Synthesis of retinyl phosphate mannose and dolichyl phosphate mannose from endogenous and exogenous retinyl phosphate and dolichyl phosphate in microsomal fraction. Specific decrease in endogenous retinyl phosphate mannose synthesis in vitamin A deficiency.

Rat liver microsomal fraction synthesized Ret-P-Man (retinyl phosphate mannose) and Dol-P-Man (dolichyl phosphate mannose) from endogenous Ret-P (retinyl phosphate) and Dol-P (dolichyl phosphate). Ret-P-Man synthesis displayed an absolute requirement for a bivalent cation, and also Dol-P-Man synthesis was stimulated by bivalent metal ions. Mn2+ and Co2+ were the most active, with maximum synthesis of Ret-P-Man occurring at 5-10 mM: Mg2+ was also active, but at higher concentrations. At 5mM-Mn2+ the amount of endogenous Ret-P mannosylated in incubation mixtures containing 5 microM-GDP-mannose in 15 min at 37 degrees C was approx. 3 pmol/mg of protein. In the same assays about 7-10 pmol of endogenous Dol-P was mannosylated. Bivalentcation requirement for Ret-P-Man synthesis from exogenous Ret-P showed maximum synthesis at 2.5 mM-Mn2+ or -Co2+. In addition to Ret-P-Man and Dol-P-Man, a mannolipid co-chromatographing with undecaprenyl phosphate mannose was detected. Triton X-100 (0.5%) abolished Ret-P-Man synthesis from endogenous Ret-P and caused a 99% inhibition of Ret-P-Man synthesis from exogenous Ret-P. The presence of detergent (0.5%) also inhibited Dol-P-Man synthesis from endogenous Dol-P and altered the requirement for Mn2+. Microsomal fraction from Syrian golden hamsters was also active in Ret-P-Man and Dol-P-Man synthesis from endogenous Ret-P and Dol-P. At 5 mM-Mn2+ about 2.5 pmol of endogenous Ret-P and 3.7 pmol of endogenous Dol-P were mannosylated from GDP-mannose per mg of protein in 15 min at 37 degrees C. On the other hand, microsomal fraction from vitamin A-deficient hamsters contained 1.2 pmol of Ret-P and 14.1 pmol of Dol-P available for mannosylation. Since GDP-mannose: Ret-P and GDP-mannose: Dol-P mannosyltransferase activities were not affected, depletion of vitamin A must affect Ret-P and Dol-P pools in opposite ways.     

Characterization of three kinetically distinct forms of glutamate decarboxylase from pig brain.

Hamster liver microsomal membranes catalyse the synthesis of retinyl phosphate mannose (Ret-P-Man) from GDP-mannose and exogenous retinyl phosphate (Ret-P). We have previously shown that maximal Ret-P-Man synthesis occurs in vitro at 20-30 min, followed by a subsequent loss of mannose from Ret-P-Man, suggestive of an intermediary function of Ret-P-Man and/or Ret-P-Man breakdown [Shidoji, Silverman-Jones & De Luca (1982) Biochem. J. 208, 865-868; Creek, Morre, Silverman-Jones, Shidoji & De Luca (1983) Biochem. J. 210, 541-547). To monitor Ret-P-Man synthesis and breakdown carefully, we developed a chromatographic system in which mannose, Ret-P-Man, mannose phosphate and GDP-mannose are separated in a single analysis on a Mono Q column eluted with a gradient of NaCl. Using this chromatographic system, we have determined that 80-90% of the Ret-P-Man made in vitro by hamster liver membranes in 30 min is recovered with the membranes upon centrifugation. Subsequent incubation of Ret-P-Man-loaded membranes at 37 degrees C results in a non-enzymic breakdown of Ret-P-Man to beta-mannopyranosyl phosphate and anhydroretinol. However, incubation of the Ret-P-Man-loaded hamster liver membranes with GDP, but not GMP, ADP, CDP or UDP, results in a loss of mannose from Ret-P-Man and the formation of GDP-mannose and Ret-P. These results demonstrate that Ret-P-Man synthesized in vitro is subject to non-enzymic breakdown to beta-mannopyranosyl phosphate and anhydroretinol and that the GDP-mannose:retinyl phosphate mannosyltransferase reaction is reversible.     

Fast atom bombardment and collisional activation mass spectrometry of retinyl phosphate mannose synthesized by liver membranes

Fast atom bombardment (FAB) and collisional activation dissociation (CAD) mass-analysed ion kinetic energy (MIKE) spectra have confirmed the structures of retinyl phosphate (Ret-P), retinyl phosphate mannose (Ret-P-Man) and guanosine 5'-diphospho-D-mannose (GDP-Man). Ret-P-Man was made in vitro while Ret-P and GDP-Man were chemically synthesized. Positive ion FAB mass spectrometry of Ret-P showed an observable short-lived spectrum with a mass ion at m/z 367 [M + H]+, and a major fragment ion at m/z 269 [M + H - H3PO4]+. Negative ion FAB mass spectrometry of Ret-P showed a strong stable spectrum with a parent ion at m/z 365 [M - H]-, a glycerol (G) adduct ion at m/z 457 [M - H + G]- and a dimer ion at m/z 731 [2M - H]-. GDP-Man showed an intense spectrum with parent ion at m/z 604 [M - H]- and cationized species at m/z 626 [M + Na - 2H]- and 648 [M + 2Na - 3H]-. Negative ion FAB mass spectrometry of Ret-P-Man showed a parent ion at m/z 527 [M - H]- and a fragment ion at m/z 259 [C6H12PO9]-. The CAD-MIKE spectra showed structurally significant fragment ions at m/z 442 and 361 for the [M - H]- ion of GDP-Man, and at m/z 509, 406, 364 and 241 for the [M - H]- ion of Ret-P-Man. FAB and CAD-MIKE spectra have been applied successfully to confirm the structure of Ret-P-Man made in vitro from Ret-P and GDP-Man.     

The conversion of exogenous retinol and related compounds into retinyl phosphate mannose by adult Brugia pahangi in vitro.

Adult Brugia pahangi took up and incorporated beta-carotene and free retinol in vitro. The uptake of retinol was 50 times greater than that of beta-carotene under similar incubation conditions. beta-Carotene was almost entirely metabolized, primarily to retinol. The metabolism of retinol by B. pahangi in vitro was less extensive, with a variety of retinoids tentatively identified, including retinyl phosphate (Ret-P), retinyl phosphate mannose (Ret-P-Man) and anhydroretinol as minor metabolites. B. pahangi microsomes were also shown to biosynthesize Ret-P-Man from exogenous Ret-P and GDP-mannose, but not from endogenous lipid acceptors alone. In this circumstance an unidentified lipid appeared to be mannosylated by B. pahangi. The rate of mannose transfer to exogenous Ret-P by B. pahangi microsomes was 150 pmol X min -1. (mg of protein) -1. Ret-P-Man synthetase activity from both B. pahangi and rat liver microsomes had an absolute requirement for bovine serum albumin and MnCl2, and occurred in the absence of detergent. The results suggest a biochemical role for vitamin A in B. pahangi, possibly in filarial glycoprotein synthesis.     

Mannosylation of endogenous and exogenous phosphatidic acid by liver microsomal membranes. Formation of phosphatidylmannose

Hamster liver post-nuclear membranes catalyze the transfer of mannose from GDP-mannose to endogenous dolichyl phosphate and to a second major endogenous acidic lipid. This mannolipid was believed to be synthesized from endogenous retinyl phosphate and was tentatively identified as retinyl phosphate mannose (Ret-P-Man) (De Luca, L. M., Brugh, M. R. Silverman-Jones, C. S. and Shidoji, Y. (1982) Biochem. J. 208, 159-170). To characterize this endogenous mannolipid in more detail, we isolated and purified the mannolipid from incubations containing hamster liver membranes and GDP-[14C]mannose and compared its properties to those of authentic Ret-P-Man. We found that the endogenous mannolipid was separable from authentic Ret-P-Man on a Mono Q anion exchange column, did not exhibit the absorbance spectrum characteristic of a retinol moiety, and was stable to mild acid under conditions which cleave authentic Ret-P-Man. The endogenous mannolipid was sensitive to mild base hydrolysis and mannose was released from the mannolipid by snake venom phosphodiesterase digestion. These properties were consistent with the endogenous acceptor being phosphatidic acid. Addition of exogenous phosphatidic acid, but not phospholipids with a head group blocking the phosphate moiety, to incubations containing hamster liver membranes and GDP-[14C]mannose resulted in the synthesis of a mannolipid with chromatographic and physical properties identical to the endogenous mannolipid. A double-labeled mannolipid was synthesized in incubations containing hamster liver membranes, GDP-[14C]mannose, and [3H]phosphatidic acid. Mannosyl transfer to exogenous phosphatidic acid was saturable with increasing concentrations of phosphatidic acid and GDP-mannose and specific for glycosyl transfer from GDP-mannose. Class E Thy-1-negative mutant mouse lymphoma cell membranes, which are defective in dolichyl phosphate mannose synthesis, also fail to transfer mannose from GDP-mannose to exogenous phosphatidic acid or retinyl phosphate. Amphomycin, an inhibitor of dolichyl phosphate mannose synthesis, blocked mannosyl transfer to the endogenous lipid, and to exogenous retinyl phosphate and phosphatidic acid. We conclude that the same mannosyltransferase responsible for dolichyl phosphate mannose synthesis can also utilize in vitro exogenous retinyl phosphate and phosphatidic acid as well as endogenous phosphatidic acid as mannosyl acceptors.     

Mannosyl carrier functions of retinyl phosphate and dolichyl phosphate in rat liver endoplasmic reticulum

Of the subcellular fractions of rat liver the endoplasmic reticulum was the most active in GDP-mannose: retinyl phosphate mannosyl-transfer activity. The synthesis of retinyl phosphate mannose reached a maximum at 20-30 min of incubation and declined at later times. Retinyl phosphate mannose and dolichyl phosphate mannose from endogenous retinyl phosphate and dolichyl phosphate could also be assayed in the endoplasmic reticulum. About 1.8 ng (5 pmol) of endogenous retinyl phosphate was mannosylated per mg of endoplasmic reticulum protein (15 min at 37 degrees C, in the presence of 5 mM-MnCl2), and about 0.15 ng (0.41 pmol) of endogenous retinyl phosphate was mannosylated with Golgi-apparatus membranes. About 20 ng (13.4 pmol) of endogenous dolichyl phosphate was mannosylated in endoplasmic reticulum and 4.5 ng (3 pmol) in Golgi apparatus under these conditions. Endoplasmic reticulum, but not Golgi-apparatus membranes, catalysed significant transfer of [14C]mannose to endogenous acceptor proteins in the presence of exogenous retinyl phosphate. Mannosylation of endogenous acceptors in the presence of exogenous dolichyl phosphate required the presence of Triton X-100 and could not be detected when dolichyl phosphate was solubilized in liposomes. Dolichyl phosphate mainly stimulated the incorporation of mannose into the lipid-oligosaccharide-containing fraction, whereas retinyl phosphate transferred mannose directly to protein.     

Retinyl phosphate mannose synthesis in rat liver membranes. Phospholipase sensitivity and phospholipid requirement.

A remarkable and immediate decrease in GDP-mannose:retinyl phosphate mannosyltransferase activity was found on pre-incubation of rat liver postnuclear membranes with phospholipase A2 or phospholipase C. Under the same conditions of pre-incubation (1 min at 37 degrees C) trypsin did not affect the enzyme activity, whereas pre-incubation for 30 min with trypsin and Pronase abolished enzyme activity. The lipid extract of untreated rat liver membranes partially restored enzyme activity after phospholipase treatment. Sphingomyelin was as active as the endogenous lipids. Other phospholipids were less active in the following order: phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidylinositol = phosphatidylserine. Dolichyl phosphate mannose synthesis was inhibited less (33%) by phospholipase C than was Ret-P-Man synthesis (98.5%) under identical conditions of incubation, which included 0.025% Triton. However, retinyl phosphate mannose synthesis by purified endoplasmic reticulum was found to be resistant to phospholipase C. Mixing experiments failed to demonstrate an inhibitory effect of the phospholipase-treated postnuclear membrane fraction on the synthetic activity of the endoplasmic reticulum, thus excluding the release of an inhibitory factor from the postnuclear membranes.     

Rat liver microsomes catalyse mannosyl transfer from GDP-d-mannose to retinyl phosphate with high efficiency in the absence of detergents

In the absence of detergent, the transfer of mannose from GDP-mannose to rat liver microsomal vesicles was highly stimulated by exogenous retinyl phosphate in incubations containing bovine serum albumin, as measured in a filter binding assay. Under these conditions 65% of mannose 6-phosphatase activity was latent. The transfer process was linear with time up to 5min and with protein concentration up to 1.5mg/0.2ml. It was also temperature-dependent. The microsomal uptake of mannose was highly dependent on retinyl phosphate and was saturable against increasing amounts of retinyl phosphate, a concentration of 15mum giving half-maximal transfer. The uptake system was also saturated by increasing concentrations of GDP-mannose, with an apparent K(m) of 18mum. Neither exogenous dolichyl phosphate nor non-phosphorylated retinoids were active in this process in the absence of detergent. Phosphatidylethanolamine and synthetic dipalmitoylglycerophosphocholine were also without activity. Several water-soluble organic phosphates (1.5mm), such as phenyl phosphate, 4-nitrophenyl phosphate, phosphoserine and phosphocholine, did not inhibit the retinyl phosphate-stimulated mannosyl transfer to microsomes. This mannosyl-transfer activity was highest in microsomes and marginal in mitochondria, plasma and nuclear membranes. It was specific for mannose residues from GDP-mannose and did not occur with UDP-[(3)H]galactose, UDP- or GDP-[(14)C]glucose, UDP-N-acetyl[(14)C]-glucosamine and UDP-N-acetyl[(14)C]galactosamine, all at 24mum. The mannosyl transfer was inhibited 85% by 3mm-EDTA and 93% by 0.8mm-amphomycin. At 2min, 90% of the radioactivity retained on the filter could be extracted with chloroform/methanol (2:1, v/v) and mainly co-migrated with retinyl phosphate mannose by t.l.c. This mannolipid was shown to bind to immunoglobulin G fraction of anti-(vitamin A) serum and was displaced by a large excess of retinoic acid, thus confirming the presence of the beta-ionone ring in the mannolipid. The amount of retinyl phosphate mannose formed in the bovine serum albumin/retinyl phosphate incubation is about 100-fold greater than in incubations containing 0.5% Triton X-100. In contrast with the lack of activity as a mannosyl acceptor for exogenous dolichyl phosphate in the present assay system, endogenous dolichyl phosphate clearly functions as an acceptor. Moreover in the same incubations a mannolipid with chromatographic properties of retinyl phosphate mannose was also synthesized from endogenous lipid acceptor. The biosynthesis of this mannolipid (retinyl phosphate mannose) was optimal at MnCl(2) concentrations between 5 and 10mm and could not be detected below 0.6mm-MnCl(2), when synthesis of dolichyl phosphate mannose from endogenous dolichyl phosphate was about 80% of optimal synthesis. Under optimal conditions (5mm-MnCl(2)) endogenous retinyl phosphate mannose represented about 20% of dolichyl phosphate mannose at 15min of incubation at 37 degrees C.     

Differential regulation of the synthesis of mannosylphosphoryl derivatives of dolichol and retinol in HeLa cells

We have shown earlier that in HeLa S3G cells, glucocorticoids stimulate the synthesis of dolichyl phosphorylmannose (Dol-P-Man) with a concomitant increase in the glycosylation of proteins (Ramachandran, C.K., Gray, S.L. and Melnykovych G. (1982) Biochem. J. 208, 47-52). Although controversial, there have been several lines of evidence suggesting that the synthesis of retinyl phosphorylmannose (Ret-P-Man) and Dol-P-Man may be carried out by the same enzyme. We examined this possibility and conclude that in HeLa S3G cells the syntheses of Dol-P-Man and Ret-P-Man are catalyzed by two different enzymes located in the same microenvironment. Our conclusion is based on the following observations: exogenously added dolichyl phosphate and retinyl phosphate did not compete with each other; when the cells were grown in the presence of 1 microM dexamethasone, the microsomal synthesis of Dol-P-Man was stimulated, without affecting the Ret-P-Man synthesis; Arrhenius plots on Ret-P-Man and Dol-P-Man synthesis showed breaks at 22 and 37.7 degrees C.     

Enzymatic synthesis of mannosyl retinyl phosphate from retinyl phosphate and guanosine diphosphate mannose.

A study was conducted to determine whether retinyl phosphate would act as substrate for the enzymatic synthesis of mannosyl retinyl phosphate. Retinyl phosphate, prepared chemically, supported the growth of vitamin A-deficient rats at the same rate as retinol. It also stimulated the uptake of [14C]mannose from GDP-[14C]mannose into total chloroform-methanol extractable lipid. This reaction occurred in the presence of ATP, Mn2+, detergent (Zonyl A), and a membrane-rich enzyme preparation from the livers of vitamin A-deficient rats, provided that a lipid extract of the membrane preparation of alpha-L-lecithin was also added. Total chloroform-methanol-extractable, labeled mannolipid was separated into two principal labeled mannolipids by thin-layer or column chromatography or by differential solvent extraction. The properties of these mannolipids identified them as glycophospholipids: one was identical with authentic synthetic dolichyl mannosyl phosphate, and the other was concluded to be mannosyl retinyl phosphate because of its incorporation of radioactivity from [3H]retinyl phosphate, its rapid hydrolysis by dilute acid, and the formation of substance that cochromatographed with retinol upon its acid hydrolysis. The presence of ATP or GTP was essential for the stimulation of mannolipid synthesis, probably because of their protective action on the substrates against phosphatases present in the crude enzyme fraction. A pH of 6.0-6.2 favored the formation of dolichyl mannosyl phosphate; a higher pH (6.7-7.0) that of mannosyl retinyl phosphate.     

Effect of phospholipids,Triton X-100 and biological membranes on redox systems involving tetrazolium salt reduction. Its implications for the assay of enzymatic activities

The influence of phospholipids and Triton X-100 on the time course of chemical and enzyme-mediated reductions of a commonly used tetrazolium salt, MTT, was studied. MTT reduction was followed by the absorbance changes at 570 nm. With ascorbate as reducing agent, a 3-fold increase in the initial rates of the absorbance changes and a 24 % increase in the final absorbance values were observed in the presence of Triton X-100 micelles or phospholipid vesicles. The enzyme-mediated reduction of MTT with NADH generated by the NAD-dependent lactate dehydrogenase was also enhanced in the presence of Triton X-100, phospholipids or erythrocyte membranes. No enhancement was observed following the enzymatic generation of NADH at 340 nm in the absence of MTT. The above findings were interpreted as arising from: a) solubilization or reduced MTT in the detergent micelles or phospholipid vesicles which favors the redox reaction occurring in the aqueous fase, and b) changes in the spectral properties of reduced MTT in aqueous and lipid-like media.     

INVOLVEMENT OF MANNOSYL-PHOSPHORYL-DOLICHOLS IN MANNOSE TRANSFER TO BRAIN GLYCOPROTEINS

Abstract— Mannose was transferred from GDP-[¹⁴C]mannose by homogenates of embryonic chick and adult rat brain to mannolipids with properties identical to manriosyl-phosphoryl-dihydropolyisoprenols. Embryonic chick brain formed six-fold larger quantities of mannolipid than adult rat brain. The reaction was stimulated by Mn²⁺ ions and Triton X-100 but inhibited by EDTA. Phosphoenolpyruvic acid had no effect on the reaction. A crude mitochondrial fraction was two to three times more active than the microsomal fraction. All radioactivity in the mannolipid could be displaced by the addition of non-radioactive GDP-mannose. The endogenous lipid acceptor in brain was readily labelled in vivo by injection of [³H]mevalonate into the amniotic sac of 7-day-old embryos. The mannolipid formed had the properties of an acidic phospholipid on column and TLC, was stable to dilute alkali but readily cleaved by dilute acid. Synthesis was markedly stimulated by the addition of pig liver or calf brain dolichol phosphate in the presence of Triton X-100 and Mn²⁺. The mannolipid so formed displayed chemical characteristics identical to the endogenous lipid acceptor. Incubation of the purified radioactive mannolipid with the 'post-nuclear' fraction from 14-day-old embryonic chick brain in the presence of EDTA and Triton X-100 resulted in the transfer of 40-50 per cent of the radioactive mannose to protein and 40-45 per cent to water soluble compounds. The efficiency of transfer of radioactivity from endogenously formed mannolipid with or without the addition of dolichol phosphate was similar to exogenously added highly purified mannolipid. These results are compatible with the hypothesis that synthesis of the mannose core of brain glycoproteins involves the synthesis first of mannosyl-phosphoryl-dolichols followed by transfer of the mannose to glycoprotein.     

Formation of alpha-1,2- and alpha-1,3-linked mannose disaccharides from dolichyl mannosyl phosphate by rat liver membrane enzymes

Dolichyl mannosyl phosphate and GDPmannose were active substrates for the transfer of mannose to methyl-alpha-D-mannose, p-nitrophenyl-alpha-D-mannose, and free mannose with rat liver microsomal membranes. The products formed during dolichyl mannosyl phosphate incubation with methyl-alpha-D-mannose or with mannose were alpha-linked. The disaccharides formed by incubation of dolichyl mannosyl phosphate or GDPmannose with mannose were identified by paper chromatography and electrophoresis as mannose-alpha-1,2-mannose and mannose-alpha-1,3-mannose. synthesis of each product was dependent on the assay conditions used and was most markedly affected by the presence of detergent. Transfer of mannose from either substrate to form mannose-alpha-1,3-mannose was severely inhibited by Triton X-100.     

Effects of metal ions on sphingomyelinase activity of Bacillus cereus

Some divalent metal ions were examined for their effects on sphingomyelinase activity of Bacillus cereus. The enzyme activity toward mixed micelles of sphingomyelin and Triton X-100 proved to be stimulated by Co2+ and Mn2+, as well as by Mg2+. Km's for Co2+ and Mn2+ were 7.4 and 1.7 microM, respectively, being smaller than the Km for Mg2+ (38 microM). Sr2+ proved to be a competitive inhibitor against Mg2+, with a Ki value of 1 mM. Zn2+ completely abolished the enzyme activity at concentrations above 0.5 mM. The concentration of Zn2+ causing 50% inhibition of the enzyme activity was 2.5 microM. Inhibition by Zn2+ was not restored by increasing concentrations of Mg2+ when the concentration of Zn2+ was above 10 microM. Ba2+ was without effect. When sphingomyelinase was incubated with unsealed ghosts of bovine erythrocytes at 37 degrees C, the enzyme was significantly adsorbed onto the membrane in the presence of Mn2+, Co2+, Sr2+ or Ba2+. Incubation with intact or Pronase-treated erythrocytes caused enzyme adsorption only in the presence of Mn2+. In the course of incubation, the enzyme was first adsorbed on the membranes of intact bovine erythrocytes in the presence of Mn2+; then sphingomyelin breakdown proceeded with ensuing desorption of adsorbed enzyme. Hot-cold hemolysis occurred in parallel with sphingomyelin breakdown. In this case, the hydrolysis of membranous sphingomyelin as well as the initial enzyme adsorption took place in the following order: unsealed ghosts greater than Pronase-treated erythrocytes greater than intact erythrocytes.     

Involvement of dolichol phosphates as intermediates in the mannosyl and galactosyl transferases of rat testicular germ cell Golgi apparatus membranes

Isolated Golgi apparatus membranes from the germinal elements (spermatocytes and early spermatids) of rat testis were examined for their ability to incorporate [14C]mannose and [14C]galactose into glycolipid and glycoprotein fractions. Transfer of mannose from GDP-[14C]mannose into a Lipid I fractions (GPD:MPP mannosyl transferase activity), identified as mannosyl phosphoryl dolichol, showed optimal activity at 1.5 mM manganese and at pH 7.5. Low concentrations of Triton X-100 (0.1%) stimulated transferase activity in the presence of exogenous dolichol phosphate (Dol-P); however, inhibition occurred at Triton X-100 concentrations greater than 0.1%. Maximal activity of this GDP:MPP mannosyl transferase occurred at 25 microM Dol-P. Activity using endogenous acceptor was 2.34 pmole/min/mg, whereas in the presence of 25 microM Dol-P the specific activity was 284 pmole/min/mg, a stimulation of 125-fold. Incorporation of mannose into a Lipid II (oligosaccharide pyrophosphoryl dolichol) and a glycoprotein fraction was also examined. In the absence of exogenous Dol-P, rapid incorporation into Lipid I occurred with a subsequent rise in Lipid II and glycoprotein fractions suggesting precursor-product relationships. Addition of exogenous Dol-P to galactosyl transferase assays showed only a minor stimulation, less than twofold, in all fractions. Over the concentration range of 9.4 to 62.5 micrograms/ml Dol-P, only 1% of radioactive product accumulated in the combined lipid fractions. These observations suggest that the mannose transfer involves Dol-P intermediates and also that spermatocyte Golgi membranes may be involved in formation of the oligosaccharide core as well as in terminal glycosylations.     

Enzymatic synthesis of polyprenol monophosphate mannose in insects.

The microsomal fraction of insects was found to contain an enzyme which transfers mannose from guanosine diphosphate mannose to an endogenous or exogenous insect lipid and to other acceptors such as dolichol monophosphate or ficaprenol monophosphate. This activity depended on the presence of Triton X-100 and magnesium ions, the optimal concentration of the latter being 10mM. The optimal temperature of the reaction was 25 degrees C and the maximal activity was obtained at pH 7.9. The mannolipid formed behaved as a monophosphodiester when chromatographed on DEAE-cellulose. Weak acid treatment of the product liberated mannose. Its behaviour both on thin layer and Sephadex G-150 chromatography would indicate the presence of a number of isoprenyl units similar to the dolichol and different from the ficaprenol derivative. Stability to phenol treatment indicated that the lipid fraction of the mannolipid is an alpha-saturated polyprenol phosphate similar to dolichol monophosphate.     

Formation of α-1,2- and α-1,3-linked mannose disaccharides from dolichyl mannosyl phosphate by rat liver membrane enzymes

Dolichyl mannosyl phosphate and GDPmannose were active substrates for the transfer of mannose to methyl-α-d-mannose, p-nitrophenyl-α-d-mannose, and free mannose with rat liver microsomal membranes. The products formed during dolichyl mannosyl phosphate incubation with methyl-α-d-mannose or with mannose were α-linked. The dissaccharides formed by incubation of dolichyl mannosyl phosphate or GDPmannose with mannose were identified by paper chromatography and electrophoresis as mannose-α-1,2-mannose and mannose-α-1,3-mannose. Synthesis of each product was dependent on the assay conditions used and was most markedly affected by the presence of detergent. Transfer of mannose from either substrate to form mannose-α-1,3-mannose was severely inhibited by Triton X-100.     

Error in assay due to time dependency of carbon monoxide difference spectrum of reduced yeast cytochrome P-450: Slow reduction caused by Triton X-100 present

Triton X-100, added to yeast Saccharomyces cerevisiae for the purpose of stabilization or solubilization affects the carbon monoxide difference spectrum of reduced cytochrome P-450 and consequently the measurement of cytochrome P-450. Eight minutes is needed for 450-nm peak to reach its maximum height. Triton X-100 is shown to behave as a Type II substrate (absorption maximum at 418 nm and minimum at 390 nm) and to modulate the spin state of cytochrome P-450 from high to low form. Low-spin yeast cytochrome P-450 is reduced more slowly than the high-spin form.     

Subunit interactions of vesicular stomatitis virus envelope glycoprotein influenced by detergent micelles and lipid bilayers.

The envelope glycoprotein (G protein) of vesicular stomatitis virus is a transmembrane protein that exists as a trimer of identical subunits in the virus envelope. We have examined the effect of modifying the environment surrounding the membrane-spanning sequence on the association of G protein subunits using resonance energy transfer. G protein subunits were labeled with either fluorescein isothiocyanate or rhodamine isothiocyanate. When the labeled G proteins were mixed in the presence of the detergent octyl glucoside, mixed trimers containing both fluorescent labels were formed as a result of subunit exchange, as shown by resonance energy transfer between the two labels. In contrast when fluorescein- and rhodamine-labeled G proteins were mixed in the presence of Triton X-100, no resonance energy transfer was observed, indicating that subunit exchange did not occur in Triton X-100 micelles. However, if labeled G proteins were first mixed in the presence of octyl glucoside, energy transfer persisted after dilution with buffer containing Triton X-100. This result indicates that the G protein subunits remained associated in Triton X-100 micelles and that the failure to undergo subunit exchange was due to lack of dissociation of G protein subunits. Chemical cross-linking experiments confirmed that G protein was trimeric in the presence of Triton X-100. The efficiency of resonance energy transfer between labeled G protein was higher when G proteins were incorporated into dimyristoylphosphatidylcholine liposomes compared to detergent micelles. This result indicates that the labels exist in a more favorable environment for energy transfer in membranes than in detergent micelles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic and saturation magnetization properties of the manganese- and cobalt-substituted Fur (ferric uptake regulation) protein from Escherichia coli.

The Fur apoprotein has been purified and reconstituted with Co2+ and Mn2+ ions. These samples have been analyzed by UV-visible, EPR, and 1H NMR spectroscopies, by XAS, and by magnetization measurements. The apo-Fur protein is able to bind one metal dication (Co2+ or Mn2+) per monomer. A saturation magnetization study confirms the presence of a high-spin metal dication [Mn(II) S = 5/2 and Co(II) S = 3/2]. The two metal ions per Fur dimer are not in magnetic interaction (|J| < 0.1 cm-1 ). The UV-visible spectrum of the cobalt-substituted form (Co-Fur) presents two main bands at 660 nm and 540(br) nm with epsilon540 nm = 65 M-1 cm-1. The EPR spectrum gives the following g values: gx = 5.0(5), gy = 4.0(2), and gz = 2. 3(1), which are in accordance with a nearly axial (E/D < 0.11) site. The value of 55 cm-1 for the splitting (Delta) between the ground and the first excited state has been derived from an EPR saturation study and is in agreement with magnetization data. The EXAFS data of Co-Fur indicate a metal environment comprising five nitrogen/oxygen atoms at 2.11 A, the absence of sulfur, and the presence of histidines as ligands. 1H NMR of Co-Fur in H2O and D2O shows at least two exchangeable signals coming from histidine NH protons and shows the signature of carboxylate group(s). The combined spectroscopic data allow us to propose that the main metal site of Fur in Co-Fur contains at least two histidines, at least one aspartate or glutamate, and no cysteine as ligands and is in an axially distorted octahedral environment.