglycerol acyltransferase from microspore-derived cultures of oilseed rape.

Particulate fractions prepared from microspore-derived (MD) embryos of oilseed rape (Brassica napus L. cv. Reston) and an embryogenic MD cell suspension culture of oilseed rape (B. napus L. cv. Jet Neuf) were used as a source of diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) for enzyme characterization and development of a solubilization procedure. DGAT activity in the 1500-100,000 g fraction from MD embryos was stimulated 4-5-fold by 3 to 4 mg of BSA/ml of reaction mixture. DGAT activity from MD embryos was stimulated 2-3-fold by fluoride salts and 1.4-fold by NaCl, whereas iodide salts caused substantial inhibition of enzyme activity. The effect of the various 1:1 electrolytes on enzyme activity appeared to be related more to their differential effects on solution structure rather than ionic strength. DGAT was solubilized from membranes of MD embryos and the cell suspension culture by about 80 and 50% respectively, using 2 M NaCl in 1% (w/v) octanoyl-N-methyl-glucamide (MEGA-8) (pH 8.0 buffer) at a detergent to protein ratio of 2:1. The specific activity of solubilized DGAT was about 2-fold greater than that of the particulate enzyme. The mechanism of solubilization appeared to be related to the lowering of the critical micellar concentration of MEGA-8 in the presence of NaCl. DGAT, solubilized from MD embryos, eluted with an M(r) of about 2 x 10(6) during gel-filtration chromatography on a Superose 6 column equilibrated in buffer containing 0.1% (w/v) MEGA-8. The solubilized enzyme exhibited optimal activity at pH 7. At concentrations above 2 microM acyl-CoA, the specificity of solubilized DGAT for oleoyl-CoA and palmitoyl-CoA was considerably greater than for stearoyl-CoA.

Identification of type-2 phosphatidic acid phosphohydrolase (PAPH-2) in neutrophil plasma membranes

Plasma membrane phosphatidic acid phosphohydrolase (PAPH) plays an important role in signal transduction by converting phosphatidic acid to diacylglycerol. PAPH-2, a Mg 2+-independent, detergent-dependent enzyme involved in cellular signal transduction, is reportedly absent from the plasma membranes of neutrophilic leukocytes, a cell that responds to metabolic stimulation with abundant phospholipase d-dependent diacylglycerol generation. The present study was designed to resolve this discrepancy, focusing on the influence of cellular disruption techniques, detergenta availability and cation sensitivity on the apparent distribution of PAPH in neutrophil sub-cellular fractions. The results clearly indicate the presence of two distinct types of PAPH within the particulate and cytosolic fractions of disrupted cells. Unlike the cytosolic enzyme, the particulate enzyme was not potentiated by magnesium and was strongly detergent-dependent. The soluble and particulate enzymes displayed dissimilar pH profiles. Separation of neutrophil particulate material into fractions rich in plasma membranes, specific granules and azurophilic granules by high speed discontinuous density gradient centrifugation revealed that the majority of the particulate activity was confined to plasma membranes. This activity was not inhibited by pretreatment with n-ethyl-maleimide in concentrations as high as 25 mM. PAPH activity recovered in the cytosolic fraction of disrupted neutrophils was almost completely inhibited by 5.0 mM n-ethylmaleimide. We conclude that resting neutrophils possess n-ethylmaleimide-resistant PAPH (type 2) within their plasma membranes. This enzyme may markedly influence the kinetics of cell activation by metabolizing second messengers generated as a result of activation of plasma membrane phospholipase D.

Degradation of angiotensin I to [des-Asp 1]angiotensin I by a novel aminopeptidase in the rat hypothalamus

The particulate enzyme that degrades angiotensin I (ANG I) to [des-aspartate 1]angiotensin I ([des-Asp 1]ANG I) in the hypothalamic homogenate of the rat has been established as a specific aminopeptidase. The major characteristic is its resistance to inhibition by 10 −4 M amastatin, bestatin and EDTA. Among the four amino acyl-β-naphthylamides (aspartyl-, glutamyl-, arginyl- and leucyl-β-napthylamide), aspartyl-β-naphthylamide is the most susceptible substrate of the enzyme; being degraded at twice the rate of arginyl- and leucyl-β-naphthylamide, and six times that of glutamyl-β-naphthylamide. Although the precise role of this aminopeptidase has yet to be determined, its presence establishes the existence of a specific pathway for the degradation of ANG I that bypasses the formation of ANG II. The relationship between degradation and hypertension is shown by our recent findings that the formation of [des-Asp 1]ANG I form ANG I in the hypothalamic homogenate of the spontaneously hypertensive rat (SHR) is significantly enhanced, and the findings of other investigators that the production of ANG II by neuronal cultures of the SHR is significantly decreased.

Subcellular localization and characterization of neuronal nitric oxide synthase.

In contrast to the predominantly particulate, Ca2+/calmodulin-dependent nitric oxide (NO) synthase in endothelial cells, the corresponding neuronal isoenzyme is considered to be mainly soluble, presumably owing to the lack of a posttranslational myristoylation. However, preliminary findings from this and other laboratories suggest that a substantial portion of the neuronal NO synthase activity may in fact be membrane bound. We have therefore investigated the distribution of this enzyme among subcellular fractions of the rat and rabbit cerebellum in more detail. Up to 60% of the total NO synthase activity was found in the particulate fraction and, according to density gradient ultracentrifugation, associated mainly with the endoplasmic reticulum fraction. There was no apparent difference between the soluble and particulate enzymes with respect to their specific activity, Ca2+ and pH dependency, inhibitor sensitivity, or immunoreactivity, suggesting that both rat and rabbit cerebella contain a single Ca2+/calmodulin-dependent NO synthase. The inhibition by the cytochrome P450 inhibitor SKF-525A of the NO synthase activity in these subcellular fractions (IC50 = 90 microM) and the fact that mammalian cytochrome P450 enzymes are endoplasmic reticulum-bound proteins support the notion that the cerebellar NO synthase is a cytochrome P450-type hemoprotein. Moreover, the aforementioned findings suggest that posttranslational myristoylation may not be the only factor determining the intracellular localization of NO synthase.

Barbiturates modulate the activity of the pituitary 3α-hydroxysteroid oxidoreductases and inhibit their production of 3α,5α-tetrahydroprogesterone

The effects of sodium phenobarbital and sodium barbital on the activity of the particulate and cytosolic 5α-dihydroprogesterone 3α-hydroxysteroid oxidoreductases (3α-HSORs) of female rat anterior pituitary were investigated. By altering the 3α-HSOR catalyzed conversion of 5α-dihydroprogesterone (5α-DHP) to 3α,5α-tetrahydroprogesterone (3α,5α-THP), these barbiturates could influence the in situ production of 3α,5α-THP. 3α,5α-THP has potent barbiturate-like effects on brain GABA A receptors. Both phenobarbital and 3α,5α-THP can affect gonadotropin release in female rats. In vitro incubations of each 3α-HSOR activity were assayed in the presence of sodium phenobarbital (0.1 to 10.0 mM) or sodium barbital (1.0 to 10.0 mM). Since both 3α-HSOR activities catalyze the reversible oxidoreduction of 5α-DHP and 3α,5α-THP, we examined the effect of these barbiturates not only on the conversion of 5α-DHP to 3α,5α-THP (reductive reaction) but also on the “back conversion” of 3α,5α-THP to 5α-DHP (oxidative reaction). The results indicate that both phenobarbital and, to a lesser extent barbital, significantly affected the activities of the two 3α-HSORs in both reductive and oxidative directions. In the reductive direction, phenobarbital inhibited the activity (33%) of both cytosolic and particulate enzymes which would presumably decrease the levels of 3α,5α-THP. In the oxidative direction, a pattern of stimulation was observed (20 to 100%). Thus, this stimulatory effect on the oxidative conversion of 3α,5α-THP to 5α-DHP, which would presumably also decrease 3α,5α-THP levels, appears correlated with the inhibitory effect of these barbiturates on the reductive conversion of 5α-DHP to 3α,5α-THP. Sodium barbital exhibited somewhat similar effects. These changes suggest that barbiturates can lower 3α,5α-THP levels in the anterior pituitary. The results also suggest the possibility that lowered 3α,5α-THP levels may be involved, at least in part, in the reduction of gonadotropin release by barbiturates.

Characterization of phosphatidic acid phosphohydrolase in neutrophil subcellular fractions

This investigation was designed to confirm the presence of PA phosphohydrolase in human neutrophils and to determine the distribution and characteristics of the enzyme in soluble and particulate subcellular fractions of disrupted neutrophils. Enzyme activity was detected in unseparated extracts of sonicated neutrophils. The majority of the recovered activity was recovered in a particulate fraction rich in neutrophil plasma-membrane markers; moderate levels (20%) of the total activity were recovered in the cytosol. While Mg 2+ markedly potentiated the cytosolic but not the particulate activity, Ca 2+ moderately inhibited both the cytosolic and particulate enzymes. The plasma-membrane-associated activity was absolutely dependent on detergent (0.5% Triton X-100) and displayed an apparent K m of 62 μM for phosphatidic acid. Enzyme activity was markedly inhibited by NaF, not influenced by excess glycerophosphate and slightly attenuated by propranolol, an inhibitor of PA phosphohydrolase in other systems. Preincubation of plasma membranes with N-ethylmaleimide at concentrations up to 25 mM had little effect on enzyme activity. However, activity in cytosolic and microsomal fractions of neutrophils were completely abolished by preincubation with N-ethylmaleimide at concentrations of less than 5 mM. We conclude that neutrophils possess a potent PA phosphohydrolase localized in their plasma membranes. Metabolism of cellular second-messengers by this enzyme may exert a profound effect on the functions of stimulated neutrophils.

Mannosyl transfer in Cryptococcus neoformans.

A particulate enzyme preparation from Cryptococcus neoformans transferred the mannosyl residue from GDP-mannose to an acceptor consisting of a commercial preparation of methyl 3-O-alpha-mannopyranosyl-alpha-mannopyranoside (containing 10% 2-O-alpha-mannopyranosyl-alpha-mannopyranoside). The configuration of the new bond was alpha by its susceptibility to alpha-mannosidase; the amount of product was dependent on the concentration of enzyme, of GDP-mannose, and of acceptor. The optimal temperature and pH were 37 degrees C and 7.0, respectively. Manganous ion was required for activity and acetyl coenzyme A was stimulatory. Studies suggested that dolichyl phosphate intermediates were not involved in this mannose transfer. The fact that none of the several acapsular mutants tested were deficient in this mannosyltransferase suggested that this enzyme was not involved in synthesis of backbone mannan linkages in capsular polysaccharide. NMR analysis of the methylmannotriose product showed only alpha(1-->2) linkages between sugar moieties. This mannosyltransferase evidently extends alpha(1-->2) mannan by adding another alpha(1-->2)-linked mannosyl residue. Its activity is appropriate for a role in synthesis of "high mannose" oligosaccharide moieties of glycoproteins.

Overexpression of human alpha-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion.

Human lysosomal alpha-galactosidase A (alpha-Gal A) was stably overexpressed in CHO cells and its biosynthesis and targeting were investigated. Clone AGA5.3-1000Mx, which was the highest enzyme overexpressor, produced intracellular alpha-Gal A levels of 20,900 U/mg (approximately 100 micrograms of enzyme/10(7) cells) and secreted approximately 13,000 U (or 75 micrograms/10(7) cells) per day. Ultrastructural examination of these cells revealed numerous 0.25-1.5 microns crystalline structures in dilated trans-Golgi network (TGN) and in lysosomes which stained with immunogold particles using affinity-purified anti-human alpha-Gal A antibodies. Pulse-chase studies revealed that approximately 65% of the total enzyme synthesized was secreted, while endogenous CHO lysosomal enzymes were not, indicating that the alpha-Gal A secretion was specific. The recombinant intracellular and secreted enzyme forms were normally processed and phosphorylated; the secreted enzyme had mannose-6-phosphate moieties and bound the immobilized 215-kD mannose-6-phosphate receptor (M6PR). Thus, the overexpressed enzyme's selective secretion did not result from oversaturation of the M6PR-mediated pathway or abnormal binding to the M6PR. Of note, the secreted alpha-Gal A was sulfated and the percent of enzyme sulfation decreased with increasing amplification, presumably due to the inaccessibility of the enzyme's tyrosine residues for the sulfotransferase in the TGN. Overexpression of human lysosomal alpha-N-acetylgalactosaminidase and acid sphingomyelinase in CHO cell lines also resulted in their respective selective secretion. In vitro studies revealed that purified secreted alpha-Gal A was precipitated as a function of enzyme concentration and pH, with 30% of the soluble enzyme being precipitated when 10 mg/ml of enzyme was incubated at pH 5.0. Thus, it is hypothesized that these overexpressed lysosomal enzymes are normally modified until they reach the TGN where the more acidic environment of this compartment causes the formation of soluble and particulate enzyme aggregates. A significant proportion of these enzyme aggregates are unable to bind the M6PR and are selectively secreted via the constitutive secretory pathway, while endogenous lysosomal enzymes bind the M6PRs and are transported to lysosomes.

Endothelial nitric oxide synthase is myristylated

The enzyme responsible for the synthesis of endothelium-derived relaxing factor and/or nitric oxide in the endothelium has been described as a particulate enzyme, whereas other isoforms of nitric oxide synthase are soluble enzymes. Here we are reporting that endothelial cells metabolically incorporate myristate (C14), but not palmitate (C16), into nitric oxide synthase. We are postulating that the endothelial-derived nitric oxide synthase is a particulate enzyme because of the fatty acid acylation of the protein which ‘anchors’ the enzyme into the membrane either directly or via another membrane-bound protein.

Alpha-melanocyte-stimulating hormone stimulates protein kinase C activity in murine B16 melanoma.

The effect of alpha-melanocyte-stimulating hormone (alpha-MSH) on protein kinase C activity and distribution was investigated in murine B16 F1 melanoma cells. alpha-MSH was found to induce an increased association of protein kinase C (PKC) activity with the particulate fraction of the cells, with an associated loss of enzyme activity from the soluble fraction. The peak response to alpha-MSH occurred between 20 and 60 min of incubation time, and enzyme activities redistributed to those seen in the control cells over the following 12 to 24 h. The average response to alpha-MSH (1 nmol/l) was an approximate 2.5-fold increase in the percentage of enzyme activity associated with the membrane within 1 h of exposure to alpha-MSH; the particulate enzyme activity represented 19.2 +/- 4.4% of total activity in the absence of alpha-MSH and 50.7 +/- 4.7% (means +/- S.E.M., n = 9, P less than 0.005) in the presence of alpha-MSH (1 nmol/l). Cells which had a relatively small percentage of their PKC activity on the membrane initially were significantly (P less than 0.01) more responsive to alpha-MSH stimulation than cells which initially had a relatively large percentage of PKC activity on the membrane. The association of PKC activity with the membrane showed some evidence of being dose-related to alpha-MSH. This is the first report, to the best of our knowledge, of alpha-MSH activating PKC.

Synthesis of β-glucans by Bradyrhizobium japonicum and Rhizobium fredii

Particulate enzyme preparations from Rhizobium and Bradyrhizobium synthesize β-glucans when incubated with uridine diphosphate glucose (UDP-glucose) and a divalent cation. Synthesis of β-1,2-linked glucans in Rhizobium fredii involves the product of the ndvB gene, a 319-kDa membrane protein, which is labeled with [14C]glucose from UDP-[14C]glucose as previously demonstrated in Rhizobium meliloti and Agrobacterium sp. Bradyrhizobium japonicum synthesize β-1,3- and β-1,6-linked glucans of a lower molecular weight than those synthesized by R. meliloti. In comparative experiments, no evidence was found for a protein-bound intermediate in B. japonicum. The ndvB gene of R. fredii was mobilized to B. japonicum and the gene was expressed, as evidenced by appearance of a large membrane protein (ca. 319 kDa) which was labeled with UDP-[14C]glucose in vitro. Key words: soybean, nitrogen fixation, β-glucan, Bradyrhizobium, Rhizobium.

On the stimulation of soluble and particulate guanylate cyclase in the rat brain and the involvement of nitric oxide as studied by cGMP immunocytochemistry.

The localization of the particulate and soluble guanylate cyclase in the rat brain was studied using cGMP-immunocytochemistry. The cGMP was fixed to tissue protein using a formaldehyde fixative, and an antibody against cGMP was used which was raised against a cGMP-formaldehyde-thyroglobulin conjugate. We used the atrial natriuretic factor (ANF) as a model compound to stimulate the particulate enzyme and sodium nitroprusside (SNP) to stimulate the soluble enzyme. Sequential immunostaining for cGMP and glial fibrillary acidic protein (GFAP) showed that the great majority of the ANF-responsive, cGMP-producing cells were astrocytes. These ANF-responsive cells were found in discrete parts of the CNS; not all astrocytes in these regions were responsive to ANF. SNP stimulated cGMP in abundantly present neuronal fibres throughout the CNS; few neuronal cell bodies showed increased cGMP production after SNP. Moreover, SNP also raised cGMP in astrocytes, however, not all astrocytes showed the response to SNP. These results suggest that cells might be present in the CNS which contain both the soluble and the particulate guanylate cyclase. It was demonstrated that in the immature cerebellum, the cGMP was raised in glial structures in response to N-methyl-D-aspartate (NMDA), ANF, SNP, and kainic acid. The response to NMDA and kainic acid was sensitive to inhibition of the nitric oxide synthesis from L-arginine by NG-methyl-L-arginine. Surprisingly the response to ANF localized in the molecular layer and the granular layer was also sensitive to inhibition by NG-methyl-L-arginine, whereas the response to ANF in the deep nuclei was not. A small depolarization induced by 10 to 20 mmol/l K+ induced an increase in cGMP in chopped hippocampus tissue which showed a biphasic temporal characteristic. The initial, fast (30 sec), peak was shown to be localized in varicose fibres throughout the hippocampus, whereas the slower response (10 min) was localized in astrocytes. These studies demonstrate that the different enzymes which synthesize cGMP are differently localized. However, there is also a time dependency in the activation of the guanylate cyclases, which becomes apparent in different structures at different times. The possible role of cGMP as a regulator of ion homeostase is discussed.

Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells.

The particulate enzyme responsible for the synthesis of endothelium-derived relaxing factor has been purified from cultured and native (noncultured) bovine aortic endothelial cells. Purification of the solubilized particulate enzyme preparation by affinity chromatography on adenosine 2',5'-bisphosphate coupled to Sepharose followed by Superose 6 gel filtration chromatography resulted in a single protein band after denaturing polyacrylamide gel electrophoresis that corresponded to approximately 135 kDa. The enzyme activity in the various fractions was assayed by its stimulatory effect on soluble guanylyl cyclase of rat fetal lung fibroblasts (RFL-6 cells), by the formation of L-citrulline from L-arginine, by measuring nitrite/nitrate formation, and by bioassay on endothelium-denuded vascular strips. Endothelium-derived relaxing factor synthase was purified 3419-fold from the crude particulate fraction of cultured bovine aortic endothelial cells with a 12% recovery (RFL-6 assay). Purified endothelium-derived relaxing factor synthase required L-arginine, NADPH, Ca2+, calmodulin, and 5,6,7,8-tetrahydrobiopterin for full activity.

Biosynthetic regulation of monobutyrin, an adipocyte-secreted lipid with angiogenic activity.

1-Butyrylglycerol (monobutyrin) is a novel angiogenic compound that is synthesized and secreted during the differentiation of 3T3-F442A preadipocytes into adipocytes. To study the regulation of monobutyrin biosynthesis we have developed an assay utilizing the lgycerol kinase enzyme from Cellulomonas to quantitate the levels of this compound in cell-conditioned medium. Analysis of several cultured cell types, including tumor cell lines, indicated that monobutyrin production is detectable only from adipocytes, reaching a steady-state concentration of approximately 1.0 microM in conditioned medium. Monobutyrin synthesis was demonstrated in vitro using [14C]butyryl-CoA with total homogenate or particulate fractions from adipocytes. Similar fractions from non-adipocyte cell lines failed to synthesize monobutyrin. This biosynthetic activity was shown to be distinct by substrate competition studies from the microsomal sn-glycerol-3-phosphate acyltransferase, whose activity is known to increase during adipocyte differentiation. The production of monobutyrin was hormonally regulated, as the addition of epinephrine to adipocytes caused a 10-fold increase in the amount of monobutyrin secreted. These results indicate that monobutyrin synthesis is adipocyte specific, occurs through an apparently novel particulate enzyme system, and is regulated in a hormone-dependent manner. The implications of these results for adipose physiology and angiogenesis are discussed.

Biochemical characteristics of cytosolic and particulate forms of protein tyrosine kinases from N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinoma

Protein tyrosine kinase (PTK) activities in methyl nitrosourea (MNU)-induced rat mammary carcinoma has been investigated by using poly (glu: tyr; 4:1) as an exogenous substrate. The PTK activity of the mammary carcinoma was almost equally distributed between the particulate and soluble (cytosolic) fractions at 110,000 X g. The activity of the particulate enzyme was stimulated by non-ionic detergent Triton X-100 by about 2-fold whereas the detergent had no effect on the cytosolic form. More than 60% of the particulate enzyme could be solubilized by 5% Triton X-100. Although, both particulate and cytosolic PTKs catalyzed the phosphorylation of several tyrosine containing synthetic substrates to various degrees, poly (glu: tyr; 4:1) was the best substrate (apparent Km. 0.7 mg/ml). Both forms of enzymes utilized ATP as the phosphoryl group donor, with an apparent Km of 40 microM. Among various divalent cations tested, Co2+, Mn2+ and Mg2+ were able to fulfill the divalent cation requirement of both forms of the PTKs. All these cations exerted biphasic effects on the kinase activities, however, Mg2+ was the most potent cation. Agents such as epidermal growth factor, insulin and platelet derived growth factor which stimulate their respective receptor-PTK activities were without effect on PTK activities of mammary carcinoma. On the other hand, though heparin and quercetin inhibited both enzyme activities in a concentration dependent manner, the particulate form was more sensitive to inhibition than the cytosolic form. These data indicate that MNU-induced rat mammary carcinoma expresses both particulate and cytosolic forms of PTKs and that there are significant differences in the properties of the two forms of PTKs. Differential effects of some agents on mammary carcinoma PTKs suggest that these enzymes may be acutely regulated in vivo and could play an important role in mammary carcinogenesis.

Endothelial cells have a particulate enzyme system responsible for EDRF formation: Measurement by vascular relaxation

Endothelium-derived relaxing factor (EDRF) released from endothelial cells (EC) has been shown to be nitric oxide (NO) or a closely related molecule. In cultured EC, the enzyme responsible for the formation of EDRF, EDRF-synthase, was initially described as being cytosolic, but more recently we have found it to be predominantly particulate. In view of this discrepancy we have investigated the EDRF synthesizing activity of cytosolic and particulate fractions isolated from native bovine aortic EC. EDRF was measured by cGMP formation in rat fetal lung cultured fibroblasts (RFL-6) and by the ability of cell fractions to relax endothelium-denuded, preconstricted rabbit aortic strips. Cytosolic fractions from native EC (100 μg) had no effect on the tone of rabbit aortic strips and little effect on cGMP levels in RFL-6 cells in the presence of L-arginine and NADPH (100 μM). However, under the same conditions the 100,000 × g pellet fractions relaxed rabbit aortic strips and increased cGMP levels in RFL-6 cells. Thus EDRF synthase from native EC, like those grown in culture, is located mainly in the particulate fraction.

Involvement of phosphorylated compounds in stimulation of 1,4-β glucan synthases ofSaprolegnia monoica

The stimulation by ATP of β glucan synthase activities of cell-free extracts of Saprolegnia monoica was enhanced by the presence of glucose-phosphates. These effects were obtained in conditions of 1,4-β glucan synthesis; on the contrary, strong inhibition was observed during 1,3-β glucan production. This stimulation was suppressed when particulate enzymes were pretreated with phosphatase. These results suggest that phosphorylation is involved in regulation of β glucan synthesis.

Calmodulin-dependent endothelium-derived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells.

Endothelium-derived relaxing factor/nitric oxide (EDRF/NO) synthesized by bovine aortic endothelial cells and subcellular fractions thereof was assayed by its stimulating effect on soluble guanylyl cyclase of rat fetal lung fibroblasts (RFL-6 cells). The release of EDRF/NO by intact endothelial cells could be stimulated with bradykinin, thrombin, or ADP and was abolished in Ca2(+)-free medium. When subcellular fractions were analyzed, some EDRF/NO-synthesizing activity was found in the cytosolic fraction, but most of the activity was associated with the particulate fraction. Both enzyme activities required L-arginine and NADPH for EDRF/NO synthesis, both were inhibited by NG-nitro-L-arginine and NG-methyl-L-arginine, and hemoglobin or methylene blue abolished the effect of the EDRF/NO produced by both enzymes. Both enzymes were highly sensitive to Ca2+; the major increase in activity occurred between 100 and 500 nM free Ca2+. Exposure of the particulate enzyme activity to 1 M KCl removed 39% of the protein and reduced total activity by 46%, but the activity was restored when exogenous calmodulin (CaM) was added. Further KCl washes caused little further loss of protein or EDRF/NO synthase activity. The KCl-washed particulate enzyme could be solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The CaM antagonists calmidazolium and trifluoperazine as well as the CaM-binding protein calcineurin inhibited the EDRF/NO synthesis by both the cytosolic and the particulate enzyme. These effects were partially reversed with exogenous CaM. Partial purification of the cytosolic and solubilized particulate enzymes by affinity chromatography on adenosine 2',5'-bisphosphate-Sepharose resulted in EDRF/NO synthase activities dependent on exogenous CaM. We conclude that endothelial cells contain both cytosolic and particulate enzymes that synthesize EDRF/NO. Both enzymes are regulated by free Ca2+ and, at least in part, by CaM.

Pathway for Biodegradation of p -Nitrophenol in a Moraxella sp

A Moraxella strain grew on p-nitrophenol with stoichiometric release of nitrite. During induction of the enzymes for growth on p-nitrophenol, traces of hydroquinone accumulated in the medium. In the presence of 2,2'-dipyridyl, p-nitrophenol was converted stoichiometrically to hydroquinone. Particulate enzymes catalyzed the conversion of p-nitrophenol to hydroquinone in the presence of NADPH and oxygen. Soluble enzymes catalyzed the conversion of hydroquinone to gamma-hydroxymuconic semialdehyde, which was identified by high-performance liquid chromatography (HPLC)-mass spectroscopy. Upon addition of catalytic amounts of NAD, gamma-hydroxymuconic semialdehyde was converted to beta-ketoadipic acid. In the presence of pyruvate and lactic dehydrogenase, substrate amounts of NAD were required and gamma-hydroxymuconic semialdehyde was converted to maleylacetic acid, which was identified by HPLC-mass spectroscopy. Similar results were obtained when the reaction was carried out in the presence of potassium ferricyanide. Extracts prepared from p-nitrophenol-growth cells also contained an enzyme that catalyzed the oxidation of 1,2,4-benzenetriol to maleylacetic acid. The enzyme responsible for the oxidation of 1,2,4-benzenetriol was separated from the enzyme responsible for hydroquinone oxidation by DEAE-cellulose chromatography. The results indicate that the pathway for biodegradation of p-nitrophenol involves the initial removal of the nitro group as nitrite and formation of hydroquinone. 1,4-Benzoquinone, a likely intermediate in the initial reaction, was not detected. Hydroquinone is converted to beta-ketoadipic acid via gamma-hydroxymuconic semialdehyde and maleylacetic acid.