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E S Kempner

Publications and source records attributed to E S Kempner.

At least 73 records · Page 4Linked to original sources
PubMed1986

Radiation target analysis of glycoproteins.

The radiation sensitivity of glycoproteins is shown to depend only on the protein portion of the molecule. An artificially created glycoprotein containing glucose-6-phosphate dehydrogenase observes this rule as well as natural enzymes and receptors containing from 2 to 50% carbohydrate. No exceptions have been found. Radiation damage to carbohydrates occurs close to the site of the primary ionization, with little spread of damage into attached polypeptides.

Binding Sites↗
PubMed1985

Functional size of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase as determined by radiation inactivation.

The functional molecular weight of rat liver 3-hydroxy-3-methylglutaryl-CoA reductase was determined by radiation inactivation. Both isolated hepatic microsomes and primary hepatocytes were irradiated with high energy electrons at -135 degrees C, and the residual microsomal enzyme activity was subsequently determined. The loss of enzyme activity in both irradiated microsomes and microsomes isolated from irradiated hepatocytes followed a single exponential decay which corresponded to a molecular mass of 200 kDa. This minimal molecular size of the functional enzyme was unaffected by either addition of cholestyramine to the rat diet or addition of 25-hydroxycholesterol plus mevalonate to the isolated rat hepatocytes. In addition, surviving enzyme protein was determined by immunoprecipitation of radiolabeled enzyme from hepatocytes that had been incubated with [35S]methionine before irradiation. The target size for loss of the monomer subunits was 98 kDa. The simplest interpretation of these results is that rat liver 3-hydroxy-3-methylglutaryl-CoA reductase in situ is a noncovalently linked dimer of the Mr = 97,200 enzyme subunit.

Animals↗
PubMed1985

Molecular size of bovine lipoprotein lipase as determined by radiation inactivation.

We have determined the size of the functional unit of bovine lipoprotein lipase by radiation inactivation. This was done in five different situations: 1) in a buffer with high salt concentration. In this situation the enzyme is relatively soluble and stable. 2) For an enzyme-heparin complex. This may reflect the physiological state of the enzyme at the vascular endothelium, where it is believed to be bound to a heparin-like molecule. 3) In the presence of lipid substrate and 4) with lipid substrate and activator protein. Here most of the enzyme is adsorbed to the substrate droplets. 5) For an enzyme-detergent complex; another model for enzyme-lipid interaction. In all five situations the enzyme activity decayed as an exponential function of radiation dose, and the target sizes were similar. The target size did not vary with the concentration of lipase protein. The combined data for bovine lipoprotein lipase yield a functional size of 72 kDa which is close to that expected for a dimer, 77 kDa.

Animals↗
PubMed1985

Functional unit size of the neurotoxin receptors on the voltage-dependent sodium channel.

Radiation inactivation was used in situ to determine the functional unit sizes of the neurotoxin receptors of the voltage-dependent sodium channel from rat brain. Frozen or lyophilized synaptosomes were irradiated with high energy electrons generated by a linear accelerator and assayed for [3H]saxitoxin, 125I-Leiurus quinquestriatus quinquestriatus (alpha-scorpion toxin), 125I-Centruroides suffusus suffusus (beta-scorpion toxin), and batrachotoxinin-A 20 alpha-[3H]benzoate binding activity. The functional unit size of the neurotoxin receptors determined in situ by target analysis are 220,000 for saxitoxin, 263,000 for alpha-scorpion toxin, and 45,000 for beta-scorpion toxin. Analysis of the inactivation curve for batrachotoxinin-A 20 alpha-benzoate binding to the channel yields two target sizes of Mr approximately 287,000 (50%) and approximately 51,000 (50%). The results are independent of the purity of the membrane preparation. Comparison of the radiation inactivation data with the protein composition of the rat brain sodium channel indicates that there are at least two functional components.

Animals↗
PubMed1985

Radiation inactivation of ricin occurs with transfer of destructive energy across a disulfide bridge.

The ionizing radiation sensitivity of ricin, a disulfide-linked heterodimeric protein, was studied as a model to determine the ability of disulfide bonds to transmit destructive energy. The radiation-dependent loss of A chain enzymatic activity after irradiation of either intact ricin or ricin in which the interchain disulfide bond was disrupted gave target sizes corresponding to the molecular size of dimeric ricin or monomeric A chain, respectively. These results clearly show that a disulfide bond can transmit destructive energy between protein subunits.

Disulfides↗
PubMed1985

Radiation inactivation studies of the benzodiazepine/gamma-aminobutyric acid/chloride ionophore receptor complex.

Radiation inactivation was used to estimate the molecular weight of the benzodiazepine (BZ), gamma-aminobutyric acid (GABA), and associated chloride ionophore (picrotoxinin/barbiturate) binding sites in frozen membranes prepared from rat forebrain. The target size of the BZ recognition site (as defined by the binding of the agonists [3H]diazepam and [3H]flunitrazepam, the antagonists [3H]Ro 15-1788 and [3H]CGS 8216, and the inverse agonist [3H]ethyl-beta-carboline-3-carboxylate) averaged 51,000 +/- 2,000 daltons. The presence or absence of GABA during irradiation had no effect on the target size of the BZ recognition site. The apparent molecular weight of the GABA binding site labelled with [3H]muscimol was identical to the BZ receptor when determined under identical assay conditions. However the target size of the picrotoxinin/barbiturate binding site labelled with the cage convulsant [35S]t-butylbicyclophosphorothionate was about threefold larger (138,000 daltons). The effects of lyophilization on BZ receptor binding activity and target size analysis were also determined. A decrease in the number of BZ binding sites (Bmax) was observed in the nonirradiated, lyophilized membranes compared with frozen membranes. Lyophilization of membranes prior to irradiation at -135 degrees C or 30 degrees C resulted in a 53 and 151% increase, respectively, in the molecular weight (target size) estimates of the BZ recognition site when compared with frozen membrane preparations. Two enzymes were also added to the membrane preparations for subsequent target size analysis. In lyophilized preparations irradiated at 30 degrees C, the target size for beta-galactosidase was also increased 71% when compared with frozen membrane preparations. In contrast, the target size for glucose-6-phosphate dehydrogenase was not altered by lyophilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Structure of the thyrotropin receptor and thyroid adenylate cyclase system as determined by target analysis.

Bovine thyroid plasma membranes were irradiated with high-energy electrons. Analysis of the target size of the thyrotropin (TSH) receptor revealed a complex pattern composed of a TSH binding component of 71 000 daltons and a large component (several hundred thousand daltons) that masked some of the binding. Both components were also observed when binding was assayed in the presence of 50 mM NaCl. Membranes preincubated with Mg2+ and 10 microM guanosine 5'-(beta,gamma-imidotriphosphate) [Gpp(NH)p], a persistent activator of adenylate cyclase, also showed the presence of these same components. Although the receptor for TSH has been reported to have some similarities to the receptor for cholera toxin, target analysis of [125I]iodocholera toxin binding was consistent with a single small component about the size of a ganglioside. Measurement of the target size of ground-state, i.e., not preactivated, adenylate cyclase was also carried out. The basal (Mn2+) adenylate cyclase yielded a Mr of 85 000, the smallest unit capable of producing cAMP. The Gpp(NH)p-responsive adenylate cyclase has a Mr of 150 000, which may reflect the contribution of the guanine nucleotide regulatory component to the mass of the active enzyme. A similar size was previously measured for the Gpp(NH)p-preactivated, detergent-solubilized thyroid enzyme [Asbury, R.F., Cook, G.H., & Wolff, J. (1978) J. Biol. Chem. 253, 5286-5292]. Radiation inactivation of the NaF-responsive enzyme indicated two or more components to this activity, the smaller of which (140 000 daltons) was similar in size to the ground-state Gpp(NH)p-responsive enzyme and the larger of which was greater than 10(6) daltons.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1984

The functional and physical form of mammalian cytochrome c oxidase determined by gel filtration, radiation inactivation, and sedimentation equilibrium analysis.

When solubilized in laurylmaltoside, cytochrome oxidases from beef heart and rat liver mitochondria exist as monodisperse populations that are stable, highly active, and have apparent molecular weights of 300,000 to 350,000, as measured by gel filtration. To determine whether these are monomeric (2 heme A, 2 Cu) or dimeric forms of the enzyme, we performed radiation inactivation and sedimentation equilibrium analyses. From radiation inactivation experiments under two different sets of conditions, we obtained estimates for the functional molecular weight of beef heart cytochrome oxidase of 114,000 and 99,000, much less than a dimer and significantly smaller than a 200,000 molecular weight monomer containing one copy of each of the 12 subunits normally present in the complex. The same functional size is obtained for a rat liver oxidase preparation depleted of subunit III. The physical molecular weight of cytochrome oxidase was determined by sedimentation equilibrium measurements in solvents of different densities using mixtures of H2O and D218O. Estimates of Mr = 194,000 +/- 9,000 for the beef heart oxidase and Mr = 152,000 +/- 6,000 for the rat liver enzyme were obtained, consistent with the size predicted for monomers of their subunit composition. From these results we conclude that mammalian cytochrome oxidases from beef heart and rat liver exist in laurylmaltoside as monomers capable of high rates of electron transfer and normal substrate binding. Further, these functions appear to be associated with a subset of the peptides present in the monomer, mainly composed of subunits I and II.

Animals↗
PubMed1984

Functional size of the human asialoglycoprotein receptor as determined by radiation inactivation.

The radiation target size of the functional asialoglycoprotein receptor (ASGP-R) in purified plasma membranes was determined for human liver and the human hepatoma cell line, Hep G2, by evaluation of the binding of 125I-asialoorosomucoid. Identical inactivation curves were observed for ASGP-R from normal human liver and post-mortem human liver and exhibited a functional unit of 70,000 daltons. The human hepatoma Hep G2 ASGP-R, in contrast, showed a target size of 140,000 daltons. These results suggest that the functional ligand-binding unit of the human ASGP-R is a multimer of the approximately 34,000-dalton unglycosylated receptor polypeptide (Schwartz, A. L., and Rup, D. (1983) J. Biol. Chem. 258, 11249-11255).

Asialoglycoprotein Receptor↗
PubMed1984

Minimal functional unit for transport and enzyme activities of (Na+ + K+)-ATPase as determined by radiation inactivation.

Frozen aqueous suspensions of partially purified membrane-bound renal (Na+ + K+)-ATPase have been irradiated at -135 degrees C with high-energy electrons. (Na+ + K+)-ATPase and K+-phosphatase activities are inactivated exponentially with apparent target sizes of 184 +/- 4 kDa and 125 +/- 3 kDa, respectively. These values are significantly lower then found previously from irradiation of lyophilized membranes. After reconstitution of irradiated (Na+ + K+)-ATPase into phospholipid vesicles the following transport functions have been measured and target sizes calculated from the exponential inactivation curves: ATP-dependent Na+-K+ exchange, 201 +/- 4 kDa; (ATP + Pi)-activated Rb+-Rb+ exchange, 206 +/- 7 kDa and ATP-independent Rb+-Rb+ exchange, 117 +/- 4 kDa. The apparent size of the alpha-chain, judged by disappearance of Coomassie stain on SDS-gels, lies between 115 and 141 kDa. That for the beta-glycoprotein, though clearly smaller, could not be estimated. We draw the following conclusions: (1) The simplest interpretation of the results is that the minimal functional unit for (Na+ + K+)-ATPase is alpha beta. (2) The inactivation target size for (Na+ + K+)-dependent ATP hydrolysis is the same as for ATP-dependent pumping of Na+ and K+. (3) The target sizes, for K+-phosphatase (125 kDa) and ATP-independent Rb+-Rb+ exchange (117 kDa) are indistinguishable from that of the alpha-chain itself, suggesting that cation binding sites and transport pathways, and the p-nitrophenyl phosphate binding site are located exclusively on the alpha-chain. (4) ATP-dependent activities appear to depend on the integrity of an alpha beta complex.

Animals↗
PubMed1984

Characterization of the glucocorticoid receptor. Comparison of wild type and variant receptors.

We have measured the size of the glucocorticoid receptors from two murine lymphoid cell lines, one displaying a wild type cytolytic response to hormone, the other a resistant variant. Using radiation inactivation and target analysis, we first compared the nuclear and cytoplasmic forms of the steroid receptors in a wild type line, WEHI 7.1 (W7). Within the variation expected for this type of measurement (+/- 14%), the nuclear and cytoplasmic forms have the same size, 75,000 and 79,000 daltons, respectively. We have also measured the size of the receptor in a hormone-insensitive "nuclear transfer-increased" (nti) variant (S49 143R). In contrast to reports indicating that the nti phenotype is associated with a much smaller cytoplasmic receptor, we found little or no difference in sizes of translocated receptor in wild type and nti cells. We have found significant differences, however, in the release of wild type and nti receptors from nuclei by nuclease digestion, salt, and spermidine. Approximately 80% of the nti receptor was readily released from nuclei incubated with micrococcal nuclease, while only 40-50% of the wild type receptor was released under similar conditions. The wild type nuclei also contained a fraction of receptor (approximately 30%) which was resistant to extraction by NaCl and spermidine. This fraction was greatly diminished in the nti nuclei. Thus, a portion of the wild type receptors appears to be stabilized within the nuclei, possibly through a type of interaction which cannot be sustained by the nti receptor.

Animals↗
PubMed1984

Functional molecular weight of the lac carrier protein from Escherichia coli as studied by radiation inactivation analysis.

Cytoplasmic membrane vesicles prepared from Escherichia coli containing multiple copies of the lac y gene were frozen in liquid nitrogen before or after generation of a proton electrochemical gradient (interior negative and alkaline) and irradiated with a high-energy electron beam at -135 degrees C. Subsequently, the lac carrier protein was extracted into octyl beta-D-glucopyranoside, reconstituted into proteoliposomes, and assayed for transport activity. Under all conditions tested, activity decreased as a single exponential function of radiation dosage, allowing straightforward application of target theory for determination of functional molecular mass. When lac carrier activity solubilized from nonenergized vesicles was assayed, the results obtained were consistent with a functional molecular size of 45-50 kDa, a value similar to the size of the protein as determined by other means. Similar values were obtained when the octyl beta-D-glucopyranoside extract was irradiated, and the target size observed for D-lactate dehydrogenase was in good agreement with the molecular size of this enzyme. Strikingly, when the same procedures were carried out with vesicles that were energized with appropriate electron donors prior to freezing and irradiation, a functional molecular size of 85-100 kDa was obtained for the lac carrier with no change in the target size of D-lactate dehydrogenase. In contrast, when the vesicles were energized under conditions in which the proton electrochemical gradient was collapsed, the target mass of the lac carrier returned to 45-50 kDa. The results indicate that the functional mass of the lac carrier protein is no greater than a dimer and suggest that the proton electrochemical gradient may cause an alteration in subunit interactions.

Escherichia coli↗
PubMed1983

Radiation inactivation of glutamate dehydrogenase hexamer: lack of energy transfer between subunits.

The effects of ionizing radiation on glutamate dehydrogenase and on fluorescein isothiocyanate--tagged glutamate dehydrogenase were analyzed by target theory. Enzymatic activity, fluorescence, and the survival of the 56,000-dalton monomer subunit were determined on frozen samples irradiated at -135 degrees C and on lyophilized samples irradiated at either -135 degrees C or +30 degrees C. The effects of temperature were the same for all three parameters. Enzymatic activity was lost after small doses of high-energy electrons, whereas fluorescence and monomer subunits survived much larger doses of radiation. Target analysis revealed that the functional unit size for enzymatic activity was the hexamer, confirming both the earlier radiation study and conventional biochemical analyses. Target sizes obtained from fluorescence and subunit structure measurements were close to that of the monomer. These results indicate that the primary ionization caused by electron bombardment results in damage to a single polypeptide strand and that there is no massive transfer of radiation energy to other units in the hexamer. The large target size observed for enzymatic activity appears to be a structural requirement for the simultaneous presence of six intact subunits rather than the result of the spread of energy from the initial site to adjacent chains with consequent damage to other subunits.

Animals↗
PubMed1983

Molecular weight of the functional unit of human leukocyte, fibroblast, and immune interferons.

There is good agreement between the target molecular weight and the known molecular weight of human leukocyte interferons (about 20,000). The target molecular weight of fibroblast interferon, 31,000 to 42,000, is significantly larger than the monomer molecular weight of 21,000 to 24,000, suggesting that the dimer may be the predominant active functional unit in solution. A range from 63,000 to 73,000 for the target molecular weight of several different fractions of immune interferon (including natural crude as well as the recombinant form) indicates that the functional form of the immune interferon may be a trimer or tetramer. Thus, these studies indicate that the functional unit of leukocyte interferon is the monomer, that of fibroblast interferon is a dimer, and that of immune interferon is probably a tetramer (or trimer).

Humans↗
PubMed1983

Lipoprotein lipase: size of the functional unit determined by radiation inactivation.

Radiation inactivation was used to determine the functional molecular weight of lipoprotein lipase (LPL) in rat heart and adipose tissues. This technique reveals the size of the smallest unit required to carry out the enzyme function. Supernatant fractions of the tissue homogenates were exposed to high energy electrons at -135 degrees C. LPL activity showed a simple exponential decay in all samples tested. Because changes in nutritional state shift the distribution of LPL between the capillary endothelial and parenchymal cells within heart and adipose tissues, fasted and refed rats were used for the radiation studies. The functional molecular weight was calculated to be 127,000 +/- 15,000 (mean +/- SD) daltons for heart and adipose. Thus, the smallest unit required for enzyme function was the same in both of these tissues and did not vary with nutritional state. The data suggest that, compared with LPL monomer sizes reported in the range 55,000 to 72,000, this active unit constitutes a dimer.

Adipose Tissue↗
PubMed1982

The influence of low temperature on the radiation sensitivity of enzymes.

When enzymes are exposed to ionizing radiation at low temperatures there is a progressive decrease in radiation sensitivity: considerably more enzymatic activity remains after the same dose of radiation at low temperature compared to room temperature. Detailed studies of five enzymes reveals the quantitative relationship between radiation sensitivity and temperature during exposure. Although 25 enzymes are shown to display this same relationship, recent reports have denied this effect in three enzymes. In this paper, we investigate two possible artifacts that could cause these discrepancies: 1) inaccurate determination of the temperature of the sample during irradiation, and 2) use of temperature-sensitive dosimeters to measure radiation dose. Procedures are described that carefully control these parameters. Thermoluminescent dosimeters are shown to be independent of temperature effects. These methods are used to investigate one of the enzymes, malate dehydrogenase, that has been reported to have a temperature-insensitive radiation inactivation. The radiation sensitivity of this enzyme is found to show the same temperature dependence as 24 other enzymes.

Cold Temperature↗