Brain Dolichol Research Articles

Brain and Liver Dolichol in Chronic Alcoholism: A Necropsy Study

Cerebral gray and white matter and liver dolichol levels were measured in postmortem samples from chronic alcoholics and nonalcoholic controls following recent suggestions that dolichol levels may be used as a marker for alcoholism. No significant differences in brain dolichol were found between the control and alcoholic groups. A significant reduction in the liver dolichol was observed in the alcoholic group. This was most marked in those alcoholics with liver disease.

Separation of Brain Dolichol Kinase from Endogenous Activating Factors: Evidence That Phospholipid Enhances the Interaction Between Enzyme and Dolichol

Porcine brain dolichol kinase activity is effectively solubilized by extracting salt-washed microsomes with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). When the detergent-solubilized activity is chromatographed on Sepharose CL-6B, a low amount of dolichol kinase activity is recovered in the void volume, and a dolichol kinase activator (DKA) is eluted (Ve/Vo = 1.9-2.2) with the bulk of the membrane phospholipids. Although only approximately 20% of the activity applied to the Sepharose CL-6B column is detected in the column fractions, virtually all of the original activity is restored when the Vo fraction is recombined with DKA. Endogenous DKA, isolated from brain microsomes, is heat-stable, is extractable with CHCl3/CH3OH (2:1), and has the chemical and chromatographic properties of phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Moreover, approximately 50% of the stimulatory activity is lost when the PC present in the DKA fraction is degraded by purified phospholipase C from Clostridium perfringens. Also consistent with a phospholipid co-factor requirement, the dolichol kinase activity recovered in the partially phospholipid-depleted fraction (Vo) is markedly stimulated by various molecular species of exogenous purified PC or PE, but not by phosphatidylinositol, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, or sphingomyelin. A comparison of defined molecular species shows that PCs containing oleoyl or linoleoyl groups in the 1 and 2 positions are the most stimulatory, suggesting that the fatty acyl moieties are involved in the enzyme-phospholipid interaction. Kinetic analyses indicate that PC enhances the interaction between dolichol kinase and dolichol, the lipophilic substrate, but does not alter the apparent Km for CTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Dolichyl phosphate: rapid increase and predominant form of brain dolichol compounds during early brain development.

Of brain dolichol compounds, dolichyl phosphate is most critical since it serves as the carrier of saccharide moieties to be utilized for N-linked protein glycosylation. The objectives of the current study were to determine (1) whether the proportion of dolichyl phosphate to dolichol in brain is relatively large and/or changes with development, and (2) whether the subcellular distribution of these isoprenoid compounds differs and/or changes with development. A recently described, improved technique for isolation and quantitation of dolichyl phosphate was employed. The data show that in the first 4 weeks of postnatal life in the rat dolichyl phosphate is, in fact, the predominant form of total dolichol compounds in brain. Subsequently, concentrations of dolichol exceeded those of dolichyl phosphate in adult brain, particularly with aging. Dolichyl phosphate was found to be localized primarily in microsomes; dolichol was found in only small amounts in microsomes but was enriched in the fraction that contained lysosomes. The observations indicate that during early brain development, an active time period for differentiating events, many of which are mediated by glycoproteins, dolichyl phosphate is present in high concentration at the subcellular site of N-linked glycoprotein synthesis, i.e., endoplasmic reticulum.

Dolichol-linked glycoprotein synthesis in developing mammalian brain: maturational changes of the N-acetylglucosaminylphosphotransferase

The enzyme UDP- N-acetylglucosamine:dolichyl phosphate, N-acetylglucosamine-1-phosphate transferase (GlcNAc-1-P transferase), the first committed step in the dolichol-linked oligosaccharide pathway for glycoprotein biosynthesis, has been studied in developing rat brain. The enzyme was shown to be localized in microsomes, particularly heavy microsomes, and to be activated by Mg 2+ and inhibited by tunicamycin. Study of the enzyme with brain development demostrated two prominent findings. First, the accentuation of enzymatic activity caused by addition of a saturating concentration of dolichyl phosphate was greater in brain of older (3–4 weeks of age and subsequently) animals (25-fold) than in brain of younger (less than two weeks of age) animals (10-fold). This difference suggests that dolichyl phosphate may be limiting for GlcNAc-1-P transferase activity in endoplasmic reticulum of the older animals. Second, a marked (3.5-fold) increase in activity occurred over a discrete time period (3–4 weeks of postnatal life) during brain development. That this increase reflected an increase in enzyme amount rather than in catalytic efficiency was suggested by kinetic studies. Coupled with our previous demonstrations of increases in brain dolichol, dolichol kinase activity, and dolichyl phosphate levels during approximately the same developmental period (Sakakihara, Y. and Volpe, J.J., Dev. Brain Res., 14 (1984) 225–262; Volpe, J.J. et al., Dev. Brain Res., in press), the data suggest a temporally discrete period of activation of the dolichol-linked pathway to glycoproteins. Whether the pathway is regulated coordinately or sequentially is a fertile topic for future study.

Zn2+, not Ca2+, is the most effective cation for activation of dolichol kinase of mammalian brain.

The cation specificity of dolichol kinase of mammalian brain and the potential involvement of a Ca2+-calmodulin system in regulation of this enzyme have been studied. Among 10 divalent cations examined, Zn2+ was found to be most effective for the activation of dolichol kinase of rat and calf brain and cultured C-6 glial cells. The activations with Ca2+, Co2+, and Mg2+ were 53%, 32%, and 18% of the full activation with Zn2+, respectively. No combinations of the cations could activate the enzyme as much as Zn2+ alone. A role for a Ca2+-calmodulin system in the regulation of brain dolichol kinase was not supported by our data. First, the concentration of free Ca2+ required for the maximum activation of dolichol kinase was two to three orders of magnitude greater than the concentration required by typical calmodulin-dependent enzymes. Second, neither the depletion of calmodulin from the microsomal fraction nor the addition of exogenous calmodulin caused an alteration in the activation of dolichol kinase by Ca2+ (or Zn2+). Third, antagonists of calmodulin failed to suppress the activation of the enzyme by Ca2+ (or Zn2+). The data raise the possibility that Zn2+ is involved in the regulation of dolichol kinase in brain.

Dolichol deposition in developing mammalian brain: Content of free and fatty-acylated dolichol and proportion of specific isoprenologues

Dolichol serves a critical role in N-linked glycoprotein synthesis as the carrier of saccharide moieties. In this study the deposition of dolichol was investigated in developing rat brain. The contents of both the free and fatty-acylated forms of dolichol as well as the specific isoprenologues of dolichol were determined. Two distinctive phases of rapid dolichol deposition were observed; the first during brain development, and the second, with aging. The predominant form of brain dolichol was shown to be the free derivative at all ages; however, during the two phases of rapid deposition of total dolichol, fatty-acylated dolichol became detectable and increased transiently. The rapid rate of increase of brain dolichol during development occurred later than that of brain cholesterol, which shares a common biosynthetic pathway with dolichol. Thus, the peak rate of increase of dolichol occurred at 25 days postnatal, and of cholesterol, at 18 days postnatal. A distinct ontogenetic alteration in the proportion of the individual molecular species (isoprenologues) of dolichol also was defined. These observations suggest that dolichol is particularly involved in developmental events of the third to sixth postnatal weeks in rat brain, e.g. myelination and synaptogenesis.

Age-associated increase of free dolichol levels in mice

Free dolichol levels were determined by high-performance liquid chromatography in liver, brain, heart, lung, testis and kidney of C57 B1/6 mice at 3, 6, 12 and 30 months of age. In all the tissues, free dolichol levels were increased with aging. The largest increase in dolichol was in testis and the smallest increase was in the liver. Among the various tissues, aging testis and kidney showed high levels of free dolichol, and represented more than twice the level of dolichol in brain. The major dolichols among the various tissues were of the 17, 18, 19 and 20 isoprene unit containing types and the proportions were unaffected by aging. Tissues such as brain and testis showed a tendency toward high proportions of longer-chain dolichols, whereas lung and heart showed high proportions of shorter-chain dolichols. This study shows that the age-associated increase in dolichol levels is a general phenomenon and the increases vary widely with tissues.

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.