Effects of dietary paraffin, squalane and sucrose polyester on residue disposition and elimination of hexachlorobenzene in rats

Previous studies have shown addition of light liquid paraffin to enhance the elimination of organochlorine xenobiotics. In the present study the effect of paraffin on the elimination of [¹⁴C]hexachlorobenzene (HCB) was compared with the effect of possible alternative compounds, squalane and sucrose polyester (SPE). Four groups of 7 rats were fed a diet containing 1.5 ppm [¹⁴C]HCB for 4 days followed by 10 days on HCB-free diet. Thereafter one group (control) remained on this diet whereas the other 3 groups received a diet supplemented with 8% (w/w) paraffin, squalane or SPE, respectively. Radioactivity in urine and faeces was measured daily and at the end of the experiment in samples of abdominal fat, muscle, liver, kidney and blood. Dietary treatment with either paraffin, squalane or SPE markedly enhanced faecal excretion of [¹⁴C]HCB, whereas urinary excretion was not affected. Both the time course as well as the extent of faecal [¹⁴C]HCB elimination were similar in the treated groups. After 3 weeks of treatment the amount of [¹⁴C]HCB excreted with faeces was about three times higher in treated animals than in controls. The half-life (t_1/2) of [¹⁴C]HCB elimination from the body was markedly decreased in treated animals (mean 34–38 days) compared to controls (110 days). [¹⁴C]HCB concentrations in some major tissues were significantly reduced to the same extent by all three dietary regimens. Thus squalane and SPE are as effective as paraffin in removing HCB from contaminated animals.     

Bradykinin effects on phospholipid metabolism and its relation to arachidonic acid turnovers in neuroblastoma × glioma hybrid cells (NG 108-15)

In neuroblastoma × glioma hybrid cells (NG 108-15) labelled with [³²P]-trisodium phosphate, [³H]-inositol and [¹⁴C]-arachidonic acid, bradykinin stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) while it had no effect on the release of [¹⁴C]-arachidonic acid (AA). The effect on PIP, was time- and dose-dependent with a maximal effect on [³H]-inositol- and [³²P]-labelled cells after 10–30 s of stimulation with 10⁻⁶ M bradykinin. However, the hydrolysis of [¹⁴C]-AA labelled PIP₂ was delayed compared to the effect on [³H]- and [¹⁴C]-PIP₂ and was not detectable until after 60 s of stimulation. Bradykinin stimulation resulted in an increased formation of [³H]-inositol phosphates (IP) and [³²P]- and [¹⁴P]- and [¹⁴C]-phosphatidic acid (PA) but the time course for PA formation did not allow the time-course for PIP₂ hydrolysis. A reduced labelling of [²³P]- and [¹⁴C]-phosphatidylcholine was also found in stimulated cells suggesting that PA may derive from other sources than PIP₂. In conclusion, our results indicate that bradykinin activates phospholipase C, but not phospholipase A₂, in NG 108-15 cells.     

Differential production of prostaglandins D2 and E2 and of unusual prostanoid by chick spinal cord and meninges in vitro

In order to specify the source of locally synthesized prostaglandin (PG) E₂ which is able to saturate the large class of low affinity PGE₂ receptors in chick spinal cord, bioconversion of [1-¹⁴C]arachidonic acid into prostanoids was studied in homogenates of chick spinal cord and meninges first without addition of exogenous glutathione (GSH). Homogenates of spinal cord produced ¹⁴C-labeled PGE₂, PGD₂ and PGF₂. Homogenates of meninges accumulated much larger amounts of [¹⁴C]PGE₂ than spinal cord and surprisingly a ¹⁴C-labeled arachidonate metabolite referred to as compound Y. Compound Y generation, which was inhibited by indomethacin and enhanced by esculetin, was therefore mediated through the cyclooxygenase pathway. The fact that no labeled compound Y was detected in homogenates incubated with [³H]PGD₂ or [³H]PGE₂ indicated that compound Y was not degradation product of PG_s. Secondly, after addition of exogenous GSH, ¹⁴C-labeled compound Y was totally converted into [¹⁴C]PGE₂. The compound Y which is converted into PGF_s after a strong reduction with NaBH₄ and into PGE₂ after a mild reduction with GSH-hemin system or SnCl₂ was therefore assumed to be a 15 hydroperoxy-PGE₂ (15 HP-PGE₂). These results suggest that PGE₂ can be synthesized in meninges either by the classical isomerization of PGH₂ or by isomerization of PGG₂ followed by a GSH-sensitive reaction.     

Synaptic vesicles from hog brain—their isolation and the coupling between synthesis and uptake of γ-aminobutyrate by glutamate decarboxylase

Purified synaptic vesicles were isolated from hog cerebral cortex by a rapid procedure consisting of homogenization of cerebral cortex slices in iso-osmotic sucrose, differential centrifugation and sucrose density-gradient centrifugation. The purity of the vesicles was evaluated both biochemically and morphologically. The vesicles contained high amounts of γ-aminobutyrate (GABA) and acetylcholine at specific concentrations of 390 nmol/mg protein and 7.2 nmol/mg protein respectively.

Glutamate decarboxylase, the enzyme which catalyses GABA formation, binds to the synaptic vesicles in a calcium-dependent manner. The percentage of glutamate decarboxylase bound to the vesicles increases from about 5% without calcium, reaching a plateau of about 60% at 4 mM Ca²⁺. Magnesium in concentrations 0.2–10 mM has no significant effect on glutamate decarboxylase binding. Also in phospholipid vesicles (small unilamellar phosphatidylserine-phosphatidylcholine. 2:1 liposomes) Ca²⁺, but not Mg²⁺, induced the binding of glutamate decarboxylase, reaching a plateau of 50% at 2 mM Ca²⁺. Both in synaptic vesicles and in phospholipid vesicles the calcium-dependent glutamate decarboxylase binding seems to be specific, and not caused by unspecific association of proteins, since the specific binding (bound enzyme activity/mg bound protein) increases 3-fold from 0 to 4 mM Ca²⁺.

The functional role of this binding was studied in GAD containing vesicles by measuring the relationship between the accumulation of [³H]GABA, newly synthetized from [³H]glutamate, and the uptake of added [¹⁴C]GABA. No significant uptake of [¹⁴C]GABA was found under the experimental conditions used, whereas large amounts of [³H]GABA were found within the vesicles. It appears that the [³H]GABA accumulation process is functionally linked to [³H]GABA synthesis and is mediated by the membrane-bound glutamate decarboxylase. This synthesis-coupled uptake of GABA into synaptic vesicles possibly serves to bring about a plasticity effect in previously stimulated GABAergic nerve endings.     

Activation of [C]chlorobiphenyls to protein-binding metabolites by rat liver microsomes

The irreversible binding of [¹⁴C]2,2′-di- and [¹⁴C]2,4,5,2′,4′,5′-hexachlorobiphenyl ([¹⁴C]DCB and [¹⁴C]HCB) to protein was studied in the presence of rat liver microsomes and a NADPH-generating system. Protein-bound radioactivity was found with [¹⁴C]DCB but not with [¹⁴C]HCB. The binding of ¹⁴C-metabolites was increased by pretreatment of the rats with phenobarbital or polychlorinated biphenyls. Protein binding was linear for 80 min. In contrast, monohydroxy-metabolites of DCB were formed and degraded within 40 min. Inhibition of secondary oxidation of DCB by scavening superoxide anions or by glucuronidation of the monophenols markedly decreased the protein binding. Addition of trichloropropene oxide or styrene oxide, both inhibitors of epoxide hydrase, did not significantly stimulate the binding. The results suggest that the majority of reactive metabolites of DCB arise from secondary metabolism, i.e., the subsequent oxidation of the phenolic metabolites. Arene oxides, the primary products, appear to play a minor role in the protein binding of DCB.     

Transport in C4 mesophyll chloroplasts. Characterization of the pyruvate carrier

1. Evidence is presented for high rates of carrier-mediated uptake of pyruvate into the stroma of intact mesophyll chloroplasts of the C₄ plant Digitaria sanguinalis, but not the chloroplasts of the C₃ plant Spinacea oleracea. Uptake of pyruvate in the dark with the C₄ mesophyll chloroplasts was followed using two techniques: uptake of [¹⁴C]pyruvate as determined by silicon oil centrifugal filtration and uptake as indicated by absorbance changes at 535 nm (shrinkage/swelling) after addition of 0.1 M pyruvate salts.

2. Uptake of the pyruvate anion by an electrogenic carrier is suggested to be the major mode of transport. Chloroplast swelling was observed in potassium pyruvate plus valinomycin and uptake of [¹⁴C]pyruvate was inhibited by membrane-permeant anions. Valinomycin reduced uptake in the absence of external potassium and the inhibition could be reversed by addition of external potassium.

3. Uptake of pyruvic acid (or a pyruvate ⁻/OH⁻ antiport) is ruled unlikely since [¹⁴C]pyruvate uptake was relatively independent of the pH gradient across the envelope and addition of pyruvate to chloroplasts did not result in an alkalization of the medium. The low rate of swelling observed in ammonium pyruvate may be due to non-mediated permeation of pyruvic acid, which is possible only at high pyruvate concentrations.

4. The concentration of pyruvate in the stroma increased with external concentration over the range tested (up to 40 mM) but the concentration ratio (internal/external) was always less than one. The steady-state concentration of [¹⁴C]pyruvate in the stroma was dependent on the ionic strength of the medium, with saturation at roughly I = 0.04 M, while accumulation of the membrane-permeant cation tetraphenylmethylphosphonium decreased with increasing ionic strength. This suggests that ionic strength modifies a membrane potential (inside negative) across the envelope and that pyruvate uptake responds to the magnitude and direction of that potential (−80 mV at low ionic strength).

5. Chloride and inorganic phosphate were potent inhibitors of [¹⁴C]pyruvate uptake. Of the sulfhydryl reagents tested, N-ethylmaleimide was not inhibitory while mersalyl completely blocked [¹⁴C]pyruvate uptake and swelling in potassium pyruvate plus valinomycin. Pyruvate uptake, as measured by valinomycin induced swelling in potassium pyruvate, was highly temperature sensitive, with an energy of activation of 39 kcal/mol above 9 °C.

6. Phenylpyruvate, -ketoisovalerate, -ketoisocaproate, -cyano-4-hydroxycinnamic acid and -cyanocinnamic acid inhibited [¹⁴C]pyruvate but not [¹⁴C]-acetate uptake in the dark and also reduced pyruvate metabolism by the chloroplasts in the light.     

Retrograde axonal transport after radioactive hydroxyindole injections into the olfactory bulb—an autoradiographic study

Light microscope autoradiography was used to study the retrograde transport of labelled material after injection of [³H]serotonin ([³H]5-HT), [³H]5-hydroxytryptophan ([³H]5-HTP) and [¹⁴C]5-hydroxyindoleacetic acid ([¹⁴C]5-HIAA) into the olfactory bulb (OB) of rat. A perikaryal labelling was clearly visualized in the Raphe Dorsalis (RD) and the Raphe Centralis (RC) 24 h after injection of [³H]5-HT (but not after injection of [³H]5-HTP or [¹⁴C]5-HIAA) into the OB of rats without monoamine-oxidase inhibitor (MAOI). In the OB, the labelled cells (mitral, granular, periglomerular and tufted cells) and the varicosities (dispersed in granular, plexiform and glomerular layers) were greater in number and intensity at 8 h than at 24 h after [³H]5-HT (10⁻³ M) injection. Five hours after injection of [¹⁴C]5-HIAA (10⁻³ M) some mitral, granular and tufted cells were labelled in the cytoplasm, nuclei and dendrites. A few varicosities were also observed. In contrast, after [³H]5-HTP injection no clear labelling was visualized in axonal processes. A net autoradiographic reaction was seen, however, in the capillary walls and some granular cells.

After injection of [³H]5-HT at various concentrations (10⁻² M to 10⁻⁵ M) into the OB of rats pretreated with MAOI, a selectivity in the pattern of labelling in the injection site and the afferent cell bodies was found at 10⁻⁴ M and 10⁻⁵ M. At these concentrations, the serotoninergic RD and RC neurons were clearly labelled, but the non-serotoninergic neurons such as those originating in the Locus Coeruleus, prepiriform cortex were devoid of label. In the OB, only varicosities and fiber-like structures were reactive. In the RD cell bodies, the intensity of labelling as well as the number of labelled cells were greater at higher concentrations of injected [³H]5-HT and when rats were pretreated with a MAOI.     

The mechanism of acceptor reactions of leuconostoc mesenteroides B-512F dextransucrase

Reactions of dextransucrase and sucrose in the presence of sugars (acceptors) of low molecular weight have been observed to give a dextran of low molecular weight and a series of oligosaccharides. The acceptor reaction of dextransucrase was examined in the absence and presence of sucrose by using d-[¹⁴C]glucose, d-[¹⁴C]fructose, and ¹⁴C-reducing-end labeled maltose as acceptors. A purified dextransucrase was pre-incubated with sucrose, and the resulting d-fructose and unreacted sucrose were removed from the enzyme by chromatography on columns of Bio-Gel P-6. The enzyme, which migrated at the void volume, was collected and referred to as “charged enzyme”. The charged enzyme was incubated with ¹⁴C-acceptor in the absence of sucrose. Each of the three acceptors gave two fractions of labeled products, a high molecular weight product, identified as dextran, and a product of low molecular weight that was an oligosaccharide. It was found that all three of the acceptors were incorporated into the products at the reducing end. Similar results were obtained when the reactions were performed in the presence of sucrose, but higher yields of labeled products were obtained and a series of homologous oligosaccharides was produced when d-glucose or maltose was the acceptor. We propose that the acceptor reaction proceeds by nucleophilic displacement of glucosyl and dextranosyl groups from a covalent enzyme-complex by a specific, acceptor hydroxyl group, and that this reaction effects a glycosidic linkage between the d-glucosyl and dextranosyl groups and the acceptor. We conclude that the acceptor reactions serve to terminate polymerization of dextran by displacing the growing dextran chain from the active site of the enzyme; the acceptors, thus, do not initiate dextran polymerization by acting as primers.     

μ-opioid receptor-mediated inhibition of the release of radiolabelled noradrenaline and acetylcholine from rat amygdala slices

Presynaptic modulation by opioids of electrically-evoked neurotransmitter release from superfused rat amygdala slices prelabelled with [³H]noradrenaline (NA) and [¹⁴C]choline was examined. Both [³H]NA and [¹⁴C]acetylcholine release were strongly inhibited by morphine, the mixed δ/μ-receptor agonist [ -Ala², -Leu⁵]enkephalin (DADLE) and the highly selective μ-agonist [ -Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO), whereas the highly selective δ-agonist [ -Pen², -Pen⁵]enkephalin and the κ-agonist bremazocine were without effect. The inhibitory effects were potently antagonized by naloxone but not by the selective δ-receptor antagonist fentanylisothiocyanate. When the selective uptake inhibitor desipramine was used to prevent uptake of [³H]NA into noradrenergic nerve terminals, but sparing the uptake into dopaminergic nerve terminals, the electrically evoked release of tritium was strongly inhibited by bremazocine but not by DADLE or DAMGO.

The data indicate, that in the amygdala transmitter release from dopaminergic nerve fibres is inhibited only via activation of κ-receptors, whereas transmitter release from noradrenergic and cholinergic nerve fibers is subjected to inhibition by opioids via activation of μ-receptors only. Regional differences and similarities of modulation of neurotransmitter release by opioids in the rat brain are briefly discussed.     

CAPABILITY OF TUBULIN AND MICROTUBULES TO INCORPORATE AND TO RELEASE TYROSINE AND PHENYLALANINE AND THE EFFECT OF THE INCORPORATION OF THESE AMINO ACIDS ON TUBULIN ASSEMBLY

Abstract— Incorporation of [¹⁴C]tyrosine into the C-terminal position of α-tubulin of rat brain cytosol was 10-fold higher for non-assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non-assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.
[¹⁴C]Tyrosine was released from assembled and non-assembled [¹⁴C]tyrosinated tubulin by the action of an endogenous carboxypeptidase. Release from non-assembled tubulin was shown by incubating a tubulinyl-[¹⁴C]tyrosine preparation in the presence of CaCl₂ at a concentration that abolished microtubule formation. Release from microtubules was inferred from the observation that the percentages of [¹⁴C]tyrosine released and the decrease of the specific radioactivity of the recovered microtubules were practically identical and did not change after a 10-fold dilution of the incubated microtubules.
[³H]Phenylalanine was released from a preparation of tubulinyl-[³H]phenylalanine also by an enzymatic activity.
The capacity of a tubulin preparation to incorporate tyrosine was increased 43% by pre-treatment with endogenous carboxypeptidase.
Tubulin tyrosinated in vitro was assembled to the same extent as native tubulin. After a mixture of tubulinyl-[¹⁴C]tyrosine and tubulinyl-[³H]phenylalanine was partially assembled, the ratio of ¹⁴C/³H found in the microtubules was the same as in the non-assembled tubulin fraction.     

HYDROLYTIC AND TRANSGLUCOLYTIC ACTIVITIES OF A PARTIALLY PURIFIED CALF BRAIN β-GLUCOSIDASE

Abstract— Properties of both a transglucosylation reaction and the hydrolytic activity of a partially purified calf brain β -glucosidase were investigated. Sodium taurocholate and a 'Gaucher factor' stimulated both activities. A purified 'stimulatory' factor from human liver did not appear to significantly affect the hydrolytic activity towards either 4-methylumbelliferone- β - d -glucoside or [¹⁴C]glucosyl ceramide. Several compounds were found to be competitive inhibitors of the hydrolytic activity, conduritol B epoxide and norjirimycin being the most effective. Glucosyl ceramide hydrolysis was more sensitive to inhibition by p -chloromercuribenzenesulfonate than 4-methylumbelliferone- β -glucoside cleavage. The partially purified enzyme preparation catalyzed the formation of [¹⁴C]glucosyl ceramide with N -[¹⁴C]oleoyl sphingosine as the acceptor and several β -glucosides as the donor.     

Photosynthetic CO2 fixation by chloroplast populations isolated by a polymer two-phase technique

Preparations of spinach chloroplasts, essentially free from contamination by other cellular material or whole cells, incorporated ¹⁴C almost entirely into glycolate, a polyglucan (probably starch) and intermediates of the Calvin cycle and starch synthesis. About 70% of the ¹⁴C was found in dihydroxyacetone phosphate, 3-phosphoglycerate and glycolate. Only small amounts were found in sucrose and amino acids.

On the other hand, preparations consisting of particles containing chloroplasts surrounded by a membrane-bound cytoplasmic layer including mitochondria and microbodies, gave a much broader spectrum of ¹⁴C-labelled products. Much less of the ¹⁴C was found in dihydroxyacetone phosphate and 3-phosphoglycerate. Instead, sucrose, malate, aspartate, alanine and some other amino acids contained about 40% of the ¹⁴C incorporated. It is concluded that sucrose is synthesized by cooperation between the chloroplast and the surrounding layer.     

INCORPORATION OF PHENYLALANINE AS A SINGLE UNIT INTO RAT BRAIN PROTEIN: RECIPROCAL INHIBITION BY PHENYLALANINE AND TYROSINE OF THEIR RESPECTIVE INCORPORATIONS

Abstract— Of seven amino acids studied, glutamic acid and phenylalanine were incorporated in highest amounts into the hot-TCA-insoluble material of the 100,000 g supernatant fraction of rat brain homogenate. The system for incorporation of phenylalanine was RNase-insensitive and required ATP (apparent K_m = 0.64 m m ), KC1 (apparent K_m = 14 m m ) and MgCl₂ (optimal concentration range 4-15 m m ). The apparent K_m for phenylalanine was 2.9 m m . [¹⁴C]Phenylalanine did not undergo modification before incorporation. Tyrosine and phenylalanine inhibited the incorporation, respectively, of [¹⁴C]phenylalanine and [¹⁴C]tyrosine when incubated simultaneously or successively. The K_m and K_t (3.3 m m ) values for phenylalanine in the incorporation reaction and as inhibitor of the incorporation of [¹⁴C]tyrosine were similar. We suggest that both the enzyme and the acceptor for the incorporation of these two amino acids are the same. [¹⁴C]Phenylalanine and [¹⁴C]tyrosine entered into COOH-terminal positions in the reactions described. Brain exhibited a 25- to 100-fold higher capacity to incorporate phenylalanine than that of liver, kidney or thyroid. The acceptor capacity in rat brain rapidly decreased from day 5 to day 15 of postnatal age and then slowly until age 150 days.     

Lumiflavin and Lumichrome Transport in the Central Nervous System

Abstract: The transport of the lipid-soluble sugarless flavins, [¹⁴C]lumiflavin and [¹⁴C]lumichrome, into and from the isolated choroid plexus and brain slices was studied in vitro. The isolated choroid plexus accumulated both [¹⁴C] flavins by a saturable, energy-requiring process that did not depend on binding or intracellular metabolism of the [¹⁴C] flavins. Both sugar-containing and sugarless flavins, as well as cyclic organic acids, significantly inhibited [¹⁴C]lumiflavin and [¹⁴C]Iumichrome uptake by the isolated choroid plexus. Within 2.5 min, 75% of the [¹⁴C]lumiflavin accumulated by the isolated choroid plexus was released into the medium. Brain slices accumulated [¹⁴C]lumiflavin by a saturable process that did not meet all the criteria for active transport. Ninety-five percent of the [¹⁴C]lumiflavin accumulated by brain slices was released into the medium within 7.5 min. In vivo , 2 h after the intraventricular injection of 6.5 nmol [¹⁴C]lumiflavin, almost all of the [¹⁴C]flavin was cleared from the CNS. Addition of 3.5 μmol FMN to the intraventricular injectate significantly decreased the clearance of [¹⁴C]lumiflavin from the CNS. These studies document that the sugarless flavins are transported by the flavin transport systems in the CNS.     

Purification of pea nodule symbiosomes using an aqueous polymer two-phase system

Symbiosomes were obtained from mature pea (Pisum sativum cv. Argona) root nodules infected with a Rhizobium leguminosarum strain (biov. viciae 3841) and purified using an aqueous polymer two-phase system (APS). The APS consists of a mixture of polymers, usually dextran T500 and poly(ethylene glycol) 3350, prepared as aqueous solutions on a weight per weight basis, where each fraction distributes according to their surface characteristics. Results of ATPase activity, cytochrome c oxidase activity, glucan synthase II activity, NAD(P)H-cytochrome c reductase activity, NO₃⁻-sensitive ATPase activity, transport of [¹⁴C]malate vs. [¹⁴C]glutamate and MAC 57 antigen analysis showed that the APS method provided intact symbiosomes with low bacteroid, plasma membrane, endoplasmic reticulum and/or mitochondria contamination. No complicated equipment is needed and the method was simple and fast, compared with other purification techniques.     

Effects of vesamicol on acetylcholine metabolism and synaptic transmission in the electric organ of Torpedo

Vesamicol [2-(4-phenylpiperidino)cyclohexanol, formerly AH5183] at a concentration of 10 μM reduced by 16–20% the amount of vesicle-bound ACh in intact pieces of Torpedo electric organ (isolated prisms). When [¹⁴C]acetate was applied to prisms in the presence of 10 μM vesamicol, vesicular translocation of newly synthesized [¹⁴C]ACh was inhibited by 40%. During short trains of field shocks given at 10 Hz to the tissue, vesamicol inhibited by 93% the release of [¹⁴C]ACh, but left the release of prestored ACh unaltered. In spite of these alterations, 10 μM vesamicol did not impair nerve-electroplaque transmission, even after prolonged electrical stimulation and during a recovery period. It is concluded that in the Torpedo electric organ the actions of vesamicol on ACh metabolism have apparently little or no effect on the efficiency of synaptic transmission.     

The mechanism of dextransucrase action: Biosynthesis of branch linkages by acceptor reactions with dextran

Dextransucrase from Leuconostoc mesenteroides B-512 catalyzes the polymerization of dextran from sucrose. The resulting dextran has 95% α-1 → 6 linkages and 5% α-1 → 3 branch linkages. A purified dextransucrase was insolubilized on Bio-Gel P-2 beads (BGD, Bio-Gel-dextransucrase). The BGD was labeled by incubating it with a very low concentration of [¹⁴C]sucrose or it was first charged with nonlabeled sucrose and then labeled with a very low concentration of [¹⁴C]sucrose. After extensive washings with buffer, the ¹⁴C label remained attached to BGD. This labeled material was previously shown to be [¹⁴C]dextran and was postulated to be attached covalently at the reducing end to the active site of the enzyme. When the labeled BGD was incubated with a low molecular weight nonlabeled dextran (acceptor dextran) all of the BGD-bound label was released as [¹⁴C]dextran whereas essentially no [¹⁴C]dextran was released when the labeled BGD was incubated in buffer alone under comparable conditions. The released [¹⁴C]dextran was shown to be a slightly branched dextran by hydrolysis with an exodextranase. Acetolysis of the released dextran gave 7.3% of the radioactivity in nigerose. Reduction with sodium borohydride, followed by acid hydrolysis, gave all of the radioactivity in glucose, indicating that the nigerose was exclusively labeled in the nonreducing glucose unit. These results indicated that [¹⁴C]dextran was being released from BGD by virtue of the action of the low molecular weight dextran and that this action gave the formation of a new α-1 → 3 branch linkage. A mehanism for branching is proposed in which a C₃-OH on an acceptor dextran acts as a nucleophile on C₁ of the reducing end of a dextranosyl-dextransucrase complex, thereby displacing dextran from dextransucrase and forming an α-1 → 3 branch linkage. It is argued that the biosynthesis of branched linkages does not require a separate branching enzyme but can take place by reactions of an acceptor dextran with a dextranosyl-dextransucrase complex.     

Biosynthesis of monogalactosyl diglyceride by chloroplasts from spinacia oleracea and from some gramineae

Synthetic diglycerides which differed in unsaturation of fatty acids gave the same incorporation of [¹⁴C]galactose from UDP-[¹⁴C]galactose when added to acetone powders of spinach chloroplasts up to about 0·6 mg diglyceride/20 mg acetone powder. Diolein and the endogenous diglyceride isolated from the acetone extract of chloroplasts stimulated galactolipid biosynthesis to a similar extent. With all diglycerides used, monogalactosyl diglyceride was the main product with little accompanying synthesis of digalactosyl diglyceride. The radioactivity in the monogalactosyl diglyceride synthesized from UDP-[¹⁴C]galactose by whole chloroplasts was distributed widely among the monogalactosyl diglycerides with different fatty acid composition. It is concluded that the enzyme which catalyses the transfer of galactose from UDP-galactose to diglyceride is not specific for polyunsaturated diglycerides and that the polyunsaturated monogalactosyl diglycerides arise either by desaturation of the fatty acyl residues after monogalactolipid synthesis or by transacylation. Acetone powders of chloroplasts prepared from several Gramineae did not exhibit transferase activity although whole chloroplasts were active.     

Oligodendroglial Glycerophospholipid Synthesis: Incorporation of Radioactive Precursors into Ethanolamine Glycerophospholipids by Calf Oligodendroglia Prepared by a Percoll Procedure and Maintained in Suspension Culture

Abstract: Oligodendroglia prepared from minced calf cerebral white matter by trypsinization at pH 7.4, screening, and isosmotic Percoll (polyvinylpyr-rolidone-coated silica gel) density gradient centrifugation survived in culture on polylysine-coated glass, extending processes and maintaining phenotypic characteristics of oligodendroglia. In the present study, ethanolamine glycerophospholipid (EGP) metabolism of the freshly isolated cells was examined during short-term suspension culture by dual label time course and substrate concentration dependence experiments with [2-³H]glycerol and either [1,2-¹⁴C]ethanolamine or L-[U-¹⁴C]serine. Rates of incorporation of ³H from the glycerol and of ¹⁴C from the ethanolamine into EGP were constant for 14 h. In medium containing 3 mM-[1,2-¹⁴C]ethanolamine and 4.8 mM-[2-³H]glycerol, rates of incorporation of ¹⁴C and ³H into diacyl glycerophosphoethanolamine (diacyl GPE) were similar. Under the same conditions, ³H specific activities of alkylacyl GPE and alkenylacyl GPE were much lower than ¹⁴C specific activities, likely as a result of the loss of tritium during synthesis of these forms of EGP via dihydroxyacetone phosphate. L-[U-¹⁴C]serine was incorporated into serine glycerophospholipid (SGP) by base exchange rather than de novo synthesis. ¹⁴C from L-[U-¹⁴C]serine also appeared in EGP after an initial lag period of several hours. Methylation of oligodendroglial EGP to choline glycerophospholipid (CGP) was not detected.