Uptake of galacturonic acid in Erwinia chrysanthemi EC16.

Uptake of [14C]galacturonic acid in Erwinia chrysanthemi was found to be stimulated during growth on pectin and its degradation products, saturated digalacturonic acid and galacturonic acid. Cells isolated from macerated potato tissue also showed increased levels of uptake activity for this molecule compared with those showed by glycerol-grown cells. Uptake was found to be an active process, and it displayed saturation kinetics. An Escherichia coli galacturonic acid transport mutant harboring the E. chrysanthemi exuT gene(s) for galacturonic acid uptake was able to transport galacturonic acid but unable to take up the dimer [3H]digalacturonic acid.

Synthesis of [35S]thiophosphoryl adenylic acid, utilizing a general procedure for [35S]thiophosphoryl chloride production.

Elemental [35S]sulfur was shown to equilibrate with the sulfur of thiophosphoryl chloride when these materials are heated together. This isotopic exchange reaction is the basis of a convenient, microscale synthesis of high specific activity [35S]PSCl3. [35S]Thiophosphoryl chloride is otherwise not commercially available except through custom synthesis. The labeled thiophosphoryl chloride was used in a novel procedure for the preparation of [35S]adenosine 5'-phosphorothioate. This isotopic exchange method should find wide application in the synthesis of many radiolabeled thiophosphoryl esters which utilize PSCl3 as the source of the thiophosphoryl group.

Effects of fire ant venom alkaloids on platelet and neutrophil function.

About 95% of venom of the imported fire ant Solenopsis invicta is composed of dialkyl piperidines. These alkaloids produce a distinct pustule at the site of injection. The formation of this pustule may involve the activation of platelets and neutrophils. The purpose of this paper was to characterize the effects of fire ant venom alkaloids (FAVA) on certain physiological and biochemical functions of human platelets and neutrophils. In platelets, FAVA caused a rise in intracellular [Ca2+], secretion of dense granules as measured by ATP release, and aggregation as measured by light transmission through a suspension of platelets. Aggregation response was less complete with FAVA than with thrombin or PAF. However, secretion response was greater with FAVA than thrombin. One of our most significant findings was that pretreatment of platelets with subthreshold concentrations of FAVA produced enhanced PAF-induced increase in [Ca2+]cyt, suggesting that synergism between the two agonists might play an important role in the physiological response to FAVA. In neutrophils, FAVA produced a rise in intracellular [Ca2+] and aggregation, although the responses were more moderate than those observed in platelets. These results suggest that FAVA activation of platelets and neutrophils may occur in vivo as a response to stings by red fire ants.

Dolichol kinase in rat sarcoplasmic reticulum membrane preparations.

A dolichol kinase (EC 2.7.1.108) was found in sarcoplasmic reticulum membrane fractions from rat leg muscle. This enzyme specifically required CTP as a phosphoryl donor and relatively little activity was found in the absence of exogenous detergent-suspended dolichol. Unlike other reported dolichol kinases, the kinase from skeletal muscle was activated almost equally well by Ca2+, Zn2+, or Mg2+, but not Mn2+. No effect of calmodulin was seen. The kinase exhibited a single pH optimum at pH 7-8 in contrast to kinases from certain other tissues. Despite the low level of dolichol present in skeletal muscle, the kinase in the sarcoplasmic reticulum fraction had an activity comparable to that of microsomal preparations from tissues such as brain and liver, which may indicate that skeletal muscle has a high capacity for dolichol phosphorylation and protein glycosylation.

Disposition of homovanillic acid in the primate.

Prior studies have shown that homovanillic acid is the principal metabolite of dopamine in the primate central nervous system (CNS). In studies of primates given deuterated homovanillic acid systemically, however, only 50% of the administered amounts have been recovered in the urine over the next 4-48 hr. These findings have left it unclear whether there is a slowly turning-over compartment of homovanillic acid, conversion of homovanillic acid to another compound, or excretion of homovanillic acid from the body by a nonrenal route. We synthesized [3H]homovanillic acid and administered it intravenously to four rhesus monkeys. Over the subsequent 4 hr, 94.9 +/- 8.9% (SD) of the administered radioactivity was recovered in the urine, almost entirely as homovanillic acid. These results are consistent with the interpretation that, in primates, there is not a major body pool of homovanillic acid with slow turnover, nor is metabolism to other compounds significant, nor is there evidence for nonrenal excretion.

Preparation and characterization of radioactive castanospermine.

A procedure for the preparation of tritiated castanospermine is described. The tritiated alkaloid was shown to be chromatographically identical to the native material and exhibited the same inhibitory properties. Radiolabeled castanospermine tightly bound to purified intestinal sucrase. Following gel chromatography, each mole of enzyme was shown to have bound 1 mol of the radioactive alkaloid. Cultured MDCK cells were also shown to take up the labeled castanospermine. This compound should be a useful tool in the investigation of enzymes that are responsible for the processing of glycoprotein oligosaccharides.

The preparation of tritiated tunicamycin.

A relatively simple and inexpensive procedure was devised for the radiolabeling of the glycoprotein biosynthesis inhibitor, tunicamycin. The procedure is based on hydrogen exchange in alkaline solutions of tritiated water. It was noted that the antibiotic was much more alkali labile than model compounds such as uridine. The alkali stability of the inhibitor was studied to determine conditions for optimum labeling and yield. The effects of alkaline incubation on the inhibitory properties of the antibiotic were also investigated and it was found that the breakdown products are not effective inhibitors of the reaction that transfers N-acetylglucosamine-1-phosphate to dolichyl phosphate. The isolated radioactive tunicamycin homologs, however, retained all their inhibitory action. Incubation of tunicamycin in the presence of deuterated water and mass spectral analysis showed that under the conditions used for the tritiation of tunicamycin the major product exchanged six hydrogen atoms. The position of the tritium atoms in labeled tunicamycin was not determined. The radioactive label in these compounds was shown to be stable under physiological conditions and should be useful for investigations involving the action of these antibiotics.

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.

Residual complement activity after incubation with myelin isolated from both the central and peripheral nervous tissues.

The relative ability of isolated central and peripheral nervous system myelin to interact with the complement system of plasma proteins was studied. The myelin used was a highly pure form, devoid of contamination by any subcellular organelles or membranes. Residual complement activity was a linear function of increasing quantities of myelin from 10 to 40 micrograms of myelin protein. Central and peripheral nervous system myelin showed identical residual complement activity at various temperatures above 7 degrees C and also after various time periods of incubation. The results show that central and peripheral nervous system myelin show equal ability to interact with complement, in spite of their different origin and differences in morphology and protein composition.

Calmodulin-dependent enzymic phosphorylation of dolichol in Tetrahymena pyriformis.

The Protozoan, Tetrahymena pyriformis, is capable of phosphorylating dolichol in the presence of CTP. Other nucleotides (ATP, UTP and GTP) were ineffective. The enzyme was activated independently by the bivalent cations Mg2+, Mn2+ and Ca2+. The Ca2+ stimulation of the enzyme activity was calmodulin-dependent. A substantial increase in the enzyme activity was seen in the presence of UTP. The apparent Km values for CTP and dolichol, as calculated from the Lineweaver-Burk plot, were 3 mM and 70 microM respectively. Although the presence of detergent was essential, at higher concentrations there was a decrease in the enzyme activity. The enzyme had two pH optima (pH 7.4 and 9.0) and at both the activity was Ca2+-calmodulin-dependent.

The role of calmodulin in the regulation of dolichol kinase.

A calcium ion-requiring CTP-dependent kinase that phosphorylates dolichol was found in particulate enzyme preparations from the protozoa Tetrahymena pyriformis. This enzyme and an analogous enzyme present in rat brain microsomes were both shown to be inactivated following washing with EGTA-containing buffers. The activity could be restored by the addition of calcium and the calcium-binding protein calmodulin. In addition, both enzymes were strongly inhibited by trifluoperazine, chlorpromazine, and antiserum against brain calmodulin. These results are evidence that the dolichol kinase from these two sources is regulated by a system involving calmodulin. Dolichol kinase is the enzyme that is believed to be important in the maintenance of the cellular levels of dolichyl phosphate, the factor which is likely to exert the most control over the rate of glycoprotein biosynthesis. On the other hand, microsomal preparations from rat liver which were shown to contain a dolichol kinase that does not require Ca2+ for activity showed no inactivation by EGTA treatment, trifluoperazine, chlorpromazine, or preincubation with antiserum against calmodulin. These findings indicate that the liver enzyme and thus the level of dolichol phosphate is controlled by a different mechanism than that of brain and T. pyriformis.

Synthesis of [32P]dolichyl phosphate, utilizing a general procedure for [32P]phosphorus oxychloride preparation.

Equilibration of the phosphorus in radioactive phosphoric acid with the phosphorus in phosphorus oxychloride occurs if these compounds are refluxed together for approximately 24 h. This observation led us to develop a method for the preparation of radioactive phosphorus oxychloride on a small scale with high specific radioactivity. The labeled phosphorus oxychloride may be utilized directly in a one-pot reaction for the preparation of labeled phosphate esters or for the synthesis of more selective phosphorylating agents such as cyanoethyl phosphate. Since the method is very simple and capable of yielding highly labeled radioactive phosphate esters on a small scale, it is applicable to a number of different problems. The preparation of 32P-labeled dolichyl phosphate is described utilizing this procedure.

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.

A micromethod for the determination of ceramide.

An enzymatic micromethod for the determination of ceramide is presented. The enzyme, E. coli diglyceride kinase was used to phosphorylate ceramide, as well as diglyceride with high specific activity gamma-[32P]ATP, and the two products are differentiated by their alkali stability. This method was applied to the detection of endogenous phospholipases and sphingolipases in several membrane systems and could have wide application.

Specific localization of 2',3'-cyclic nucleotide 3'-phosphohydrolase, (Ca2+/Mg2+)-ATPase, and acetylcholinesterase in human erythrocyte membrane.

A procedure was developed for the detection of 2',3'-cyclic nucleotide 3'-phosphohydrolase in myelin. This assay was sufficiently to detect the low levels of 2',3'-cyclic nucleotide 3'-phosphohydrolase in human erythrocytes. The 2',3'-cyclic nucleotide 3'-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghosts and resealed ghosts were assayed for 2',3'-cyclic nucleotide 3'-phosphohydrolase (Ca2+/Mg2+)-ATPase, and acetylcholinesterase activity, and the 2',3'-cyclic nucleotide 3'-phosphohydrolase profile is the same as that of the (Ca2+/Mg2+)-ATP, an established inner membrane marker.

Dolichyl phosphate phosphatase from Tetrahymena pyriformis.

A soluble dolichyl phosphate phosphatase from Tetrahymena pyriformis was purified about 68-fold. The enzyme appeared to be specific for dolichyl phosphate and existed in two interrelated forms, one of mol.wt. about 500000 and the other of mol.wt. about 63000. The enzyme was strongly inhibited by 5 mM-Mn2+ and was strongly stimulated by Mg2+. Tetrahymena in the exponential growth phase contained more of this enzymic activity than cells in stationary or lag phase. The dolichyl phosphate phosphatase may be loosely bound to mitochondrial membranes. Two roles proposed for this enzyme are (1) that of releasing dolichol from its phosphorylated biosynthetic form for its use in the cell as unesterified dolichol or dolichyl ester and/or (2) that of regulation of synthesis of glycoproteins or some other glycosylated compound.

Biological activities of isolated tunicamycin and streptovirudin fractions.

The nucleoside antibiotics tunicamycin and streptovirudin were separated by high-performance liquid chromatography into a series of 256-nm-absorbing peaks. Most of the streptovirudin peaks eluted from a Biosil ODS column earlier than those of tunicamycin, indicating that they were less hydrophobic. With the exception of the first peak, 17 other tunicamycin peaks were potent inhibitors of the formation of dolichylpyrophosphoryl-N-acetylglucosamine with 50% inhibition of the solubilized GlcNAc-1-P transferase requiring about 10 ng of antibiotic per mL. These fractions also inhibited the synthesis of dolichylphosphorylglucose, but in these cases about 500 ng/mL was necessary to achieve 50% inhibition. In MDCK cells in culture, the four major tunicamycin peaks inhibited the incorporation of [2-(3)H]mannose into protein by 50% at about 0.2-0.5 microgram/mL, but [3H]leucine incorporation into protein was unaffected, except at high levels of antibiotic (5-10 microgram/mL). Essentially the same results were observed with the streptovirudin fractions except that they were somewhat less active and some inhibition of protein synthesis was observed with several of these peaks.

Isolation and characterization of dolichols from Tetrahymena pyriformis.

Dolichols of Tetrahymena pyriformis were isolated and characterized by TLC, HPLC and mass spectrometry. Four strains of Tetrahymena were studied and found to have relatively small amounts of dolichol, from 0.26 to 2.60 mg dolichol/kg wet weight. All four strains had approximately the same relative proportions of isoprenologs, dolichol-13 (2%), dolichol-14 (74%), dolichol-15 (23%), and dolichol-16 (less than 1%). Tetrahymena dolichols were found mainly in the mitochondrial subcellular fraction (86%). The pellicle fraction contained 9% and the microsomal fraction, 5% of the remaining dolichol. Free dolichol has also been found in the mitochondrial fraction of four other organisms. We were not able to demonstrate dolichyl esters in these organisms, but their presence is inferred, because reduced yields of dolichol were obtained if the lipid extracts were not saponified prior to HPLC assay.