Purification and properties of UDP-GlcNAc:dolichyl-phosphate GlcNAc-1-phosphate transferase. Activation and inhibition of the enzyme

The GlcNAc-1-P transferase was solubilized from pig aorta microsomal fractions using 0.5% Nonidet P-40. The activity of the solubilized enzyme was stimulated by exogeneously added phospholipids in the order phosphatidylglycerol greater than phosphatidylinositol greater than phosphatidylserine. When the enzyme was stored in 20% glycerol containing 20 micrograms of phosphatidylglycerol/mg of protein, more than 80% of the activity remained after storage for 6 days at 0-4 degrees C. On the other hand, in the absence of the stabilizers, the enzyme lost most of its activity within 24 h. The transferase was purified about 68-fold using ammonium sulfate and DEAE-cellulose fractionation. The DEAE-cellulose chromatography separated a heat-stable factor from the enzyme, which when added back to the partially purified enzyme stimulated about 5-fold. With this partially purified enzyme, the Km for UDP-GlcNAc was found to be 1 X 10(-7) M, and that for dolichyl-P about 1 X 10(-6) M. The stimulatory factor increased the Vmax for both UDP-GlcNAc and dolichyl-P 5-10-fold, but the Km values remained the same. The pH optimum for the enzyme was between 7.4 and 7.6, and either Mn2+ (1 mM) or Mg2+ (10 mM) was required for optimum activity. The GlcNAc-1-P transferase was also stimulated by the addition of GDP-mannose (or other purine sugar nucleotides) or dolichyl-phosphoryl-mannose to the incubation mixtures. These two compounds acted in different ways on the enzyme since their stimulatory effects were additive. The effect of GDP-mannose was found to be due to protection of the substrate, UDP-GlcNAc, from degradation, but the effect of dolichyl-P-mannose remains to be established. In addition, the stimulations shown by phosphatidylglycerol, GDP-mannose, and factor, or phosphatidylglycerol, dolichyl-P-mannose, and factor, were all additive, indicating that they were acting at different sites on the enzyme. The transferase was quite sensitive to the action of sulfhydryl reagents such as N-ethylmaleimide or p-chloromercuribenzene sulfonate, and was rapidly inactivated in their presence. The enzyme could be protected to the extent of about 50% when all of the substrates (UDP-GlcNAc, dolichyl-P, Mn2+) were added before the addition of the sulfhydryl reagents.     

Phospholipase C-delta3 binds with high specificity to phosphatidylinositol 4,5-bisphosphate and phosphatidic acid in bilayer membranes.

In order to acquire an understanding of phospholipase C-delta3 (PLC-delta3) action on substrate localized in lipid membrane we have studied the binding of human recombinant PLC-delta3 to large, unilamellar phospholipid vesicles (LUVs). PLC-delta3 bound weakly to vesicles composed of phosphatidylcholine (PtdCho) or PtdCho plus phosphatidylethanolamine (PtdEtn) or phosphatidylinositol (PtdIns). The enzyme bound strongly to LUVs composed of PtdEtn + PtdCho and phosphatidylinositol 4,5-bisphosphate (PtdInsP2). The binding affinity (molar partition coefficient) of PLC-delta3 to PtdEtn + PtdCho + PtdInsP2 vesicles was 7.7 x 105 m-1. High binding of PLC-delta3 was also observed for LUVs composed of phosphatidic acid (PA). Binding of PLC-delta3 to phosphatidylserine (PtdSer) vesicles was less efficient. Calculated molar partition coefficient for binding of PLC-delta3 to PA and PtdSer vesicles was 1.6 x 104 m-1 and 9.4 x 102 m-1, respectively. Presence of PA in the LUVs containing PtdInsP2 considerably enhanced the binding of PLC-delta3 to the phospholipid membrane. Binding of PLC-delta3 to phospholipid vesicles was not dependent on Ca2+ presence. In the liposome assay PA caused a concentration-dependent increase in activity of PLC-delta3. The stimulatory effect of PA on PLC-delta3 was calcium-dependent. At Ca2+ concentrations lower than 1 microm, no effect of PA on the activity of PLC-delta3 was observed. PA enhanced PLC-delta3 activity by increasing the Vmax and lowering Km for PtdInsP2. As the mol fraction of PA increased from 0-40 mol% the enzyme Vmax increased 2.3-fold and Km decreased threefold. Based on the results presented, we assume that PA supports binding of PLC-delta3 to lipid membranes by interaction with the PH domain of the enzyme. The stimulatory effect of PA depends on calcium-dependent interaction with the C2 domain of PLC-delta3. We propose that binding of PLC-delta3 to PA may serve as a mechanism for dynamic membrane association and modulation of PLC-delta3 activity.     

Solubilization and properties of UDP-D-glucose:N-acetylglucosaminyl pyrophosphorylundecaprenol glucosyltransferase from Bacillus coagulans AHU 1366 membranes

The glucosyltransferase which catalyzes the conversion of GlcNAc-PP-undecaprenol into Glc(beta 1----4)GlcNAc-PP-undecaprenol in the presence of UDP-glucose was solubilized from Bacillus coagulans AHU 1366 membranes by treatment with 0.1% Triton X-100 and partially purified by means of column chromatography on Sephacryl S-300 and DEAE-Sephacel. The final preparation was virtually free from other enzymes involved in the de novo synthesis of teichoic acid. The enzyme had a pH optimum of 6.6-8.0 and a Km value for UDP-glucose of 21 microM. The enzyme required 40 mM MgCl2, 0.6 M KCl, and 0.1% Nonidet P-40 for full activity.     

Purification and characterization of human placental aromatase cytochrome P-450

Aromatase cytochrome P-450, which catalyzes the conversion of androgens to estrogens, was purified from human placental microsomes. The enzyme was extracted with sodium cholate, fractionated by ammonium sulfate precipitation, and subjected to column chromatography in the presence of its substrate, androstenedione, and the nonionic detergent, Nonidet P-40. The preparation exhibits a single major band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a specific content of 11.5 nmol of P-450/mg of protein. The purified enzyme displays spectroscopic properties typical of the ferric and ferrous forms of cytochrome P-450. Full enzymatic activity can be reconstituted with rabbit liver microsomal cytochrome P-450 reductase and Nonidet P-40. Purified aromatase cytochrome P-450 displays catalytic characteristics similar to the enzyme in intact microsomes in the aromatization of androstenedione, 19-hydroxyandrostenedione and 19-oxoandrostenedione. Testosterone and 16 alpha-hydroxytestosterone are aromatized at maximal rates similar to androstenedione, and all substrates exhibit relative affinities corresponding to those observed in microsomes. We have raised rabbit antibodies to the purified enzyme which show considerable specificity and sensitivity on immunoblots.     

Partial purification and characterization of beta-mannosyltransferase from suspension-cultured soybean cells

The beta-mannosyltransferase that catalyzes the synthesis of Man-beta-GlcNAc-GlcNAc-PP-dolichol from GDP-mannose and dolichyl-PP-GlcNAc-GlcNAc was solubilized from microsomes of suspension-cultured soybean cells by treatment with 1.5% Triton X-100 and was purified about 700-fold by chromatography on DEAE-cellulose, hydroxylapatite, and a GDP affinity column. The purified enzyme was reasonably stable in the presence of 20% glycerol and 0.5 mM dithiothreitol. The enzyme required either detergent (Triton X-100 or NP-40) or phospholipid for maximum activity, but the effects of these two were not additive. Thus, either phosphatidylcholine or Triton X-100 could give maximum stimulation. In terms of phospholipid stimulation, both the head group and the acyl chain appeared to be important since phosphatidylcholines with 18-carbon unsaturated fatty acids were most effective. The purified enzyme had a sharp pH optimum of 6.9-7.0 and required a divalent cation. Mg2+ was the best metal ion with optimum activity occurring at 6 mM, but Mn2+ was reasonably effective while Ca2+ was slightly stimulatory. The Km for GDP-mannose was calculated to be 1.7 X 10(-6) M and that for dolichyl-PP-GlcNAc-GlcNAc about 9 X 10(-6) M. The enzyme was inhibited by a number of guanosine nucleotides such as GDP-glucose, GDP, GMP, and GTP, but various uridine and adenosine nucleotides were without effect. The purified enzyme was apparently free of alpha-1,3-mannosyltransferase (and perhaps other mannosyltransferases) and dolichyl-P-mannose synthase since the only product seen from dolichyl-PP-GlcNAc-GlcNAc and GDP-mannose was Man-beta-GlcNAc-GlcNAc-PP-dolichol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microsomal cytochrome P-450 from neonatal pig testis. Purification and properties of A C21 steroid side-chain cleavage system (17 alpha-hydroxylase-C17,20 lyase)

A cytochrome P-450 from neonatal pig testicular microsomes was purified to homogeneity as judged by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and by double diffusion on agar against antiserum raised in rabbits against the protein. The enzyme shows both 17 alpha-hydroxylase (Vmax = 4.6 nmol of product/min/nmol of P-450, Km = 1.5 microM) and C17,20 lyase (Vmax = 2.6 nmol of product/min/nmol of P-450, Km = 2.4 microM) activities. Both activities require NADPH and a flavoprotein P-450 reductase; microsomal P-450 reductase from pig and rat livers was used in these studies. The enzyme possesses a single subunit of molecular weight 59,000 +/- 1,000 as determined by electrophoresis on polyacrylamide with sodium dodecyl sulfate and by chromatography on sodium dodecyl sulfate-Sephadex. The enzyme is a glycoprotein and contains 8 nmol of heme/mg of protein and 40 nmol of phospholipid/mg of protein. All heme detected by pyridine hemochromogen is accounted for as P-450 by difference spectroscopy of the reduced P-450.carbon monoxide complex. This complex shows an absorbance maximum at 448 nm with no evidence of P-420. These studies raise the possibility that one microsomal protein (cytochrome P-450) may possess two enzymatic activities (hydroxylase and lyase).     

Properties of mannosyl- and N-acetylglucosamine-1-phosphate transferases towards dolichol phosphate in liver microsomes from pig embryos

1. The activity of mannosyl- and N-acetylglucosamine-1-phosphate transferases in microsomes from pig embryonic liver was linear to 1 min of incubation at 37 degrees C. 2. The activity of both enzymes was higher in the presence of Mg2+ as compared to Mn2+. A maximal stimulatory effect of Mn2+ was obtained at 2 mM concentration and greater concentrations of it inhibited the activities of both enzymes. 3. The activity of mannosyl transferase was found to be highest after treatment of microsomes with Nonidet P-40 while the activity of N-acetylglucosamine-1-phosphate transferase was greatest in the presence of sodium deoxycholate. 4. The Km for acceptor substrate was 1.6 x 10(-5)M in the reaction for dolichol phosphate mannose synthesis and 2.2 x 10(-5)M in the reaction for dolichol pyrophosphate N-acetylglucosamine formation. 5. The Km for GDP-mannose was 1.4 x 10(-5)M and for UDP-N-acetylglucosamine-6.2 x 10(-5)M. At saturating concentrations of donor substrates V values (pmol/min/mg) were 1330 and 150, respectively.     

Aldrin epoxidation catalyzed by purified rat-liver cytochromes P-450 and P-448. High selectivity for cytochrome P-450

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat liver microsomal cytochrome P-450 or P-448, NADPH-cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate. Cytochrome P-450, purified from hepatic microsomes of phenobarbital-treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observed in the absence of the lipid and sodium cholate was increased threefold by addition of dilauroylphosphatidylcholine and was further stimulated twofold by addition of sodium cholate. The apparent Km for aldrin in the complete system was 7 +/- 2 microM. SKF 525-A, at a concentration of 250 microM, inhibited aldrin epoxidation by 65%, whereas 7,8-benzoflavone had no inhibitory effect at concentrations up to 250 microM. Addition of ethanol markedly increased epoxidase activity. The increase was threefold in the presence of 5% ethanol. When cytochrome P-448 purified from hepatic microsomes of 3-methylcholanthrene-treated rats was used, a very low rate of epoxidation was observed which was less than 3% of the activity mediated by cytochrome P-450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent Km for aldrin was 27 +/- 7 microM. The modifiers of monooxygenase reactions, 7,8-benzoflavone, SKF 525-A and ethanol, inhibited the activity mediated by cytochrome P-448. The I50 was 0.05, 0.2 and 800 mM, respectively. These results indicate that aldrin is a highly selective substrate for cytochrome P-450 species present in microsomes of phenobarbital-treated animals and is a poor substrate for cytochrome P-448. The two forms of aldrin epoxidase can be characterised by their turnover number, their apparent Km and their sensitivity to modifiers, like 7,8-benzoflavone and ethanol.     

Partial purification of microsomal signal peptidase from hen oviduct

Signal peptidase has been purified approximately 600-fold from hen oviduct microsomes. Treatment of microsomes with ice-cold sodium carbonate at pH 11.5 removes soluble and extrinsic membrane proteins prior to solubilization of signal peptidase with Nonidet P-40. After dialysis to pH 8.2, the solubilized enzyme is chromatographed on diethylaminoethyl cellulose at pH 8.2. More than 90% of contaminating proteins bind to the column while signal peptidase and endogenous phospholipid are eluted in the column void volume. Enzyme activity subsequently binds to carboxymethyl cellulose at pH 5.8 and is eluted by approximately 100 to 200 mM NaCl during a NaCl gradient. Polypeptides present in partially purified hen oviduct signal peptidase have relative molecular masses ranging from 54 kD to less than 11 kD with major bands at 29, 23, 22, 19, 18 and 13 kD. The purified peptidase requires phospholipid for activity and is maximally active in the presence of 2 mg/ml phosphatidylcholine.     

Phospholipid synthesis in a membrane fraction associated with mitochondria

A crude rat liver mitochondrial fraction that was capable of the rapid, linked synthesis of phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho) labeled from [3-3H] serine has been fractionated. PtdSer synthase, PtdEtn methyltransferase, and CDP-choline:diacylglycerol cholinephosphotransferase activities were present in the crude mitochondrial preparation but were absent from highly purified mitochondria and could be attributed to the presence of a membrane fraction, X. Thus, previous claims of the mitochondrial location of some of these enzymes might be explained by the presence of fraction X in the mitochondrial preparation. Fraction X had many similarities to microsomes except that it sedimented with mitochondria (at 10,000 x g). However, the specific activities of PtdSer synthase and glucose-6-phosphate phosphatase in fraction X were almost twice that of microsomes, and the specific activities of CTP:phosphocholine cytidylyltransferase and NADPH:cytochrome c reductase in fraction X were much lower than in microsomes. The marker enzymes for mitochondria, Golgi apparatus, plasma membrane, lysosomes, and peroxisomes all had low activities in fraction X. Polyacrylamide gel electrophoresis revealed distinct differences, as well as similarities, among the proteins of fraction X, microsomes, and rough and smooth endoplasmic reticulum. The combined mitochondria-fraction X membranes can synthesize PtdSer, PtdEtn, and PtdCho from serine. Thus, fraction X in combination with mitochondria might be responsible for the observed compartmentalization of a serine-labeled pool of phospholipids previously identified (Vance, J. E., and Vance, D. E. (1986) J. Biol. Chem. 261, 4486-4491) and might be involved in the transfer of lipids between the endoplasmic reticulum and mitochondria.     

Partial purification and properties of UDP-N-acetylmannosamine:N-acetylglucosaminyl pyrophosphorylundecaprenol N-acetylmannosaminyltransferase from Bacillus subtilis

An enzyme which catalyzes the conversion of GlcNAc-PP-undecaprenol into ManNAc(beta 1----4)GlcNAc-PP-undecaprenol, a key lipid intermediate in the de novo synthesis of various teichoic acids, was partially purified from the 20,000 x g supernatant fraction of Bacillus subtilis AHU 1035 cell homogenate. By means of ammonium sulfate precipitation, gel chromatography, and ion-exchange chromatography, the enzyme was purified about 70-fold, giving a preparation virtually free from substances obstructive to measurement of the N-acetylmannosaminyltransferase reaction. The enzyme was shown to be specific to UDP-ManNAc. The Km value for UDP-ManNAc was 4.4 microM, and the optimum pH was 7.3. The enzyme required 10 mM MgCl2, 0.3 M KCl, 25% glycerol, and 0.1% Nonidet P-40 to function at full activity.     

Purification and characterization of dolichyl-P-mannose:Man5(GlcNAc)2-PP-dolichol mannosyltransferase

The dolichyl-P-mannose:dolichyl-PP-heptasaccharide alpha-mannosyltransferase (2.4.1.130), which catalyzes the transfer of mannose from dolichyl-P-mannose to the Man5(GlcNAc)2-PP-dolichol acceptor glycolipid, was solubilized from pig aorta microsomes with 0.5% NP-40 and purified 985-fold by a variety of conventional methods. The partially purified enzyme had a pH optimum of 6.5 and required Ca2+, at an optimum concentration of 8-10 mM, for activity. Mn2+ was only 20% as effective as Ca2+, and Mg2+ was inhibitory. The mannosyltransferase activity was also inhibited by the addition of EDTA to the enzyme, but this inhibition was fully reversible by the addition of Ca2+. The enzyme was quite specific for dolichyl-P-mannose as the mannosyl donor and Man5(GlcNAc)2-PP-dolichol as the mannosyl acceptor. The Km values for dolichyl-P-mannose and the acceptor lipid Man5(GlcNAc)2-PP-dolichol were 1.8 and 1.6 microM. On Bio-Gel P-4 columns and by HPLC, the radiolabeled oligosaccharide formed during incubation of dolichyl-P-[14C]mannose and unlabeled Man5(GlcNAc)2-PP-dolichol with the purified enzyme behaved like Man6(GlcNAc)2. This octasaccharide was susceptible to digestion by endoglucosaminidase H, indicating that the newly added mannose was attached to the 6-linked mannose in an alpha 1,3-linkage. This linkage was further confirmed by acetolysis of the oligosaccharide product [i.e., Man6(GlcNAc)2], which gave a labeled disaccharide as the major product (greater than 90%).     

Phosphatidylcholine and N-methylated phospholipids are nonessential in Saccharomyces cerevisiae

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in numerous eukaryotes and is generally thought to be essential for membrane structure and cellular function. We designed a specific test of this idea by using genetic and biochemical manipulation of yeast. Yeast mutants (pem1 pem2Delta) lacking the phosphatidylethanolamine (PtdEtn) methyltransferase enzymes require choline for growth and cannot make N-methylated phospholipids. When these strains are grown on a glucose carbon source supplemented with 20 mm propanolamine (Prn), the PtdCho level declines precipitously to the limits of detection (<0.6%), and the hexagonal phase-forming, primary amine-containing lipids, PtdEtn and PtdPrn, constitute approximately 60% of the total phospholipid content of the cell. When the lipids were analyzed by mass spectrometry, there was no compensatory shift in unsaturation of the PtdEtn and PtdPrn toward more bilayer-forming species. Thus the majority of the cellular amino phospholipids remained hexagonal phase-forming. The pem1 pem2Delta cells will also grow without choline, in the presence of Prn, on nonfermentable carbon sources (requiring functional mitochondria) and accumulate nearly 70% of their phospholipid as hexagonal phase-forming types. These data provide compelling evidence that the functions of PtdCho and N-methylated lipids in membranes are nonessential in Saccharomyces cerevisiae.     

Purification and characterization of UDP-N-acetyl-D-glucosamine:dolichol phosphate N-acetyl-D-glucosamine-1-phosphate transferase involved in the biosynthesis of asparagine-linked glycoproteins in the mammary gland

The GlcNAc-1-P-transferase that initiates the dolichol cycle for the biosynthesis of asparagine-linked glycoproteins has been purified from the lactating bovine mammary gland. After solubilization from microsomes with 0.25% Nonidet P-40, the enzyme activity was stabilized with 20% glycerol, 20 micrograms/ml phosphatidylglycerol, 5 microM dolichol phosphate, and 2.5 microM UDP-GlcNAc. The purification protocol involved (NH4)2SO4 precipitation, gel filtration on Sephacryl S-300, DEAE-TSK, and hydroxylapatite chromatography. The purified enzyme was devoid of several readily detectable glycosyltransferases of the dolichol cycle. It showed two bands (A, 50 kDa and B, 46 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after either Coomassie Blue or silver staining. Antisera (anti-A and anti-B) raised against individual bands A and B inhibited the enzyme activity in solubilized microsomes. Each of the partially purified antibodies recognizes both bands A and B on Western blots of the enzyme; with the solubilized microsomes, the antibodies also recognize an additional polypeptide of approximately 70 kDa. When radioiodinated microsomes were immunoprecipitated with anti-B and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, again bands of 46, 50, and 70 kDa were observed. The peptide mapping of 50 and 46 kDa bands of the purified enzyme by chemical cleavage with N-chlorosuccinimide gave similar fragmentation patterns. The results indicate that either 70 kDa band is a precursor form of the enzyme or this polypeptide, representing the native enzyme or its subunit, is proteolyzed to smaller, enzymatically active peptide(s) of 50 and 46 kDa during purification despite the inclusion of several inhibitors against serine-proteases in all buffers used for tissue homogenization and enzyme purification. A number of properties of the purified enzyme, including its specific activation by Man-P-Dol were also characterized.     

Substrate-specific forms of human platelet phospholipase A2

Purification of human platelet phospholipase A2 (PLA2) from a particulate fraction by ion-exchange chromatography at 4 degrees C yielded a single peak of enzyme activity, which catalyzed the hydrolysis of arachidonic acid from the 2-position of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). The activity toward PtdCho and that toward PtdEtn differed in stability during storage, pH optimum, Ca2+ requirement, and affinity for the substrate; however, each activity preferred phospholipid with arachidonate at the 2-position. The two activities appeared to be eluted as an aggregate in a single peak from the ion-exchange column. When the column was run at 22 degrees C, an additional PLA2 activity peak specific for PtdEtn was resolved from the original PLA2 peak. But when the particulate fraction was briefly sonicated in 0.1% octylglucoside before chromatography at 22 degrees C, a different PLA2 activity peak, specific for PtdCho, was obtained. Resolution of the two specific forms of PLA2 under different conditions probably resulted from selective solubilization of the aggregate. The specific PLA2 activities thus isolated were very labile, whereas those in the aggregate were relatively stable. These findings suggest that human platelets contain at least two substrate-specific forms of PLA2, one for PtdCho and another for PtdEtn.     

Solubilisation and reconstitution of acylcoenzyme A:estradiol-17 beta acyltransferase

Acylcoenzyme A:estradiol-17 beta acyltransferase in microsomes of bovine placenta cotyledons was strongly membrane bound. The enzyme was solubilised from microsomes by sodium cholate and was reconstituted into phospholipid vesicles. The apparent Km for estradiol-17 beta was 11 microM which was close to the value of 8 microM previously found with the membrane-bound enzyme. Testosterone was also a substrate for the reconstituted enzyme (apparent Km 62 microM) and was a competitive inhibitor (Ki 74 microM) of the acylation of estradiol-17 beta. Although various long-chained fatty acyl CoAs acted as acyl donors, these proved to have widely differing apparent Km values with palmitoleoyl CoA having the highest affinity (Km 24 microM) and arachidonoyl CoA the lowest affinity (Km 330 microM).     

Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis in rat-2 fibroblasts by cell-permeable ceramides.

Phospholipids and sphingolipids are important precursors of lipid-derived second messengers such as diacylglycerol and ceramide, which participate in several signal transduction pathways and in that way mediate the effects of various agonists. The cross-talk between glycerophospholipid and sphingolipid metabolism was investigated by examining the effects of cell-permeable ceramides on phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) synthesis in Rat-2 fibroblasts. Addition of short-chain C6-ceramide to the cells resulted in a dose- and time-dependent inhibition of the CDP-pathways for PtdCho and PtdEtn synthesis. Treatment of cells for 4 h with 50 microM C6-ceramide caused an 83% and a 56% decrease in incorporation of radiolabelled choline and ethanolamine into PtdCho and PtdEtn, respectively. Exposure of the cells for longer time-periods (>/= 16 h) to 50 microM C6-ceramide resulted in apoptosis. The structural analogue dihydro-C6-ceramide did not affect PtdCho and PtdEtn synthesis. In pulse-chase experiments, radioactive choline and ethanolamine accumulated in CDP-choline and CDP-ethanolamine under the influence of C6-ceramide, suggesting that synthesis of both PtdCho and PtdEtn were inhibited at the final step in the CDP-pathways. Indeed, cholinephosphotransferase and ethanolaminephosphotransferase activities in membrane fractions from C6-ceramide-treated cells were reduced by 64% and 43%, respectively, when compared with control cells. No changes in diacylglycerol mass levels or synthesis of diacylglycerol from radiolabelled palmitate were observed. It was concluded that C6-ceramide affected glycerophospholipid synthesis predominantly by inhibition of the step in the CDP-pathways catalysed by cholinephosphotransferase and ethanolaminephosphotransferase.     

Kinetic and structural properties of biocatalytically active sheep lung microsomal NADPH-cytochrome c reductase

NADPH-cytochrome c reductase was purified to electrophoretic homogeneity from detergent solubilized sheep lung microsomes. The specific activity of the purified enzyme ranged from 56 to 67 mumol cytochrome c reduced/min/mg protein and the yield was 48-52% of the initial activity in lung microsomes. The reductase had Mr of 78,000 and contained 1 mol each of FAD and FMN. Km values obtained in 0.3 M phosphate buffer, pH 7.8 at 37 degrees C for NADPH and cytochrome c were 11.1 +/- 0.70 microM and 20.0 +/- 2.15 microM. Lung reductase was inhibited by its substrate, cytochrome c when its concentration was above 160 microM. The lung reductase exhibited a ping-pong type kinetic mechanism for NADPH mediated cytochrome c reduction. Purified lung reductase was biocatalytically active in supporting benzo(a)pyrene hydroxylation reaction when coupled with lung cytochrome P-450 and lipid.     

Purification and biochemical characterization of a hydroxyneurosporene desaturase involved in the biosynthetic pathway of the carotenoid spheroidene in Rhodobacter sphaeroides.

Hydroxyneurosporene desaturase is involved in the carotenoid biosynthetic pathway of Rhodobacter species. The gene encoding this enzyme was expressed in Escherichia coli, purified, and biochemically characterized. The resulting protein contained an N-terminal six-histidine extension which derived from the cloning vector; this allowed for a one-step purification of the enzyme to homogeneity after solubilization with Nonidet P-40. The hydrogen acceptor in the C-3,4 desaturation reaction was molecular oxygen. NAD+, NADP+, and flavin adenine dinucleotide had no influence on enzymatic activity. Different acyclic 1-hydroxycarotenoids were tested as substrates. Very good conversion was achieved with 1-hydroxyneurosporene and 1-hydroxylycopene, whereas 1-hydroxy-gamma-carotene and 1,1'-dihydroxylycopene were much less effective. From 1'-hydroxy-3,4-didehydrolycopene only trace amounts of product were obtained, and 1-methoxyneurosporene was not converted by purified hydroxyneurosporene desaturase. A Km of 13.4 microM was determined for 1-hydroxyneurosporene.     

Comparison of highly purified sheep liver and lung NADPH-cytochrome P-450 reductases by the analysis of kinetic and catalytic properties

1. Reductase was purified to electrophoretic homogeneity from sheep liver and lung microsomes. The specific activity of both enzymes ranged from 55 to 66 mumol cytochrome c reduced/min/mg protein. 2. Liver and lung reductases appeared to have similar kinetic and spectral properties. Km (NADPH) and Km (cytochrome c) values were calculated to be 14.3 +/- 1.23 microM and 22.2 +/- 2.78 microM for liver and 11.1 +/- 0.70 microM and 20.0 +/- 2.15 microM for lung reductase, respectively. Kinetic studies showed that cytochrome c can bind the oxidized form of the enzyme as well as its reduced form and both reductases operated through a ping-pong type mechanism. 3. These reductases cannot be distinguished on the basis of monomer molecular weights (Mr 78,000) except that the liver reductase was found to be more susceptible to proteolytic attack. 4. Both reductases supported aniline 4-hydroxylation and ethylmorphine N-demethylation reactions to the same extent in the reconstituted systems. However, sheep lung reductase appeared only 36.5 and 14.8% as effective in catalyzing benzo[a]pyrene reaction as an equivalent amount of reductase from liver in the presence of liver cytochrome P-450 and 3MC-treated rat liver cytochrome P-448, respectively.