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N S Radin

Publications and source records attributed to N S Radin.

At least 55 records · Page 3Linked to original sources
PubMed1990

Glucosyceramide synthase of mouse kidney: further characterization with an improved assay method.

The synthesis of glucosylceramide from ceramide and UDP-[3H]glucose by mouse kidney homogenates is very sensitive to the concentration of tissue. This was shown to be due to the presence of a UDP-glc pyrophosphatase, which could be blocked by adding NAD to the medium. A new solvent partitioning system is described, containing t-butyl methyl ether, isopropyl alcohol, and aqueous sodium sulfate, which separates the original substrate (UDP-[3H]glc) from the enzyme product, [3H]cerebroside. A particular advantage of the solvent system is that only a single partitioning step is needed, without backwashes, and the enzyme product appears in the upper phase, making transfer to a counting vial more reliable. A novel incubation device, a thermostatically controlled ultrasonic bath, is used to produce highly uniform enzyme reaction rates. Ca2+, as well as Mg2+ and Mn2+, was found to be a good stimulator of the glucosyltransferase. The enzyme activity in kidney of 22-day old mice, approximately 240 pmol/h/mg tissue, is significantly greater than previously demonstrated. The enzyme was stable in intact kidneys stored at -70 degrees C but unstable at 4 degrees C. The enzyme, when acting on endogenous ceramides, showed no demonstrable glucosylation of the C24 family of ceramides although this family is the predominant one in kidney.

Animals↗
PubMed1990

Effect of an inhibitor of glucosylceramide synthesis on cultured rabbit skin fibroblasts.

1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an effective inhibitor of UDP-glucose:ceramide glucosyltransferase, caused growth inhibition of cultured rabbit skin fibroblasts in a dose-dependent manner. At 50 microM both threo and erythro isomers of PDMP completely suppressed the cell growth. Major gangliosides of the fibroblasts, GM3 and GD3, were greatly reduced in amounts in the presence of threo-PDMP and accumulation of ceramides was observed. Surface labeling with galactose oxidase and [3H]NaBH4 demonstrated that neural glycosphingolipids with four or more sugars present on the surface of control cells were not detectable when the fibroblasts were grown in medium containing threo-PDMP. Metabolic labeling of cellular glycosphingolipids with [14C]-galactose showed reduced incorporation of radioactivity into gangliosides and neutral glycosphingolipids when threo-PDMP was present in the medium. In contrast, the erythro isomer of PDMP did not affect the biosynthesis of glycosphingolipids, a result suggesting that the inhibitory effect of erythro-PDMP on cell growth was due to a mechanism other than the inhibition of glucosyltransferase.

Animals↗
PubMed1990

Preparative scale isolation of sphingosine.

A high-yield method is described for the preparation of sphingenine from galactosylceramide. Two successive cleavage reactions are used, alkaline butanol to form galactosylsphingosine, then acidic acetonitrile to form the sphingenine. A column purification procedure utilizing silica gel is described. The method is superior to previously described methods in its rapidity and avoidance of isomerization and contamination by racemization and rearrangement products.

Acetonitriles↗
PubMed1989

Heat-stable protein that stimulates acid alpha-glucosidase.

A hot-water extract of bovine spleen and guinea pig liver exhibited the ability to enhance acid alpha-glucosidase activity, with methylumbelliferyl alpha-glucoside, glycogen or maltose as substrate. The level of activator required for maximal stabilization was similar for all three substrates, indicating direct action on the enzyme rather than on substrate. The stimulator was partially purified by chromatography with gel-permeation (apparent Mr 20,000-24,000), ion-exchange and C4 reverse-phase columns. It was retained by a narrow-pore dialysis tubing and destroyed by treatment with Pronase, and is presumably a protein. The stimulating protein protected the enzyme against denaturation by heat or incubation with a buffer of high ionic strength in the absence of substrate. RNA inhibited the enzyme, and the activator protein was able to counteract the effect. Activating material was found in a variety of mouse and rat tissues, as well as human urine.

Animals↗
PubMed1989

Effects of D-threo-PDMP, an inhibitor of glucosylceramide synthetase, on expression of cell surface glycolipid antigen and binding to adhesive proteins by B16 melanoma cells.

Incubating B16 melanoma cells with an inhibitor of glucosylceramide (GlcCer) synthetase, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP), led to a considerable decrease in the levels of GlcCer and lactosylceramide (LacCer). The content of ganglioside GM3 was little affected, but the ability to bind a monoclonal antibody against the ganglioside (M2590) was greatly reduced, suggesting that the reduction in the simple glycolipids led to encryption of the membrane antigen. This interpretation is supported by the observation that permeabilization of the treated cells with Triton X-100 restored immunological reactivity. Specificity of the PDMP effect was shown by its lack of effect on the reactivity of two other surface antigens to anti-melanoma monoclonal antibodies M562 and M622, and of the major histocompatibility antigens to anti-H-2KbDb monoclonal antibody. The ability of the treated cells to attach to laminin or type IV collagen was lost but that to fibronectin was not. The effects of the enzyme inhibitor were counteracted by including GlcCer in the culture medium. This indicates that the lipid was absorbed by the cells and utilized like endogenously-formed GlcCer. Cells preattached to laminin or collagen could be induced to round up by addition of inhibitor. In contrast, L-threo-PDMP (which does not block the synthesis of GlcCer) had no effect on the immunologic reactivity of GM3 or the adhesion properties of the cells. However, it did produce considerable accumulation of LacCer. These data suggest that the simple glycolipid, GlcCer, is an essential factor for antigenic expression of the more complex glycolipids on cell surfaces and that there is a close association and interaction between glycolipids and adhesive receptors on the cell surface.

Animals↗
PubMed1988

The activator protein for glucosylceramide beta-glucosidase from guinea pig liver. Improved isolation method and complete amino acid sequence.

beta-Glucosidase activator (SAP-2) is a family of heat-stable, acidic glycoproteins which stimulate enzymatic hydrolysis of glucosylceramide. In this study, we improved the purification method and found that SAP-2 is highly heterogeneous. A hot water extract of frozen guinea pig liver was fractionated by ammonium sulfate sedimentation, then chromatographed with DEAE-Sephacel, Sephadex G-75, and concanavalin A-Sepharose. A fraction binding to concanavalin A-Sepharose was purified further with a C4 high performance liquid chromatography reverse phase column. This yielded several peaks, the main one of which was studied. The specific activity of the purified SAP-2 was 35 units/micrograms (1 unit produces 50% stimulation of a basal glucosidase preparation). N-terminal amino acid sequencing showed that this preparation is a mixture of polypeptides differing in the presence or absence of one or two of the end amino acids. The complete amino acid sequence of the 81 residues in SAP-2 has been determined. Comparison of the sequence of guinea pig SAP-2 with the sequence of human sphingomyelinase activator revealed 58% homology and quite similar hydropathy profiles. Both proteins possess a highly hydrophilic region around Asn-22, which is glycosylated, and 6 cysteine residues, in oxidized form, located in the same positions. Comparison with the published nucleotide sequence for the precursor form of the human activator protein for sulfatide sulfatase (SAP-1) suggested that this activator also has a possibly glycosylated Asn and 6 Cys residues at similar positions, although the remainder of the molecule is somewhat different. Examination of another region of the precursor's nucleotide sequence, assuming a few changes in the identifications, revealed the presence of the sphingomyelinase activator. It appears that two or more activators are derived from a single precursor protein. Marked homologies were seen also with a lung surfactant protein and a sulfated glycoprotein from Sertoli cells.

Amino Acid Sequence↗
PubMed1988

The carbohydrate moiety of the activator protein for glucosylceramide beta-glucosidase.

SAP-2 is a family of heat-stable, acidic glycoproteins which stimulate enzymatic hydrolysis of glucosylceramide. We studied the carbohydrate moieties of a ConA-binding form of SAP-2. The protein contained glucosamine, galactose, mannose, and fucose; galactosamine and sialic acid were not detectable. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining showed three bands of 6.5, 8.5, and 10 kDa. After deglycosylation with peptide N-glycosidase, SAP-2 eluted more slowly from the C4 column and showed a single band of 4 kDa. From carbohydrate analysis it was evident that deglycosylation had removed more than 90% of the sugars. These data indicate that SAP-2 possesses N-linked complex or hybrid type oligosaccharide chains. The specific activity of the deglycosylated protein in the glucosidase stimulation assay was unaffected.

Amino Acids↗
PubMed1988

The inhibition of glucosylceramide beta-glucosidase and other acid hydrolases by nucleic acids.

During the isolation of the activator protein for glucosylceramide beta-glucosidase, we found that certain column fractions contained an inhibitor of the enzyme. After separation from the activator protein by a DEAE-Sephacel column, the inhibitor was purified further with a Spehadex G-75 column. The u.v. absorption spectrum of the purified material was similar to that of nucleic acids and the protein content of the purified material was negligible. Furthermore the purified inhibitor reacted with orcinol but not with diphenylamine, and its inhibitory activity was completely destroyed by treatment with RNAases. It seems likely that the purified inhibitor was tRNA. Authentic RNA, tRNA and DNA had similar inhibitory effects on beta-glucosidase (Ki 17 micrograms/ml for tRNA, noncompetitive toward the substrate). The inhibitory effect of nucleic acids was not fully overcome by an excess amount of the activator protein, but phosphatidylserine could restore the activity to normal. Tests with several other hydrolases revealed that the inhibitory effect of nucleic acids was fairly general.

DNA↗
PubMed1988

Phenotypic changes in 3T3 cells associated with the change of sphingolipid synthesis by a ceramide analog, 2-decanoylamino-3-morpholino-1-phenylpropanol (compound RV538).

A culture of BALB/c 3T3 cells grown in the presence of 40 microM of the ceramide analog compound RV538 (2-decanolyamino-3-morpholino-1-phenylpropanol) for several passages caused a substantial decrease in the level of all glycosphingolipids and an accumulation of ceramide and sphingomyelin. Associated with these chemical changes of sphingolipid composition and metabolism, the following phenotypic changes were observed: (i) loss of the cobblestone appearance at cell density saturation and development of fibroblastic appearance with partial overlapping of cells; (ii) reduction of cell growth rate; (iii) enhanced production of lactic acid; (iv) enhanced rate of glucose transport; and (v) higher incidence of large colony formation with infiltrating appearance in soft agar. Cell morphology changes, lactate production, and enhanced sugar uptake were reversed by co-culturing cells with gangliosides, particularly trisialogangliosides. Thus, these phenotypic changes mimicking those of oncogenically transformed cells are closely related to the blocked synthesis of glycolipids in these cells, whereas other changes may be caused by an accumulation of ceramide and sphingomyelin.

Animals↗
PubMed1988

Normalization of liver glucosylceramide levels in the "Gaucher" mouse by phosphatidylserine injection.

A model of the human genetic disorder, Gaucher disease, can be rapidly generated in mice by the injection of emulsified glucosylceramide and an inhibitor of the lipid's hydrolase, conduritol B epoxide. The liver grows rapidly as it absorbs the load of lipid but the effect disappears as new glucosidase is formed and the load is hydrolyzed. This normalization process is accelerated by treatment with phosphatidylserine, which is a known stimulator of the enzyme. It is possible that injecting the phospholipid into Gaucher patients would have a therapeutic effect since it might help them utilize their residual glucosidase to destroy stored glycolipid.

Animals↗
PubMed1988

Stimulation of liver growth and DNA synthesis by glucosylceramide.

The nature of the growth-stimulating effect of glucosylceramide was studied. Mice were injected intraperitoneally with emulsified glucosylceramide and conduritol B epoxide, an inhibitor of cerebroside glucosidase. Within one or two days, the liver grew 18-24%, as reported. Two enzymes involved in DNA synthesis also increased more than the weight. The total liver activity of thymidine kinase increased 46-73%, and the total activity of ornithine decarboxylase increased as much as 101%. It is suggested that elevated liver levels of glucocerebroside stimulate cell proliferation through a relatively direct mechanism.

Animals↗
PubMed1987

Antitumor activity via inhibition of glycosphingolipid biosynthesis.

The production by cancer cells of glycolipids, perhaps derived partly from host glycolipids, may play essential roles in malignancy, tumor growth, immunity from host immunodefense, and metastasis. The glycolipids are derived from the primary glycolipid, glucosylceramide (GlcCer), which is formed enzymatically from ceramide and uridine diphosphoglucose (UDP-glu). Injection of an inhibitor of this enzyme into mice bearing intraperitoneal Ehrlich ascites tumor cells (EATC) resulted in complete cure of about 30% of the mice and marked prolongation of life in the remainder. Almost all of the surviving mice were immune to a second inoculation of EATC. Injection of GlcCer stimulated cancer cell growth about 50% but this was largely reversed by the inhibitor. This type of inhibitor may have wide application to cancer chemotherapy.

Animals↗
PubMed1987

Preparation of the active isomer of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, inhibitor of murine glucocerebroside synthetase.

1-Phenyl-2-decanoylamino-3-morpholino-1-propanol was previously shown to be an effective inhibitor of the glucosyltransferase in liver that forms glucosylceramide. Since the inhibitor consists of four isomers, it was important for further testing to determine which isomer was most effective and to devise a method for preparation of this isomer. The mixture of isomers was synthesized as described before and separated by crystallization into two diastereomers, differing in migration rate with thin-layer chromatography (TLC) and in retention time with high performance liquid chromatography (HPLC). The slower moving diastereomer, which proved to be the active inhibitor, was separated into its enantiomers by crystallization with dibenzoyltartaric acid isomers. The inhibitory activity resided in the less soluble salt formed with the D-tartaric acid compound. The optical isomers could be characterized by TLC as their (1R)-(-)-camphanate esters. Using a second synthetic route, starting with L-threo- and DL-erythro-1-phenyl-2-amino-1,3-propanediol, we tentatively established the active form of the inhibitor to be the D-threo (1S,2R) isomer. 13C NMR spectroscopy supported the threo and erythro assignments. Kinetic analysis showed that it acted uncompetitively against UDP-glucose and by mixed competition against ceramide, with Ki of 0.7 microM. The DL-erythro and DL-threo compounds inhibited brain galactosylceramide synthetase to a small extent. Glucosylceramide glucosidase activity of liver was unaffected by the DL-threo mixture.

Animals↗
PubMed1986

Uptake by neuroblastoma cells of glucosylceramide, glucosylceramide glucosidase, its stimulator protein, and phosphatidylserine.

Serum-free cultured neuroblastoma cells (clone NlE-115) have been shown to absorb emulsified glucosylceramide, glucosylceramide glucosidase, an activator protein for the enzyme, and phosphatidylserine from a synthetic medium. Uptake of the enzyme was augmented by phosphatidylserine, and vice versa. Uptake of the enzyme-lipid complex was further augmented by the activator protein. It appears likely that the activator forms a complex only with the enzyme-lipid complex, not with the individual components. Two uptake mechanisms for the enzyme seem to be involved, one of which (the complex with activator proteins and acidic lipid) is sensitive to mannosyl phosphate groups. Hydrolysis of absorbed glucosylceramide was slow unless the medium was supplemented with the acidic phospholipid or glucosidase. The most rapid disappearance of stored glycolipid took place when the ternary mixture was added to the cell medium, enzyme + activator protein + phosphatidylserine. These findings may be relevant to enzyme replacement therapy for Gaucher disease.

Animals↗
PubMed1986

Glucosylceramide and the level of the glucosidase-stimulating proteins.

The concentration of beta-glucosidase-stimulating proteins (called cohydrolase here) was measured in mouse liver and brain by immunoassay. Factors that might influence the levels of cohydrolase were examined. Injecting mice with an inactivator of glucosidase (conduritol B epoxide) rapidly produced elevations in liver glucosylceramide (the enzyme's substrate) and in liver and brain cohydrolase. Injection of glucosylceramide emulsified with Myrj 52 produced the same two effects in liver but not in brain. The increases in cohydrolase level induced by the enzyme inhibitor persisted in both organs for at least seven days, reaching 61-70% above the normal level. Injection of emulsified galactocerebroside, sphingomyelin and mixed glucosphingolipids but not of ceramide also produced rises in cohydrolase level. An increase in cohydrolase level resulted from injection of phenylhydrazine, which produces hemolysis and consequently an increased workload for the glucosidase of liver. When the enzyme inhibitor and/or larger amounts of glucosylceramide emulsion were injected (750 mg/kg body weight), increases in liver weight of 13 to 37% appeared within one day. The increased weight was characterized by increases in the weights of protein, total lipid and DNA and a very high increase in glucosylceramide level. These procedures have produced a rapidly developing model version of Gaucher disease in mice. Injected glucocerebroside also induced an elevated level of glucosidase activity.

Animals↗
PubMed1986

A computer program for selecting animals for control and experimental groups in biochemical studies.

The standard method for selecting experimental animals for control and experimental groups is by a randomization process in which each animal is placed into one of the groups without looking at any of its individual differences. An alternative, active matching process is proposed by which three to five animals are chosen to form 'experimental units' that are closely matched with respect to average weight and with respect to distribution of weights (standard distribution values). Several such units are then randomly assigned to each control or treatment group and, at the end of the experiment, the animals within each unit are pooled prior to analysis. The merits of this approach and a computer algorithm for making the selections are described.

Algorithms↗