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Biomedical subjects

N S Radin

Publications and source records attributed to N S Radin.

At least 91 records · Page 5Linked to original sources
PubMed1981

Mechanism of activation of glucocerebrosidase by co-beta-glucosidase (glucosidase activator protein).

The nature of the stimulatory action of the protein 'coglucosidase' on glucocerebrosidase was investigated with the use of highly purified cofactor from bovine spleen, radioactive glucosyl ceramide and methylumbelliferyl-beta-glucoside. A complex between glucosidase and either substrate could not be detected under equilibrium and non-equilibrium binding conditions. Complex formation between stimulating protein and the enzyme could be shown by the binding of the enzyme to an affinity column containing coglucosidase. This binding could be blocked by adding phosphatidylserine to the enzyme. The lipid also stimulated the enzyme. Additional evidence for binding of the enzyme to the two kinds of stimulators was the finding that they protected the enzyme against inactivation by N-ethylmaleimide and chloromercuriphenylsulfonate. A role for lipids in the stimulatory action of coglucosidase was shown by extracting lipids from the enzyme; this resulted in a loss of basal enzyme activity and of sensitivity to activation by the protein. Adding back to the lipids or phosphatidylserine increased the sensitivity of the delipidated enzyme to coglucosidase. Using the crude, unextracted enzyme we could show that low concentrations of phosphatidylserine augmented the effectiveness of coglucosidase but high concentrations of the lipid blocked the effect of the protein. It is proposed that lipids, particularly acidic ones, act on solubilized glucocerebrosidase to produce an enzyme conformation which allows binding and stimulation by coglucosidase. At higher lipid concentrations, the acidic lipids bind, in competition with coglucosidase, to the latter's binding site on the enzyme.

4-Chloromercuribenzenesulfonate↗
PubMed1981

A 2-phase liquid scintillation assay for glycolipid synthetases.

Glycolipid synthetases can be assayed conveniently by incubating the lipid substrate with the radiosugar-labeled nucleotide in a small plastic scintillation vial. At the end of the incubation period, water and perchloric acid are added, then n-butanol, then a toluene-based scintillation cocktail. The radioactive lipid partitions into the scintillation fluid, leaving excess sugar nucleotide in the aqueous phase. Only a small fraction of the total radioactivity in the aqueous layer is detectable. This method is illustrated for ceramide:UDP-glucose glucosyltransferase. The approach should be applicable to other lipid synthetases that can be assayed with radioactive hydrophilic substrate.

Animals↗
PubMed1981

An improved fluorometric leukocyte beta-glucosidase assay for Gaucher's disease.

Three fluorometric leukocyte beta -glucosidase assays were compared for their ability to diagnose Gaucher's disease and identify carriers of the disorder: the acid beta-glucosidase assay of Beutler and Kuhl [2], a pH 5.5-sodium taurocholate-dependent assay and a new procedure which employs conduritol B epoxide, an active-site specific inhibitor of glucocerebrosidase. All three assays unambiguously identified patients with Gaucher's disease. With regard to identifying carriers the bile salt dependent assay of Peters et al. and the conduritol B epoxide-dependent procedure gave the greatest discrimination between the mean beta-glucosidase values for the control and heterozygote samples when evaluated using Student's t test. The most reliable assay for the identification of the carrier state was the conduritol B epoxide-dependent procedure which can be expected to provide the fewest false negative results when classifying heterozygotes (5%). However, the fact that none of these methods will completely separate control and heterozygote samples indicates that their use in screening programs will result in a significant number of incorrect assignments.

Adolescent↗
PubMed1981

The use of galactose oxidase in lipid labeling.

Galactose oxidase can be used to oxidize the terminal carbon atom of lipids containing galactose or N-acetylgalactosamine, and the resultant aldehyde group can be reduced back to the original carbinol with radioactive borohydride. The efficiency of the first reaction has been investigated systematically by using [6-3H]galactosyl ceramide as substrate and measuring the amount of radioactive water formed. This enabled us to establish that the addition of catalase and peroxidase greatly speeded the oxidation, that phosphate and PIPES buffers were the best among those tested, that the reaction continued for 24 hr without a second addition of galactose oxidase, and that the optimum concentration of organic solvent (tetrahydrofuran) was 50%. The suggestion if made that a similar set of variables be studied for each lipid or nonlipid by the same basic technique: labeling by the oxidase/borohydride method and use of the resultant compound as substrate.

Catalase↗
PubMed1980

A revised fluorometric assay for Gaucher's disease using conduritol-beta-epoxide with liver as the source of Beta-glucosidase.

To date, enzymatic diagnosis of Gaucher's disease via a fluorometric assay procedure which utilizes 4-methylumbelliferyl-beta-D-glucopyranoside as a substrate has not been possible when liver serves as the source of enzyme since currently employed fluorometric procedures cannot adequately differentiate between a broad-specificity beta-glucosidase and lysosomal glucocerebrosidase activities in crude extracts of liver. Incorporation of conduritol-beta-epoxide into the incubation medium for the fluorometric assay allows one to selectively measure the glucocerebrosidase activity present in a given liver extract. In five cases of Gaucher's disease this revised fluorometric procedure proved as effective as the assy procedure which utilizes authentic, radiolabeled glucocerebroside as the substrate in demonstrating a deficiency of glucocerebrosidase activity in liver.

Clinical Enzyme Tests↗
PubMed1980

Glucosylceramide uptake protein from spleen cytosol.

Incubation of rat red cells with liposomes containing radioactive glucosylceramide, together with unlabeled phosphatidylcholine and cholesterol, produced cells labeled with the glycolipid. Part of the bound cerebroside could be removed by further incubation with unlabeled liposomes. Both processes, uptake and release, were stimulated by adding a preparation obtained from bovine spleen. The active material was purified partially by ammonium sulfate precipitation and gel permeation with a Sephadex G-75 column. The latter step indicated a molecular weight of about 22,000. The material lost much of its activity when treated with trypsin or N-ethylmaleimide, indicating that it is a protein. The "cerebroside uptake protein" was quite stable if stored in dithioerythritol at 4 degrees C. Tests with liposomes containing labeled phosphatidylcholine or cholesterol showed that the red cells took up these lipids as well, but the transfer protein exerted little effect on this. Ghosts prepared from rat red cells also took up the three lipids from the liposomes and here, as well, the uptake protein showed high specificity in facilitating the uptake of cerebroside. This appears to be the first demonstration of the existence of a glycolipid transfer protein. Possible physiological roles for the uptake protein in red cell metabolism and in the modulation of the glycosphingolipid composition of cell membranes are discussed.

Animals↗
PubMed1980

Analogs of ceramide that inhibit glucocerebroside synthetase in mouse brain.

In a search for potent inhibitors of glucocerebroside biosynthesis, we synthesized aromatic analogs of the enzyme's substrate, ceramide, many of which have not previously been described in the literature. Mouse brain and spleen, rat brain, and human placenta and spleen were all found to be susceptible to inhibition by a variety of compounds, although to differing extents. The most potent inhibitor was 2-decanoylamino-3-morpholino-1-phenylpropanol. The dehydro version of this compound (2-decanoylamino-3-morpholinopropiophenone) was less effective but it produced inactivation of the enzyme, probably by covalent reaction with the enzyme's active site. Examination of the various effects seen leads us to suggest that the active region of the enzyme contains four recognitional sites: an anionic moiety that may bind the glucose in activated form, an oxygen-binding region oriented toward the third carbon atom of ceramide, a narrow region that binds the alkyl chain of the fatty acid moiety, and a less narrow region that binds the hydrocarbon chain of the sphingoid base moiety.

Animals↗
PubMed1979

Conversion by rat brain preparation of lignoceric acid to ceramides and cerebrosides containing both alpha-hydroxy and nonhydroxy fatty acids. Effects of compounds which affect sphingolipid metabolism.

Three synthetic compounds which affect sphingolipid metabolism in vitro were examined for their effects on the synthesis in a rat brain system of nonhydroxyceramide, hydroxyceramide, nonhydroxycerebroside, and hydroxycerebroside from [1-14C]ignoceric acid. These compounds are N-octanoyl-D-threo-p-nitrophenylaminopropanediol (Compound I), N-(2,3-epoxydecanoyl)-norephedrine (Compound II), and N-decyl-N'-glucosylthiourea (Compound III). In the presence of up to 0.5 mM Compound I, only the hydroxyceramide fromation increased, while the synthesis of the other three lipids decreased. Compound II strongly inhibited the formation of all four lipids at all concentrations tested (0.1 to 1 mM). Compound III increased the synthesis of both ceramides approximately 2-fold at 1 mM, but the conversion of lignoceric acid to both cerebrosides remained relatively unchanged. Several significant conclusions from these observations are discussed.

Animals↗
PubMed1979

Short chain ceramides as substrates for glucocerebroside synthetase. Differences between liver and brain enzymes.

In order to increase the sensitivity of the assay for ceramide: UDPGlc glucosyltransferase, the enzyme that makes glucocerebroside, we synthesized a variety of ceramide homologues that might be better substrates than the naturally occurring ceramides. N-Octanoyl sphingosine proved to be the best lipid tested in liver and brain. It could be added to the tissue homogenate in the dry form, as a thin layer coated on Celite, or in liposomes, prepared from lecithin and cerebroside sulfate. The liposomal form produced better replication of assay values. It is suggested that the addition of cerebroside sulfate to liposomal preparations might be a good, and more physiological, replacement for the commonly used dicetyl phosphate. A new homologue of DL-sphinganine, decasphinganine, was synthesized by an efficient series of steps and acylated with different fatty acids to form ceramide homologues. The best substrate in this series was the lauroyl amide and it is suggested that this lipid be used in cerebroside synthetase assays because of the convenience of preparing it, even though it is not as good as octanoyl sphingosine. Both compounds are distinctly better than natural ceramide or DL-sphinganine amides. From comparisons of enzyme activity under various conditions, the tentative conclusion is drawn that the enzymes in liver and brain have different properties, and that liver has two different synthetases.

Amides↗
PubMed1979

Destruction and resynthesis of mouse beta-glucosidases.

1. Injection of a single dose of conduritol B epoxide into mice produced almost complete destruction of glucocerebrosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) in liver, spleen, brain, and kidney within 5 h. Restoration of activity became noticeable within 1 day (2 days in the case of brain) and was about 80% of normal within 16 days. 2. The same injection produced less destruction of aryl beta-glucosidase (EC 3.2.1.21), measured at pH 5.4 with methylumbelliferyl glucoside in the absence of taurocholate. Brain showed the least amount of destruction, about 50%, but measurements of activity at lower pH values revealed complete loss of activity. This suggests that brain contains two different aryl glucosidases with differing sensitivity to the inhibitor. Liver, on the other hand, did not show differential destruction when assayed at different pH values. Resynthesis of the enzyme activities was almost complete by 16 days. 3. Injection of phenylhydrazine produced hemolysis and spleen enlargement, with concomitant increases in specific activities of glucocerebrosidase and aryl glucosidase in liver and spleen (but not in kidney). When this experiment was done in mice previously treated with conduritol B expoxide, the reappearance of cerebrosidase was found to be accelerated. This is interpreted to mean that the increased load of glucolipids from the erythrocytes had induced an enhanced synthesis of the glucohydrolase. A similar explanation may apply to aryl glucosidase and glucopeptides in the cells.

Animals↗
PubMed1979

Enzymic effects of beta-glucosidase destruction in mice. Changes in glucuronidase levels.

1. The injection into mice of a single dose of conduritol B epoxide, a covalent inhibitor of glucosidases, quickly produced changes in tissue levels of beta-D-glucuronidase (EC 3.2.1.31). The specific activity of the enzyme decreased in liver, spleen and kidney while brain showed little change. The inhibitor did not act on glucuronidase in vitro, so the effect of the inhibitor is complex, possibly a result of the loss of glucosidase activity. Since glucuronidase contains glucose, we suggest that the transport of the enzyme between subcellular regions and tissues involves loss of part of the glucose moieties. 2. Levels of glucocerebrosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) dropped very rapidly after epoxide injection, reaching a minimum at 1 h in liver. There was a noticeable restoration of activity within the next 1--2 h. Aryl beta-glucosidase (EC 3.2.1.21) decrease somewhat less than cerebrosidase, reaching a minimum within 2 h. It too showed some recovery of activity within 3 h. 3. Acid phosphatase rose slightly in liver but not in brain. alpha-L-Fucosidase and angiotensin-converting enzyme were not affected by the epoxide injection. The latter two enzymes are known to contain glucose. 4. Injection of a hemolyzing agent, phenylhydrazine, produced an increased level of glucuronidase in liver and spleen within 6 days, but not in kidney. This enhancement was a little less in mice previously injected with the glucosidase inhibitor. 5. Mice injected with the epoxide once a day eight times showed a distinct rise in brain glucuronidase level, as well as a rise in brain weight. However, the other organs showed only the same decrease in glucuronidase specific activity noted with the single injection protocol. It is suggested that the difference is due to the blood-brain barrier, which could slow the loss of brain glucuronidase from the extracellular fluid.

Acid Phosphatase↗