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R G Kemp

Publications and source records attributed to R G Kemp.

At least 73 records · Page 4Linked to original sources
PubMed1982

Murine leukemia. Proposed role for gangliosides in immune suppression.

Glycolipid-bound sialic acid levels were elevated 2 to 4-fold in the sera of two strains of mice bearing thymic lymphoma produced either spontaneously (AKR/J) or due to chemical carcinogenesis [Swiss mice injected with 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboximide]. The serum glycolipid-bound sialic acid level reflected the tumor burden of the AKR/J mice during the early stages of leukemogenesis. Furthermore, the elevation was found to coincide with the ontogenesis of thymic lymphoma and not to be simply an age-dependent phenomenon. TLC analysis of Florisil column-purified gangliosides from the sera of AKR/J and Swiss mice suggested presence of gangliosides with mobilities very close to GM2 and GM3 standards respectively. On the premise that the elevated glycolipid levels in circulation might interfere with normal lymphocyte functions, the immunoinhibitory properties of exogenously added mixed gangliosides were examined on tests of in vitro correlates of the immune response. Gangliosides inhibited concanavalin A and lipopolysaccharide-induced [3H]-thymidine, [14C]-leucine and [3H]-lysine uptake by normal AKR/J mouse thymocytes and spleen cells. Mixed gangliosides also suppressed the two-way mixed lymphocyte reaction of AKR/J X Swiss and AKR/J X DBA/2 spleen cells. These and other results strongly suggest a general immunologically relevant role for gangliosides in the ontogeny of thymic lymphoma of mice.

Animals↗
PubMed1981

Glycolipid sialyltransferases in normal and neoplastic murine thymocytes.

Total labeled glycolipids from thymocytes of leukemic AKR/J mouse thymus incubated for 24 h in the presence of the precursors [3H] galactose or [14C] glucosamine were found to exceed those from non-leukemic thymocytes. The amount of labeled compounds that co-migrated with monosialogangliosides (GM3 and GM2) and disialogangliosides (GD1a and GD1b) was higher, whereas incorporation into monosialoganglioside (GM1) and trisialoganglioside (GT1) was lower in leukemic than in non-leukemic thymocytes. Consistent with this altered pattern of ganglioside distribution was the finding of a higher activity of two of the sialyltransferase activities in homogenates of leukemic thymus as compared to normal thymus. About 2-fold higher activities of the enzymes CMP-AcNeu: lactosylceramide sialyltransferase and CMP-AcNeu: GM1 sialyltransferase were observed in leukemic thymus homogenate compared to homogenates of non-leukemic cells. The substrate affinity of sialyltransferase with GM1 as acceptor from the leukemic thymus was similar to that of the enzyme from normal thymus. Thus, our studies reveal an enzymatic basis for the enhanced rate of synthesis and altered ganglioside profile observed in malignant thymocytes, and are consistent with the general view on the pattern of acidic glycolipids in tumorigenesis.

Animals↗
PubMed1981

Studies on the phosphorylation of muscle phosphofructokinase.

Phosphorylation of rabbit skeletal muscle phosphofructokinase by the catalytic subunit of cyclic AMP-dependent protein kinase occurs with a Km of about 230 microM and Vmax approaching that seen with histone as a substrate. The rate of phosphorylation of phosphofructokinase by protein kinase is increased by allosteric activators of phosphofructokinase, whereas inhibitors of phosphofructokinase inhibit the phosphorylation. Inhibitors and activators change Vmax but not Km. The site of phosphorylation is a serine residue that is the sixth amino acid from the carboxyl terminus. Limited proteolysis by trypsin releases an octapeptide from the carboxyl terminus and a brief exposure to subtilisin releases a dodecapeptide from the carboxyl end. The sequence of the dodecapeptide is His-Ile-Ser-Arg-Lys-Arg-Ser(P)-Gly-Glu-Ala-Thr-Val. Phosphofructokinase isolated from a rabbit injected 18 h prior to killing with [32P]PO4 contained covalently bound radioactive phosphate. Approximately 80% of the phosphate was released in a trichloroacetic acid-soluble form following limited proteolysis by trypsin, under which conditions the enzyme remained with a monomer size of about 80,000 daltons. The position of elution from Sephadex G-25 of the phosphopeptide was identical with that found following limited trypsin proteolysis of in vitro labeled enzyme. Migration of the phosphopeptides on thin layer cellulose chromatography was also identical. We conclude that at least 80% of the radioactive phosphate introduced within 18 h of an intravenous injection of [32P]PO4 is found at the same site as that introduced by phosphorylation with the catalytic subunit of cyclic AMP-dependent protein kinase.

Amino Acid Sequence↗
PubMed1980

Increased ATP inhibition of liver phosphofructokinase from genetically diabetic mice.

Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was partially purified from the livers of genetically diabetic mice (C57BL/KsJ-db) and their lean littermates (C57BL/KsJ). These genetically diabetic mice have been shown to be hyperglucagonemic and to exhibit symptoms resembling those of maturity-onset diabetes in humans. Two isoenzymes of phosphofructokinase were obtained after DEAE-Sephadex chromatography of extracts of livers from either normal or diabetic animals. One of these isozymes, peak II, from the genetically diabetic mice was shown to be more sensitive to ATP inhibition at physiological pH than the peak II isozyme from the normal animals. In addition, the peak II isozyme from the diabetic mice exhibited decreased affinity for fructose 6-phosphate. The altered kinetic properties of phosphofructokinase from diabetic animals are markedly similar to those recently reported for liver phosphofructokinase isolated from normal animals after glucagon treatment. Our results suggest that increased glucagon levels in diabetes may lead to altered regulation of phosphofructokinase in this disease.

Adenosine Triphosphate↗
PubMed1979

Inhibition of the lectin-induced mitogenic response of thymocytes by glycolipids.

Exogenous gangliosides at concentrations found in serum inhibit the concanavalin A- (Con A) induced mitogenic response of mouse thymocytes. Of four gangliosides tested, the trisialoganglioside, GT1, was the most potent inhibitor. Ceramides, cerebrosides, and sialic acid were not inhibitory at any concentration tested. The inhibition by gangliosides was not due to interference with Con A binding as shown by direct binding studies with [3H]acetyl-Con A nor was it due to a nonspecific killing effect. Thymocytes exposed to a ganglioside concentration 5 times that required to inhibit mitogenesis were still capable of excluding trypan blue up to 44 hr after ganglioside addition. Furthermore, ganglioside inhibition could be reversed by washing the cells 4 hr after addition of the glycolipid. A productive interaction with Con A occurs in the presence of ganglioside as shown by a Con A-induced increase in carbohydrate metabolism. However, uridine and thymidine incorporation are inhibited by the presence of ganglioside. Complete inhibition could be achieved if the glycolipid were added as late as 24 to 28 hr after the Con A in a 48-hr mitogenic assay. The results are discussed in light of recent findings that elevated levels of gangliosides are found in in the sera of tumor-bearing animals, and it is suggested that gangliosides shed by tumor cells could be involved in the generalized immunosuppression observed in such animals.

Animals↗
PubMed1978

The A2B2 hybrid isozyme of phosphofructokinase.

The hybrid isozyme of phosphofructokinase, A2B2, was formed by incubation of rabbit muscle enzyme. A4, and rabbit liver enzyme, B4, in the presence of sodium citrate at neutral pH. The enzyme composition of the resulting mixture of A2B2 and the homoprotomeric forms was identical to that found in rabbit adipose tissue extracts. Hybrid formation, which apparently proceeds by way of dimers, can be blocked by fructose-1,6-P2, fructose-6-P, and high concentrations of MgATP. The A2B2 isozyme was separated from A4 and B4 by ion exchange chromatography. The kinetic regulatory properties of A2B2 were compared with those of A4, B4, and a 1:1 mixture of A4 and B4. ATP inhibition of A2B2 was intermediate between that observed with A4 and B4 and was clearly not identical to a simple summing of the effects of A and B subunits. Similar comparisons were made using other phosphofructokinase inhibitors, citrate, 2,3-P2-glycerate, and P-creatine. In each case the observed inhibition was intermediate between the observed with A4 and B4. The existence in a number of tissues of phosphofructokinase A2B2 provides added diversity to the regulatory mechanisms of glycolysis.

Animals↗
PubMed1977

Effect of cyclic 3':5'-AMP derivatives prostaglandins and related agents on human chorionic gonadotropin secretion in human malignant trophoblast in culture.

The secretion of human chorionic gonadotropin (hCG) is stimulated by addition of N6, O2'-dibutyryl cyclic 3':5'-AMP (dbcAMP) or theophylline to normal term placenta and human malignant trophoblast cells in vitro. To understand better the specificity of this process, malignant trophoblast cultures were incubated with 3':5'-cyclic AMP (cAMP) derivatives, prostaglandins and other agents for 1 to 3 days, and the secretion of radioimmunoassayable hCG was measured. Whereas dbcAMP was the most potent agent in stimulating secretion of hCG, the N6--and O2'-monobutyryl derivatives of cAMP and phosphodiesterase inhibitors (theophylline, papaverine, 3-isobutyl-1-methylxanthine) also increased the secretion of the hormone. A slight increase in hCG secretion was observed following addition of adenine. By contrast, butyrate, cAMP, cyclic 3':5'-GMP (cGMP), dbcGMP, 5'-AMP, adenosine, L-epinephrine and prostaglandins E1, E2, F1a and F2a were ineffective. Particulate fractions from sonicates of malignant trophoblast cultures contained adenylate cyclase activity which was stimulated more than 10-fold by NaF, but not by either catecholamines or prostaglandins. The relatively specific stimulation of hCG secretion suggested that a regulatory process involving cAMP may have physiological significance in the trophoblast.

Adenine↗
PubMed1976

Specific modification of an effector binding site of phosphofructokinase by pyridoxal phasphate.

Conditions are described for the covalent modification of rabbit skeletal muscle phosphofructokinase by pyridoxal phosphate plus sodium borohydride to produce an enzyme that appears by a number of criteria to be modified at the citrate binding site. Evidence that modification occurs at this site is as follows. (1) Protection against activity loss due to modification is provided by the combination of MgATP and citrate, whereas neither low concentrations of citrate nor MgATP alone is effective. This is consistent with the increased affinity for citrate that is observed in the presence of MgATP. (2) The extensive changes in activity and equilibrium binding result from the incorporation of only 1 mol of pyridoxal phosphate per mol of protomer. (3) Modification greatly increases sensitivity to MgATP inhibition, an effect consistent with the known synergism between MgATP and citrate. (4) The affinity of the enzyme for both MgATP and MgIPT at the catalytic site is increased by the modification. (5) The sensitivity of the enzyme to citrate inhibition is greatly diminished following covalent modification. (6) Modification abolishes the equilibrium binding of citrate. (7) Enhanced binding of MgATP is observed following modification, a result consistent with the enhancement of MgATP binding by citrate. Phosphofructokinase protected by citrate plus MgATP can also be modified by the incorporation of 1 or more mol of pyridoxal phosphate, but the enzyme so produced is capable of interacting with citrate and shows none of the properties herein described for the enzyme modified in the absence of citrate.

Adenosine Triphosphate↗
PubMed1975

Interaction of inhibitors with muscle phosphofructokinase.

The interaction of several inhibitors with muscle phosphofructokinase has been studied by both equilibrium binding measurements and kinetic analysis. At low concentrations of citrate a maximum of 1 mol is bound per mol of enzyme protomer. Tight binding requires MgATP and very weak binding is observed in the absence of either magnesium ion or ATP. ITP at low concentrations cannot replace ATP. In the presence of MgATP and at pH 7.0, the dissociation constant for the enzyme-citrate complex is 20 muM. At 50 muM citrate and excess magnesium ion, the concentration of ATP required to give half-maximal binding of citrate is approximately 3 muM . Both P-enolpyruvate and 3-P-glycerate compete for the binding of citrate and the estimated Ki values are 480 and 52 muM, respectively. Creatine-P, another inhibitor of muscle phosphofructokinase, does not compete with the binding of citrate. Measurement of the equilibrium binding of ATP shows that citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P all increase the affinity of enzyme for MgATP with the concentration required to give an effect increasing in the order given. In kinetic studies, citrate, 3-P-glycerate and P-enolpyruvate each act synergistically with ATP to inhibit the phosphofructokinase reaction. This is indicated by the observation that the three metabolites do not inhibit the enzyme with ITP as the phosphoryl donor and that they inhibit at ATP concentrations that are not themselves inhibitory. Furthermore, the sensitivity to the inhibitors increases with increasing ATP concentrations. Striking differences in the extent of inhibition can be seen by varying the order of addition of assay components. Preincubation of the enzyme with ATP and citrate, 3-P-glycerate, or P-enolpyruvate results in greater inhibition than when the inhibitor is added after the reaction is started with fructose-6-P. Furthermore, the inhibition is reversed partially 10 to 15 min after the addition of fructose-6-P. This phenomenon is particularly striking with creatine-P as the inhibitor. Very high concentrations of this inhibitor are required to show any effect if the inhibitor is added after fructose-6-P. These effects are interpreted as reflecting slow conformational changes between an active form with high affinity for fructose-6-P and an inactive, or less active, conformation that binds the inhibitors. Citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P increase the rate of the phosphofructokinase at subsaturating concentrations of MgITP. The results indicate a common binding site on the enzyme for citrate, 3-P-glycerate, and P-enolpyruvate that is distinct from the ATP inhibitory site. An additional site (or sites) for creatine-P is indicated. All four inhibitors act synergistically with ATP by increasing the affinity of the enzyme for MgATP at an inhibitory site. The inhibitors appear also to increase the affinity of the catalytic nucleoside triphosphate site for substrate.

Adenosine Triphosphate↗