Roy W. Keenan

The preparation of tritiated betulaprenols and dolichols

Abstract A rapid and simple procedure is described for the preparation of tritium-labeled polyprenols. The isoprenoid alcohols are oxidized by chromium trioxide-pyridine complex in dichloromethane and the resulting aldehydes are isolated and reduced with 3 H-labeled sodium borohydride. Radioactive betulaprenols and dolichols have been obtained in high yield and purity by using this procedure.

The binding of [3H] dolichol by plasma high density lipoproteins

Radioactive dolichol was rapidly taken up by a plasma protein fraction following the intravenous injection of detergent suspended [3H]dolichol into rats. The half-life of labeled dolichol in the plasma was found to be approx. 13 h. Density gradient centrifugation or gel filtration of samples of human or rat plasma incubated with [3H]dolichol suspensions also showed that the labeled material became associated with the same plasma protein fractions as in the in vivo studies. Evidence was obtained to indicate that dolichol is specifically associated with the high density lipoprotein fraction of plasma. The possible role of high density lipoproteins as a vehicle for dolichol transport and the problems involved in the binding of this long moleculare are discussed.

The tissue and subcellular distribution of [3H]dolichol in the rat.

Abstract Following the intravenous injection of nanomolar amounts of [ 3 H]dolichol into rats, the radioactivity rapidly appeared in the high-density lipoprotein fraction of the plasma and circulated with a half-life of about 9 h. A fraction of the injected activity was excreted in the feces, presumably through the bile, but evidence was obtained that little oxidative breakdown of dolichol occurred. All tissues assayed acquired radioactivity, but the liver attained the highest specific activity and the largest percentage of the total radioactive dolichol. Subcellular fractionation of the liver revealed that mitochondrial preparations contained the bulk of the labeled dolichol at all times tested up to 40 h after injection. Disruption of the mitochondrial structure by two different techniques permitted the isolation of inner and outer membrane fractions and it was found that the [ 3 H]dolichol was concentrated in the outer membrane fraction. The significance of these findings is discussed.

Tubulin evolution: Two major types of α-tubulin

SummaryTubulin subunits have been isolated from a variety of protists and marine invertebrates. The sources were: sperm tails of a tunicate (Ciona intestinalis), an abalone (Haliotis rufescens) and a sea anemone (Tealia crassicornis), the gill cilia of a clam (Mercenaria mercenaria), the cilia of a ciliate (Tetrahymena pyriformis) and the cytoplasm of a slime mold (Physarum polycephalum). All the β-tubulins, as characterised by their electropherograms after limited proteolytic cleavage withStaphylococcus aureus protease, were fairly similar. In contrast, two markedly different peptide patterns were found for the α-tubulins of (a) metazoan axonemes and (b) protistan axonemes, plant axonemes and slime mold cytoplasm.Metazoan axonemal α-tubulin peptide patterns could be further divided into two similar but distinct subtypes which did not correlate with the taxonomic divisions of deuterostomia and protostomia, or to different tubulins within an axoneme, or to different tubulins of flagella and cilia. We have postulated that these small differences may be accounted for by a simple glutamicaspartic acid exchange at a particular position in the α-tubulin sequence. Identical peptide patterns were observed for sea urchin and sea anemone sperm tail tubulins, proving that the metazoan type of axonemal tubulin arose before the divergence of bilateral and radial symmetric organisms.The close similarity of the slime mold cytoplasmic α-tubulin peptide pattern to protistan and plant axonemal α-tubulin patterns suggests that the same type of tubulin might be used to form both axonemal and cytoplasmic types of microtubules in protists and plants. The large structural constraints imposed upon this tubulin molecule probably allowed very little change in its primary structure, thus explaining the similarity of tubulins from organisms which diverged at such an early time in eukaryote history. Duplication and modification of the tubulin gene may then have led to the development of specific axonemal and cytoplasmic microtubules during the evolution of the metazoa.

Inositol phospholipid levels of rat forebrain obtained by freeze-blowing method

Four different techniques of handling rat brain prior to lipid extraction and assay were tested to investigate the levels of inositol phospholipids in the brain. In these four techniques, the rat forebrains were either (1) freeze-blown followed by being preserved in liquid N2, (2) subjected to microwave irradiation prior to decapitation, (3) removed and frozen in liquid N2, or (4) removed at room temperature and subjected to lipid extraction as rapidly as possible. There was little change in phosphatidylinositol levels under any of these conditions; however, higher levels of phosphatidylinositol 4-phosphate were observed in freeze-blown and microwave-irradiated samples compared to the other samples. Even more striking differences were seen in phosphatidylinositol 4,5-bisphosphate fractions. The highest level of this lipid, 763 +/- 39 nmol/g tissue, which was obtained from freeze-blown samples, was more than 2-fold higher than that of the lowest values which were obtained by extraction without prior inactivation. These results indicate that the values of phosphatidylinositol 4,5-bisphosphate in brain in situ are higher than those generally reported, and that the freeze-blowing method has an advantage for further investigation of inositol phospholipid metabolism in brain due to the rapid breakdown of these compounds.

A dolichyl phosphate-cleaving acid phosphatase from Tetrahymena pyriformis.

Tetrahymena pyriformis contains an enzyme which hydrolyzed dolichyl phosphate. This activity was solubilized from lyophilized samples of this organism and was relatively stable when stored frozen. The soluble enzyme preparation had an acid pH optimum and hydrolyzed both dolichyl and phytanyl phosphates at equivalent rates. The polyprenylphosphate phosphatase activity was compared with the acid phosphatases which hydrolyzed p-nitrophenyl phosphate and marked differences were found. Dolichyl phosphate hydrolysis required Mg2+ for maximum activity while the bulk of the phosphatase activity was not effected by the absence of this ion. Other differences were that the polyprenylphosphate phosphatase was relatively insensitive to inhibitors such as tartrate and vanadium oxide sulfate which had a pronounced effect on the rate of p-nitrophenyl phosphate hydrolysis. The two activities also appeared to have different subcellular distributions. The polyprenylphosphate phosphatase was markedly inhibited by ethoxy formic anhydride, a reagent which is active against enzymes containing a histidine residue at their active site, while p-nitrophenyl phosphate hydrolysis was unaffected. The polyprenylphosphate phosphatase may be important in regulating the level of dolichyl phosphate in T. pyriformis and thus the rate of glycoprotein synthesis. It is also a useful tool which is capable of liberating dolichol from dolichyl phosphate under mild conditions which will permit the further characterization of the polyprenols.

The role of glucolipid in the biosynthesis of glycoprotein in Tetrahymena pyriformis

Abstract Cell-free preparations from Tetrahymena pyriformis catalyze the incorporation of glucose from UDP-glucose into a glucolipid having properties which are identical to those of other dolichyl phosphoryl sugar derivatives. Kinetic and other experiments have provided evidence that this glucolipid serves as glucose donor for two other types of glucosylated substances, one of which has been tentatively identified as an oligosaccharide lipid and the other a glycoprotein or glycoproteins. In addition, the partially purified glucolipid served as a glucosyl donor to these cell components, suggesting that in this protozoan, at least part of the glycoprotein is synthesized by reactions involving lipid-linked sugars in a manner analogous to that which has been observed in glycoprotein synthesis in mammalian cells.

A rapid and simple microprocedure for myelin isolation.

In order to conduct enzymatic studies on myelin from discrete regions of brain or spinal cord, we required a method capable of yielding purified myelin from tissue samples weighing 50 mg or less. Microprocedures that have been reported such as that of Detering and Wells (1976) or Fagg et al. (1978) were not suitable for rapid and routine myelin preparations. In this communication, the results of experiments in which we were able to isolate myelin from as little as 5 mg of whole brain are reported. Data are presented to show that the myelin isolated by this procedure is comparable to the myelin isolated by the macro‐scale procedure of Norton and Poduslo (1973a).

Studies on the biosynthesis of glucolipid in Tetrahymena pyriformis

Abstract 1. 1. Cell-free preparations from the protozoan Tetrahymena pyriformis rapidly convert glucose from UDP[14C]glucose into a chloroform-soluble form. 2. 2. The reaction has been shown to require UDPglucose and Mg2+ in addition to the enzyme and is reversed in the presence of an excess of U DP but not UMP or other nucleoside diphosphates. 3. 3. Only one radioactive glucolipid has been shown to be formed under our conditions. This compound is stable to mild alkaline hydrolysis, but dilute-acid hydrolysis rapidly releases free glucose. 4. 4. Sufficient material has not been obtained to permit the characterization of the lipid moiety, but the chemical and physical properties provide indirect evidence that the compound is probably a glucosyl-phosphoryl polyprenol of the type which are active as intermediates in glycosylation reactions.

The hydrolysis of dolichyl palmitate by intestinal mucosa

A dolichyl palmitate esterase was found in cell-free extracts of both pancreas and intestinal mucosa. The substrate for the reaction was dolichyl palmitate that was synthesized with labeled fatty acid. The reaction was monitored by the liberation of the free fatty acid and HPLC. All polyprenol esters studied were hydrolyzed despite differences in chain length. The role of this enzyme might be to promote the absorption of dolichol from the diet.