Gene L. Cottam

Glycosylation of LDL decreases its ability to interact with high-affinity receptors of human fibroblasts in vitro and decreases its clearance from rabbit plasma in vivo

Incubation of human LDL in vitro at 37 degrees C for 48 h with [14C]glucose at concentrations from 5 to 200 mM resulted in a glycosylated LDL, containing 0.4-20 mol of glucose incorporated per apolipoprotein B of 250 000 daltons. The extent of glucose incorporated was proportional to the time of incubation and concentration of glucose. Glycosylation of LDL abolished its uptake and degradation by the high-affinity process for LDL in normal human skin fibroblasts. 125I-labeled glycosylated LDL was bound, internalized and degraded by the fibroblasts via a nonspecific low-affinity process. The 125I-labeled glycosylated LDL and 125I-labeled LDL were taken up and degraded at similar rates in a non-saturable, low-affinity process by peritoneal macrophages isolated from mice. When 125I-labeled glycosylated LDL or 125I-labeled LDL were injected into rabbits, the glycosylated LDL had a delayed plasma clearance in comparison to the LDL. The mean fractional catabolic rates were 0.67 day-1 and 1.70 day-1 for 125I-labeled glycosylated LDL and 125I-labeled LDL, respectively. The uptake and degradation of 125I-labeled LDL by human skin fibroblasts was decreased as the concentration of free carbohydrate, glucose, sucrose or sorbitol, in the medium was increased from 10 mM to 1 M. It is speculated that pathologic levels of plasma glucose in vivo could result in a decrease in LDL uptake as a result of glycosylation of LDL. A decrease in uptake of native or modified LDL in vivo could contribute to hypercholesterolemia and its pathophysiology.

The sulfhydryl groups of citrate cleavage enzyme

Abstract Chicken liver citrate cleavage enzyme has an apparent optimum activity at pH 8.7, similar to the enzyme isolated from rat liver. Early results indicating a lower pH optimum were due to a pH dependent disulfide inhibition of the enzyme. Both chicken liver and rat liver citrate cleavage enzymes are inhibited by oxidized glutathione, oxidized BAL, lipoate, and arsenite. Studies with the purified rat liver enzyme indicate the presence of 6–8 moles of very reactive SH groups per mole of enzyme which are essential for enzyme activity. These groups can be protected by Mg-citrate but not by Mg-ATP. The loss of enzyme activity observed upon storage and its subsequent reactivation by dithiothreitol does not result from changes in aggregation and dissociation of the enzyme as judged by gel electrophoresis and ultracentrifugation.

Proton relaxation rate and fluorometric studies of manganese and rare earth binding to concanavalin A.

Abstract The binding of Mn 2+ , Ca 2+ , and rare earth ions to apoconcanavalin A has been studied by water proton relaxation enhancement, electron paramagnetic resonance spectroscopy, and fluorescence spectroscopy. An electron paramagnetic resonance and water proton relaxation rate study of the titration of apoconcanavalin A with Mn 2+ gives evidence of two equivalent binding sites per monomer with K D = 50 μ m ± 4 μ m. When a similar Mn 2+ titration of apoconcanavalin A is performed in the presence of Ca 2+ ion, very little free Mn 2+ is detected by electron paramagnetic resonance until the two M n 2+ binding sites per monomer are filled. The substitution of a rare earth ion for Ca 2+ ion in the above experiment often resulted in a slight displacement of Mn 2+ from the transition metal site as detected by electron paramagnetic resonance. A water proton relaxation rate study of the titration of apoconcanavalin A with Gd 3+ reflects two binding sites with a K D = 40 μ m ± 4 μ m and two with a K D = 200 μ m ± 50 μ m. The fluorescence emission spectrum of concanavalin A (λ em = 340 nm) is slightly quenched by the addition of Tb 3+ while Tb 3+ fluorescence is greatly enhanced. A fluorometric titration of apoconcanavalin A with Tb 3+ also reflects two sites with a K D = 40 μ m ± 15 μ m and two with a K D = 270 μ m ± 50 μ m.

Gadolinium as a probe of the alkaline earth and ATP-metal binding sites in pyruvate kinase

Abstract The rare earth gadolinium forms a binary enzyme-metal complex with muscle pyruvate kinase which enhances the water proton relaxation rate ( ϵ b = 12 ± 2). Analysis of a Scatchard plot of the binding data indicates 3.7 ± 0.5 gadolinium binding sites with K d = 26 ± 10 μM per protein of 237,000 daltons. The transition metal ion, manganese, is displaced from the enzyme by the rare earths, gadolinium, neodymium, thulium, and lanthanum as well as the alkaline earths, magnesium and calcium suggesting all of these metal ions bind to the same site on the protein. Upon addition of ATP to a solution of gadolinium and enzyme a decrease in enhancement is observed which is consistent with the formation of a metal bridge complex. Because of the low dissociation constant for the Gd-ATP complex (0.1 μ m ) it is possible to directly measure the dissociation of the Gd-ATP complex from the ternary enzyme-Gd-ATP complex, K 2 = 13 μM ± 4 μM. However, a ternary complex of phospho-enolpyruvate-Gd-enzyme is not detected by water proton relaxation rate enhancement measurements which leads to speculation that the ionic radius of gadolinium (0.94 A) is so large that it results in a distortion of the phosphoenolypyruvate binding site on pyruvate kinase thus preventing phosphoenolpyruvate binding.

Nature of the phosphorylated residue in citrate cleavage enzyme

Abstract Citrate cleavage enzyme was phosphorylated with γ-32P-ATP. A 32P-compound was isolated by Dowex chromatography of an alkaline hydrolysate of the 32P-phosphoenzyme. The isolated compound behaved chromatographically and electrophoretically like phosphohistidine.

Research: Lipid Peroxidation Following Thermal Injury

Formation of lipid peroxides in vivo leads to a variety of normal and pathologic consequences. For instance, peroxidation of cell membrane lipids results in altered membrane properties and increased cell destruction. Following severe thermal injury, elevated plasma levels of malondialdehyde, one product of lipid peroxidation, were detected, which suggests that an elevated level of lipid peroxidation was occurring in those patients. In vivo the major mechanisms to protect cells from lipid peroxidation are the presence of vitamin E, the fat-soluble antioxidant, and destruction of peroxides by glutathione peroxidase. In four of five patients investigated, the plasma content of vitamin E was low. The erythrocyte glutathione peroxidase activity was initially low in two patients and, by 15 days postburn, was below normal values in the other three patients. The results suggest that lipid peroxidation was occurring at elevated levels in these severely burned patients. An increased rate of lipid peroxidation could contribute to the decreased levels of phosphatidylethanolamine and essential polyunsaturated fatly acids in plasma and erythrocyte lipids and the shortened erythrocyte lifetimes previously observed in severely burned patients.

Dehydroisoandrosterone prevention of autoimmune disease in NZB/W F1 mice: lack of an effect on associated immunological abnormalities

The effect of dietary dehydroisoandrosterone (DHA) on several immunological abnormalities associated with the development of systemic lupus erythematosus in New Zealand Black/New Zealand White F1 (NZB/W) female mice was examined. Despite the extraordinary benefits in prolonged survival and decreased synthesis of antibodies to double-stranded DNA obtained by adding DHA (0.4% w/v) to the diet fed to these mice (Lucas et al. (1985) J. Clin. Invest. 75, 2091-2093), remarkably small changes in the chemistry and function of the immune system were detected. DHA prevented the increases in spleen mass and in peritoneal cell number which occur with age in NZB/W female mice, but did not prevent the development of hypergammaglobulinemia. DHA did not affect peritoneal macrophage functions as measured by the phagocytosis of opsonized and non-opsonized sheep erythrocytes, or the zymosan-stimulated release of PGE2, 6-ketoPGF1 alpha, TXB2 and LTC4. In spleen, DHA delayed the loss of T-cell mitogenic responses until 5.5 months of age, but did not alter the spleen lymphocyte population.

A secreted peptidase involved in T cell β-endorphin metabolism

Abstract β-endorphin metabolism by CD4 + and CD8 + T cells, and the thymoma cell line, EL4, was investigated. In all three cell types, extracellular β-endorphin was metabolized exclusively by a secreted, metal-dependent, thiol peptidase. The enzyme activity is expressed constitutively in EL4 cells and following activation of CD4 + and CD8 + T cells with anti-CD3 antibody. The enzyme is not one of the proteinases associated with cytolytic T cells and does not appear to be identical with any previously described β-endorphin metabolizing enzyme. The enzyme cleaves β-endorphin at approximately equal rates at either of two sites to yield β-endorphin 1–17 (which is γ-endorphin), β-endorphin 1–18 , β-endorphin 18–31 and β-endorphin 19–31 . Evidence in the literature indicates that these N- and C-terminal peptides which contain, respectively, the opioid and non-opioid receptor binding domains of β-endorphin, are biologically active. Thus, it is likely that this new T cell peptidase has important immunoregulatory activity.

Reversible solubility of deoxyhemoglobin S.

Abstract The solubility of deoxyhemoglobin S in 1.96 M phosphate is sensitive to changes in oxygenation and temperature in a manner similar to the widely used in vitro gelation assay. In addition, the pH of the phosphate buffer used in the solubility determination has a profound effect on deoxyhemoglobin S solubility. It is suggested that solubility in 1.96 M phosphate may be a sensitive method of monitoring the aggregation phenomenon of deoxyhemoglobin S.

Anti-CD3 activation of human CD4+ T cells increases expression of the intracellular β-endorphin endopeptidase (IDE/γ-EpGE)

In this study, increased expression of an endopeptidase hydrolyzing β-endorphin (β-Ep) to γ-endorphin (γ-Ep, β-Ep1–17) was observed upon immobilized anti-CD3 stimulated activation of human peripheral blood CD4+ T cells (hCD4+ T cells). Although freshly isolated hCD4+ T cells are devoid of significant β-Ep endopeptidase activity (<0.1 nmol h−1 106 cells−1), activation of these cells with immobilized anti-CD3 results in a time dependent appearance of β-Ep endopeptidase activity which reaches a maximal value of 17.4±0.48 nmol h−1 106 cells−1 after 48 h of culture. Significant up-regulation of both mRNA encoding IDE/γ-EpGE and immunoreactive protein are observed in anti-CD3 stimulated hCD4+ T cells, indicating transcription and translation of IDE/γ-EpGE may be elevated. No significant hydrolysis of exogenous β-Ep is observed with intact hCD4+ T cells whether quiescent or activated or from preparations of hCD4+ T cell membranes. Therefore, this activity appears to be intracellular. Immunoreactive IDE/γ-EpGE is detected inside activated hCD4+ T cells. Analysis of metabolites generated upon hydrolysis of β-Ep with lysed activated hCD4+ T cell preparations identified the presence of: β-Ep1–18, β-Ep2–18, β-Ep1–17, β-Ep2–17, β-Ep18–31, β-Ep19–31, β-Ep1–13, β-Ep2–13, β-Ep18–26, and β-Ep20–31 as major metabolites and the majority of these are consistent with β-Ep hydrolytic activity attributable to IDE/γ-EpGE.