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.     

Enzymatic synthesis of polyprenol monophosphate mannose in insects

Summary 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 °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±-saturated polyprenol phosphate similar to dolichol monophosphate.Abbreviations DoIMP dolichol monophosphate - FMP ficaprenol monophosphate - IGAL insect glycosyl acceptor lipid Dedicated to ProfessorLuis F. Leloir on the occasion of his 70th birthday.     

The transfer of mannose from guanosine diphosphate mannose to dolichol phosphate and protein by pig liver endoplasmic reticulum

When pig liver microsomal preparations were incubated with GDP-[¹⁴C]mannose, 10–40% of the ¹⁴C was transferred to mannolipid and 1–3% to mannoprotein. The transfer to mannolipid was readily reversible and GDP was one of the products of the reaction. It was possible to reverse the reaction by adding excess of GDP and to show the incorporation of [¹⁴C]GDP into GDP-mannose. When excess of unlabelled GDP-mannose was added to a partially completed incubation there was a rapid transfer back of [¹⁴C]mannose from the mannolipid to GDP-mannose. The other product of the reaction, the mannolipid, had the properties of a prenol phosphate mannose. This was illustrated by its lability to dilute acid but stability to dilute alkali, and by its chromatographic properties. Dolichol phosphate stimulated the incorporation of [¹⁴C]mannose into both mannolipid and into protein, although the former effect was larger and more consistent than the latter. The incorporation of exogenous [³H]dolichol phosphate into the mannolipid, and its release, accompanied by mannose, on treatment of the mannolipid with dilute acid, confirmed that exogenous dolichol phosphate can act as an acceptor of mannose in this system. It was shown that other exogenous polyprenol phosphates (but not farnesol phosphate or cetyl phosphate) can substitute for dolichol phosphate in this respect but that they are much less efficient than dolichol phosphate in stimulating the transfer of mannose to protein. Since pig liver contained substances with the chromatographic properties of both dolichol phosphate and dolichol phosphate mannose, which caused an increase in transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to mannolipid, it was concluded that endogenous dolichol phosphate acts as an acceptor of mannose in the microsomal preparation. The results indicate that the mannolipid is an intermediate in the transfer of mannose from GDP-mannose to protein. Some 4% of the mannose of a sample of mannolipid added to an incubation was transferred to protein. A scheme is proposed to explain the variations with time in the production of radioactive mannolipid, mannoprotein, mannose 1-phosphate and mannose from GDP-[¹⁴C]mannose that takes account of the above observations. ATP, ADP, UTP, GDP, ADP-glucose and UDP-glucose markedly inhibited the transfer of mannose to the mannolipid.     

A mannosyl-carrier lipid of bovine adrenal meddulla and rat parotid.

1. The transfer of mannose from GDP-(U-14-C)mannose into endogenous acceptors of bovine adrenal medullla and rat parotid was studied. The rapidly labelled product, a glycolipid, was partially purified and characterized. 2. It was stable to mild alkaline hydrolysis but yielded (14-C)mannose on mild acid hydrolysis. It co-chromatographed with mannosyl phosphoryl dolichol in four t.l.c. systems and on DEAE-cellulose acetate. Addition of dolichol phosphate or a dolichol phosphate-enriched fraction prepared from pig liver stimulated mannolipid synthesis. 3. The formation of mammolipid appeared reversible, since addition of GDP to a system synthesizing the mannolipid caused a rapid loss of label from the mannolipid. UDP-N-acetylglucosamine did not inhibit mannolipid synthesis except at high concentrations (2 mM), even though in the absence of GDP-mannose, N-acetylglucosamine was incorporated into a lipid having the properties of a glycosylated polyprenyl phosphate. 4. Mannose from GDP-mannose was also incorporated into two other acceptors, (2y being insoluble in chloroform-methanol (2:1, v/v) but soluble in choloroform-methanol-water (10:10:3, by vol.) and (ii) protein. These are formed much more slowly than the mannolipid. 5. Exogenous mannolipid served as a mannose donor for acceptors (i) and (ii), and it is suggested that transfer of mannose from GDP-mannose to mannosylated protein occurs via two intermediates, the mannolipid and acceptor (i).     

Rapid Brain Uptake of Manganese(II) Across the Blood-Brain Barrier

Abstract: ⁵⁴Mn²⁺ uptake into brain and choroid plexus from the circulation was studied using the in situ rat brain perfusion technique. Initial uptake from blood was linear with time (30 s to 6 min) and extrapolated to zero with an average transfer coefficient of ∼6 × 10^-5 ml/s/g for brain and ∼7 × 10^-3 ml/s/g for choroid plexus. Influx from physiologic saline was three- to fourfold more rapid and exceeded that predicted for passive diffusion by more than one order of magnitude. The lower uptake rate from blood could be explained by plasma protein binding as the free fraction of ⁵⁴Mn²⁺ in rat plasma was ≤30%. Purified albumin, transferrin, and α₂-macroglobulin were each found to bind ⁵⁴Mn²⁺ significantly and to restrict brain ⁵⁴Mn²⁺ influx. The results demonstrate that ⁵⁴Mn²⁺ is readily taken up into the CNS, most likely as the free ion, and that transport is critically affected by plasma protein binding. The results support the hypothesis that Mn²⁺ transport across the blood-brain barrier is facilitated by either an active or a passive mechanism.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of [¹⁴C]-labeIled material into subcellular fractions of 15-day-old rat brain was studied as a function of time after intracerebral injection of [2-¹⁴C]mevalonic acid. As previously shown for adult brain, the data indicated the microsomal fraction to be the site of sterol biosynthesis. The synaptosomal fraction exhibited a marked early uptake of [¹⁴C]-nonsaponifiable material. Total radioactivity in both myelin and myelin-like fractions remained low in comparison to that in the other subcellular fractions at all time periods examined. At 2 h after injection, labelled digitonin-precipitable material was demonstrable in all subcellular fractions. Examination of the [¹⁴C]-labelled nonsaponifiable material by thin-layer chromatography indicated the rapid appearance of labelled 4-desmethyl sterol in all subcellular fractions, with the most rapid appearance in the myelin fraction, followed in decreasing order by microsomal, synaptosomal, and mitochondrial fractions. Examination of [¹⁴C] digitonin-precipitable material from each fraction by the dibromide method demonstrated that although 4-desmethyl sterol appeared quickly, the formation of cholesterol was slow in all fractions, an effect that had been reported earlier for adult brain.     

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.     

AMOUNTS AND TURNOVER RATES OF BRAIN PROTEINS IN MORPHINE-TOLERANT MICE

Abstract— Mice were injected intracerebrally with radioactive leucine 30 min before decapitation. A double-label technique was used; e.g. [³H]leucine in control, untreated mice and [¹⁴C]leucine in morphine-tolerant (dependent) mice. Proteins were extracted sequentially from mouse brain with aqueous buffer, Triton X-100, and sodium lauryl sulphate. Each extract was subjected to electrophoresis on discontinuous, porosity-gradient acrylamide gels. When the protein patterns from untreated, acutely morphinized mice were compared with those from morphine-tolerant (dependent) mice, we observed no differences in the amounts of protein or of radioactivity in any of the 55 discrete bands.     

Role of mannosyl phosphoryl polyisoprenol in biosynthesis of mammary glycoproteins

When a membrane preparation from the lactating bovine mammary gland is incubated with GDP-[¹⁴C] mannose, mannose is incorporated into a [¹⁴C] mannolipid, a [Man-¹⁴C] oligosaccharide-lipid, and metabolically stable endogenous acceptor(s). The rate of mannosyl incorporation is the fastest into [¹⁴C] mannolipid, intermediate in [Man-¹⁴C] oligosaccharide-lipid, and least into [Man-¹⁴C] endogenous acceptor(s). The [¹⁴C] mannolipid has been partially purified and characterized. Mild acid hydrolysis of this compound gives [¹⁴C] mannose, whereas alkaline hydrolysis yielded [¹⁴C] mannose phosphate as the labeled product. The t_½ of hydrolysis of the mannolipid under the acidic and basic conditions are comparable to values obtained for mannosyl phosphoryl dolichol in other systems. The mannolipid is chromatographically indistinguishable from calf brain mannosyl phosphoryl polyisoprenol and chemically synthesized β-mannosyl phosphoryl dolichol. Exogenous dolichol phosphate stimulates the synthesis of mannolipid in mammary particulate preparations 8.5-fold. Synthesis of mannolipid is freely reversible; in the presence of GDP, the transfer of mannosyl moiety from endogenously labeled mannolipid to GDP-mannose is obtained. All of these results indicate that the structure of mannolipid is mannosyl phosphoryl polyisoprenol. Even though the precise chain length of the polyisoprenol portion has not been established, it is tentatively suggested to be dolichol. Partially purified [¹⁴C] mannolipid can directly serve as a mannosyl donor in the synthesis of [Man-¹⁴C] oligosaccharide-lipid and [Man-¹⁴C] endogenous acceptor(s). Pulse and chase kinetics utilizing GDP-mannose to chase the mannosyl transfer from GDP-[¹⁴C] mannose in the mammary membrane incubations caused an immediate and rapid turnover of [¹⁴C] mannose from [¹⁴C] mannolipid while the incorporation of label in [Man-¹⁴C] oligosaccharide-lipid and radioactive endogenous acceptor(s) continued for a short period before coming to a halt. Both gel filtration and electrophoresis indicate that the endogenous acceptor(s) are a mixture of 2 or more glycoproteins since incubation with proteases releases all of the radioactivity into water soluble low-molecular-weight components, perhaps glycopeptides. All of the above evidence is consistent with the following precursor-product relationship: GDP-mannose ? mannosyl phosphoryl polyisoprenol → mannosyl-oligosaccharide-lipid → mannosyl-proteins. The exact structure of the oligosaccharide-lipid and the endogenous glycoproteins is unknown.     

Enzymatic transfer of mannose from guanosine diphosphate mannose to dolichol phosphate in yeast (Hansenula holstii) A possible step in mannan synthesis

A particulate enzyme fraction isolated from yeast (Hansenula holstii) catalyzes the transfer of mannose from GDPmannose to endogenous lipid acceptors. Kinetic studies are presented which suggest that one of the mannolipids is a precursor to cell wall mannan. The solubility and chromatographic properties, the stability to mild alkali, and the release of mannose by mild acid hydrolysis are characteristic of polyisoprenyl phosphoryl mannose. Addition of dolichol phosphate to the enzyme system stimulates the synthesis of a mannolipid with properties similar to that synthesized from endogenous lipid. That the exogenous dolichol phosphate was acting as a mannosyl acceptor was demonstrated by showing that dolichol [³²P]phosphate was converted to dolichol [³²P]phosphate mannose.     

On the Role of Long-Chain Aldehydes in Mammalian Plasmalogen Biosynthesis

Abstract: [1-³H, 1-¹⁴C]Palmitaldehyde(³H:¹⁴C= 15) was injected intracerebrally to 18-day-old rats and incorporation of radioactivity into brain lipids was followed over a 24-h period. The substrate was metabolized primarily by oxidation to palmitic acid with loss of tritium and, to a lesser extent, by reduction to hexadecanol. The alkyl moieties of the ethanolamine phospholipids showed considerably lower ³H:¹⁴C ratios than the substrate, indicating a substantial participation in ether lipid synthesis by tritium-free alcohols derived from ¹⁴C-labeled fatty acids. Virtually no ³H radioactivity was found in alkenyl moieties, indicating stereospecificity of both reduction of aldehyde and dehydrogenation of alkyl to alkenyl glycerolipid. The data are consistent with the general concept that plasmalogen biosynthesis proceeds exclusively through fatty alcohols and alkyl glycerolipids and that fatty aldehydes cannot be utilized directly.     

Some Characteristics of Glutamic Acid Decarboxylase of Chick Ampullary Cristae

Abstract: In a previous study, it was demonstrated that enzyme-mediated γ-aminobutyric acid (GABA) synthesis occurs in the vestibule of the chick inner ear. As deeper knowledge of the properties of its synthesizing enzyme might contribute to the understanding of the role of GABA in inner ear function, some characteristics of glutamate decarboxylase (GAD) were studied in chick isolated ampullary cristae under conditions in which ¹⁴CO₂ release from [1-¹⁴C]glutamate and [¹⁴C]GABA formation from [U-¹⁴C]glutamate for estimating GAD activity were equal. It was found that K _m for glutamate is 5 m M and that the enzyme pH optimum is 7.3. These values fall within the range described for the corresponding enzyme in nervous tissue of other species. Pyridoxal phosphate (PLP) activates the enzyme and aminooxyacetic acid inhibits it, the same as these agents activate or inhibit GAD from several nervous tissue sources. 2-Mercaptoethanol shows some protection from inactivation of the PLP-de-pendent enzyme and Triton X-100 exerts some inhibition of vestibular GAD activity, as previously shown in other nervous tissue preparations. Although its cellular localization is at present uncertain, these results indicate that GAD of chick vestibular tissue possesses properties resembling those of the brain enzyme and might be controlled in a manner similar to that of GAD in brain, thus possibly participating in the regulation of inner ear function.     

Phosphorylation and Fucosylation of Myelin Protein In Vitro by Sciatic Nerve from Developing Rats

Abstract: Proteins of the paniculate fraction of sciatic nerve of rats ranging from 1 to 55 days of age were analyzed by polyacrylamide gel electrophoresis. The major myelin protein, P₀, could not be detected at 1 day of age, but by 10 days it comprised from 15 to 20% of the particulate protein, the same proportion as in adult rats. Growth of nerve continued throughout the period studied. Rat sciatic nerves were incubated with [³²P]orthophosphate or [³H]fucose. Particulate matter proteins from sciatic nerve (and in certain cases proteins of myelin purified from sciatic nerve) were separated by polyacrylamide disc gel electrophoresis and the distribution of protein and of radioactivity along the gels was determined. [³²P]Phosphate appeared to label all myelin proteins. Labeling with fucose was more specific; myelin basic proteins were not fucosylated. A developmental study showed that sciatic nerves from 2-day-old rats could incorporate radioactive fucose and [³²P]-phosphate into several proteins at the P₀ region of polyacrylamide gels. Specific radioactivity of [³H]fucose in P₀ protein was highest in preparations from 5-day-old rats and declined by 80% over the next 5 days as it was diluted by accumulating myelin. The specific radioactivity of incorporated [³²P] phosphate was high at the early age points and declined as a result of the accumulation of compact myelin. The results indicate an association of fucosylation and/or phosphorylation with some step in the formation of myelin.     

The effects of triton X-100 on the transfer of mannose, glucose and n-acetylglusomine phosphate to dolichol monophosphate by preparations of rough and smooth endoplasmic reticulum and of mitochondria of rat liver.

Triton X-100 and exogenous dolichol monophosphate have been used to investigate the nature of enzymes responsible for the transfer of mannose, glucose and N-acetylglucosamine phosphate from nucleotide donors to dolichol monophosphate in vesicles derived from rough and smooth endoplasmic reticulum and mitochondria. Mitochondria were shown to contain the highest specific activities of these enzymes. The responses of the glycosyltransferases to increasing concentrations of Triton X-100 and the effect on these responses of exogenous dolichol monophosphate suggest that the enzymes for mannose and glucose transfer are less hydrophobic, and therefore less intrinsic, in the membrane than the enzyme for N-acetylglucosamine phosphate transfer. In smooth vesicles the results are consistent with mannosyl- and glucosyl-transferases being located at both inner and outer faces of the membrane. In rough vesicles and in mitochondria mannosyl- and glucosyl-transferases were confirmed at the outer face. There is, however, only one site of N-acetylglucosamine phosphate transfer, this being more hydrophobically located in the membrane than the other sites of glycosyl transfer. Mitochondrial enzyme activity closely resembled that of rough endoplasmic reticulum in response to Triton X-100 and exogenous dolichol monophosphate, and is probably associated with the outer membrane.     

Glycoprotein Biosynthesis in Peripheral Nervous System Myelin: Effect of Tunicamycin

Abstract: The effect of an inhibitor of N -glycosylation of glycoproteins, tunicamycin, on synthesis of PNS myelin proteins was investigated in vitro by using chopped sciatic nerves or spinal roots of 21-day-old Wistar rats. Tunicamycin when incubated with these nerves in the presence of ³H-labeled fucose, mannose, or glucosamine inhibited the uptake of radioactivity into myelin proteins including some high-molecular-weight proteins, P₀, 23K protein, and 19K protein by amounts ranging from 42 to 79%. Uptake of ¹⁴Camino acid mixture was inhibited much less by tunicamycin, but a new radioactive protein peak appeared when the protein mixtures had been separated by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This protein ran directly in front of the P₀ peak, did not correspond to any bands stained by Fast green, and was not labeled by fucose. This peak appeared in increasing larger proportions with progressive time of incubation of nerves with ³H amino acids in the presence of tunicamycin. The new protein, which cross-reacts with P₀ antiserum, was tentatively identified as a nonglycosylated P₀ protein that appears to be almost as well incorporated as P₀ into the subcellular fraction containing myelin. At this time it is not possible to determine whether the unglycosylated P₀ is actually assembled into a site and configuration like that of P₀.     

Evidence for the stimulation of dolichol and mannolipid synthesis by glucocorticoids in HeLa cells.

The effects of addition of 1 microM-dexamethasone on the rate of transfer of mannose from GDP-mannose into mannolipid have been studied in HeLa cell cultures. Concurrent with an increase in incorporation of mannose into glycoproteins, the incorporation of mannose from GDP-mannose in vitro into mannolipid and dolichol-linked oligosaccharides was increased after dexamethasone treatment. Stimulation of mannolipid synthesis showed a correlation with the 11 beta, 17 alpha, 21-trihydroxy structure of C21 steroids. Dexamethasone treatment also resulted in an increased incorporation of acetate into dolichol and dolichyl phosphate. The results suggest that the effect of dexamethasone on the cell-surface glycoprotein accumulation is related to increased sugar-linked dolichol synthesis.     

RAPIDLY LABELED AND SECRETED PROTEINS OF THE CHICK BRAIN

Abstract— The extracellular and cerebrospinal fluids (ECF) of the chick brain were found to contain a distinctive group of rapidly labeled proteins. Gel staining patterns suggest that most ECF protein bands correspond with components also found in either the homogenized whole brain cytoplasmic fraction or the blood serum. The valine-incorporation profiles of these three fractions, however, were entirely distinctive. Comparisons were carried out using a sensitive double-labeling method, in which ECF proteins from chicks labeled for 1 h with [³H]valine were comigrated on SDS-polyacrylamide gels with the cytoplasmic or serum proteins from a ¹⁴C-labeled animal. Analyses of the ³H- and ¹⁴C-labeling profiles from these gels showed that certain newly-synthesized proteins are heavily enriched in the ECF relative to the other two fractions. Most prominently, material with an apparent molecular weight of # 17,000 was found to incorporate nearly one-third of all the radioactivity appearing in the ECF proteins, but was not heavily labeled in either the cytoplasmic or serum fractions. The effects of a simple training experience on the pattern of chicks' brain protein synthesis were also examined.     

A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum

Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [³H]CNQX and [³H]kainic acid binding on chick cerebellar membranes. [³H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [³H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [³H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate.     

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.     

The involvement of endogenous dolichol in the formation of lipid-linked precursors of glycoprotein in rat liver

Endogenous dolichol was shown to function as a natural acceptor of mannose residues by using regenerating rat liver containing [(3)H]dolichol. When subcellular fractions from this liver were incubated with GDP-[(14)C]mannose a double-labelled lipid, which represented 30% of the total [(14)C]mannolipid, could be isolated. This lipid was shown to be identical with the dolichol phosphate mannose formed from exogenous dolichol phosphate, by chromatography, stability to alkali and by chemical cleavage to mannose and dolichol derivatives. It was formed by the rough endoplasmic reticulum and mitochondria. If it is concerned in glycoprotein synthesis this would suggest that it functions in the formation of both secreted and mitochondrial glycoproteins. When both the dolichol and retinol of rat tissue were radioactive they made similar contributions to the synthesis of the lipid by liver microsomal fractions and intestinal epithelial cells.