Biomedical subjects

R B Sisk

Publications and source records attributed to R B Sisk.

6 recordsLinked to original sources

PubMed1992

Calorimetric studies on the influence of N-methylated headgroups on the mixing behavior of diheptadecanoyl phosphatidylcholine with 1-behenoyl-2-lauroylphosphatidylcholine.

Recent studies of five different phosphatidylcholine/phosphatidylcholine (PC/PC) systems indicate that binary mixtures of phosphatidylcholines in which one component has a normalized chain length difference (delta C/CL) in the range of 0.09-0.40 and the other a delta C/CL in the range of 0.42-0.57 exhibit the phase behavior of a eutectic system. Here, delta C is the effective chain-length difference between the two acyl chains, and CL is the effective length of the longer of the two acyl chains for the same lipid molecule in the gel state. In each mixture, gel phase immiscibility occurs over a wide compositional range due to the difference in the gel phase acyl chain packing properties of each component. Although the mixtures differ in the location of their eutectic horizontal, with respect to temperature, all have a similar eutectic point that occurs at a composition of approximately 40 mol percent of the component with the delta C/CL value in the range of 0.42-0.57. Here, we extend these studies by systematically modifying the headgroup of C(17):C(17)PC and then analyzing the mixing behavior of the modified lipid with C(22):C(12)PC using DSC. Progressive demethylation of the C(17):C(17)PC headgroup leads to an increase in gel phase immiscibility and a decrease in the amount of C(22):C(12)PC that comprises the eutectic composition. The temperature defining the location of the eutectic horizontal, however, remains virtually unchanged in all three phase diagrams. Our results suggest that the eutectic composition is influenced by changes in gel phase acyl chain packing that are dependent on headgroup-headgroup interactions. In contrast, the eutectic nature of the phase diagram and the location of its solidus line are properties of acyl chain interactions that are independent of phospholipid headgroup-headgroup interactions.

Calorimetry, Differential Scanning↗

PubMed1991

Fluid-phase connectivity and translational diffusion in a eutectic, two-component, two-phase phosphatidylcholine bilayer.

In recent work [Vaz, W.L.C., Melo, E.C.C., & Thompson, T.E. (1989) Biophys. J. 56, 869-876] we have shown that translational diffusion studies using fluorescence recovery after photobleaching (FRAP) provide information concerning domain structures and fluid-phase connectivity in lipid bilayers in which solid and fluid phases coexist. In the present paper, translational diffusion of the fluid-phase-soluble, solid-phase-insoluble fluorescent lipid derivative N-(7-nitrobenzoxa-2,3-diazol-4-yl) dilauroyl-phosphatidylethanolamine and the fluid-phase connectivity are examined in lipid bilayers prepared from binary mixtures of 1-docosanoyl-2-dodecanoylphosphatidylcholine (C22:0C12:0PC) and 1,2-diheptadecanoylphosphatidylcholine (di-C17:0PC) by using FRAP. The phosphatidylcholine mixture used provides a eutectic system with a eutectic point at a composition of about 0.4 mole fraction of di-C17:0PC and a temperature of about 37 degrees C [Sisk, R.B., Wang, Z.Q., Lin, H.N., & Huang, C.H. (1990) Biophys. J. 58, 777-783]. Two regions in temperature and composition, respectively below and above 0.4 mole fraction of di-C17:0PC, where fluid and solid phases coexist in the same lipid bilayer, are available for examination of fluid-phase connectivity. In mixtures containing less than 0.4 mole fraction of di-C17:0PC the fluid phase coexists with a mixed interdigitated Lc gel phase composed mostly of C22:0C12:0PC, whereas in mixtures containing greater than 0.4 mole fraction of di-C17:0PC the fluid phase coexists with a P beta' gel phase mostly composed of di-C17:0PC. When the solid phase is a P beta' gel phase, the temperature of fluid-phase connectivity for the mixtures lies close to the fluidus, which means that a small (approximately 20%) mass fraction of solid phase can divide the large bulk of the bilayer that is fluid into nonconnected domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Diffusion↗

PubMed1990

Mixing behavior of identical molecular weight phosphatidylcholines with various chain-length differences in two-component lamellae.

It has recently been suggested that mixed-chain phosphatidylcholines with normalized chain length differences (deltaC/CL) in the range of 0.10-0.40 undergo spontaneous self-assembly in excess water at T less than Tm into the partially interdigitated bilayer and those with delta C/CL values in the range of 0.44-0.57 form, in excess water, mixed interdigitated bilayers at T less than Tm. The mixing behavior of binary mixtures of C(22):C(12)PC/C(17):C(17)PC, C(22):C(12)/C(15):C(19)PC, and C(15):C(19)PC/C(13):C(21)PC reported in this work is used to support this view. The values of delta C/CL for C(17):C(17)PC, C(15):C(19)PC, C(13):C(21)PC, and C(22):C(12)PC are 0.10, 0.15, 0.35, and 0.55, respectively. The binary mixture of C(15):C(19)PC/C(13):C(21)PC exhibits a lens-shaped phase diagram, indicating that these two identical molecular weight (MW) lipids with delta C/CL values less than 0.4 are completely miscible over the entire compositional range in both gel and liquid-crystalline phases. In contrast, the phase diagrams of C(22):C(12)PC/C(17):C(17)PC and C(22):C(12)PC/C(15):C(19)PC are eutectic, indicating immiscibility of the component lipids over a wide compositional range in the gel phase. This immiscibility of identical MW lipids in the bilayer plane can be attributed to the different packing properties of the component lipids in the bilayer at T less than Tm.

Calorimetry, Differential Scanning↗

PubMed1987

Evidence for the activation of the multifunctional Ca2+/calmodulin-dependent protein kinase in response to hormones that increase intracellular Ca2+.

The phosphorylation state of six cytoplasmic proteins is increased following treatment of isolated rat hepatocytes with hormones that elevate free intracellular Ca2+ levels (Garrison, J. C. and Wagner, J. D. (1982) J. Biol. Chem. 257, 13135-13143). Tryptic 32P-phosphopeptide maps of two of the substrates, pyruvate kinase and a 49,000-dalton protein, the major 32P-labeled protein in hepatocytes, were prepared following stimulation of cells with vasopressin, a Ca2+-linked hormone. Peptide maps of the 49,000-dalton protein phosphorylated in vitro with the recently identified multifunctional Ca2+/calmodulin-dependent protein kinase contained phosphopeptides identical to those observed in the intact cell, suggesting that this kinase is activated in response to Ca2+-mobilizing hormones. Similar in vitro phosphorylation experiments with pyruvate kinase suggested that the Ca2+/calmodulin-dependent protein kinase can phosphorylate not only the serine residues observed following vasopressin stimulation of the intact cell but also additional threonine residues. Both pyruvate kinase and the 49,000-dalton protein are also phosphorylated in the hepatocyte in response to glucagon and in vitro by the cAMP-dependent protein kinase. Both vasopressin and glucagon appear to stimulate the phosphorylation of identical serine residues in pyruvate kinase but only vasopressin enhances the phosphorylation of certain sites in the 49,000-dalton protein. Comparison of the tryptic phosphopeptide maps of these substrates phosphorylated in vitro with either the Ca2+/calmodulin-dependent protein kinase or the cAMP-dependent protein kinase suggests that the Ca2+-dependent kinase can phosphorylate unique sites in both substrates. It appears to share specificity at other sites with the cAMP-dependent protein kinase. Overall, the results suggest that the multifunctional Ca2+/calmodulin-dependent protein kinase plays an important role in the response of the hepatocyte to a Ca2+ signal.

Calcium↗

PubMed1987

A study of the mechanism of glucagon-induced protein phosphorylation in isolated rat hepatocytes using (Sp)-cAMPS and (Rp)-cAMPS, the stimulatory and inhibitory diastereomers of adenosine cyclic 3',5'-phosphorothioate.

Maximal doses of glucagon increase the phosphorylation state of 12 cytosolic proteins in isolated hepatocytes from fasted rats (Garrison, J. C., and Wagner, J. D. (1982) J. Biol. Chem. 257, 13135-13143). Incubation of hepatocytes with lower concentrations of glucagon indicates that a hierarchy of substrates exists with the concentration of glucagon required for half-maximal increases in phosphorylation varying 5-15-fold. The proteins whose phosphorylation state is most sensitive to low concentrations of glucagon are pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, both of which play key roles in the regulation of gluconeogenesis. Treatment of hepatocytes with (Sp)-cAMPS, the stimulatory diastereomer of adenosine cyclic 3',5'-phosphorothioate, mimics the response seen with glucagon. When hepatocytes are pretreated with the cAMP antagonist, (Rp)-cAMPS, the phosphorylation response is abolished at low concentrations of glucagon, and the dose of glucagon required for half-maximal stimulation of phosphorylation is increased 5-10-fold. The (Sp)-cAMPS-stimulated increases in phosphorylation state are also blunted by (Rp)-cAMPS. These results provide direct pharmacological evidence for the activation of the cAMP-dependent protein kinase in response to glucagon in the intact cell. Although low doses of glucagon appear to stimulate protein phosphorylation via the cAMP-dependent protein kinase, high doses of glucagon also cause a small increase in the concentration of free intracellular Ca2+ in hepatocytes. The glucagon-stimulated increases in the level of Ca2+ can be mimicked by (Sp)-cAMPS and inhibited by pretreatment with (Rp)-cAMPS. These results suggest that glucagon can elevate intracellular Ca2+ via cAMP and the cAMP-dependent protein kinase.

Animals↗

PubMed1986

Pertussis toxin or phorbol 12-myristate 13-acetate can distinguish between epidermal growth factor- and angiotensin-stimulated signals in hepatocytes.

Epidermal growth factor (EGF) causes rapid increases in free intracellular Ca2+ and stimulates the phosphorylation of 11 cytosolic proteins in hepatocytes. Ten of the 11 cytosolic proteins altered by EGF are identical to those affected by angiotensin II, a hormone that stimulates the breakdown of phosphatidylinositol 4,5-bisphosphate. An increase in the phosphorylation of the other protein, spot c (Mr = 36,000, pI = 5.5), is observed only with EGF. Treatment of intact rats with pertussis toxin to ADP-ribosylate Ni, the inhibitory GTP-binding protein of the adenylate cyclase complex, abolished the effect of EGF on Ca2+ mobilization and on the phosphorylation of the 10 proteins affected in common with angiotensin II. This treatment had minimal effects on the ability of EGF to stimulate the phosphorylation of its unique substrate, spot c. In marked contrast, modification of Ni did not block the ability of angiotensin II to stimulate Ca2+ mobilization or protein phosphorylation. Pretreatment of normal hepatocytes with 4 beta-phorbol 12-myristate 13-acetate blocked all responses to EGF, including the increased phosphorylation of spot c, but had no effect on the responses to angiotensin II. These results imply that Ni or a similar pertussis toxin substrate may mediate the apparent effects of EGF on phosphatidylinositol breakdown and that protein kinase C may regulate a site in the transduction pathway. Angiotensin II appears to use a different signal transduction mechanism to stimulate phosphatidylinositol metabolism in hepatocytes.

Adenylate Cyclase Toxin↗