Rat heart gap junctions as disulfide-bonded connexon multimers: Their depolymerization and solubilization in deoxycholate

Summary Unproteolyzed gap junctions isolated from rat heart and liver were analyzed for the presence of inter-subunit disulfide bonds by sodium dodecylsulfate polyacrylamide gel electrophoresis. Rat cardiac junctions contained multiple disulfide bonds connecting theM _r 47,000 subunits of the same connexon and of different connexons. Inter-subunit disulfide bonds were absent in liver junctions. Unproteolyzed rat heart gap junctions were resistant to deoxycholate in their oxidized state, but dissolved readily in the detergent when the disulfide bonds were cleaved with -mercaptoethanol. Disulfide bonding in proteolyzed cardiac junctions was limited to pairs ofM _r 29,500 subunits. These junctions were not soluble in deoxycholate even in the presence of -mercaptoethanol. These results show that heart and liver junctions differ in their quarternary organization.     

Isolation and Characterization of Pyrophosphate:D-Fructose-6-phosphate 1-Phosphotransferase from Cucumber Seeds

Pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase waspurified over 700-fold from germinating cucumber (Cucumis sativuscv. Fletcher) seeds. The purified enzyme has a specific activityof 5.2 µmol.min^–1.mg protein^–1 in the presenceof 1 µM fru-2,6-P₂. The pH optima is similar for boththe forward and reverse reactions (pH 7.5–7.8). Magnesium,manganese and cobalt activate the enzyme, with the highest affinitybeing for magnesium. The enzyme exhibits normal Michaelis-Mentenkinetics in both the presence and absence of fru-2,6-P₂. Half-maximumactivation of the enzyme was obtained with 35 nM fru-2,6-P2.Fru-2,6-P₂ stimulates activity by increasing V_max and increasingthe affinity for fru-6-P, fru-1,6-P₂ and PPi. Phosphate causesnoncompetitive inhibition with respect to both fru-6-P and PPi.On the basis of the steadystate substrate interaction and Piinhibition data a sequential ternary complex mechanism is proposed. (Received April 28, 1986; Accepted July 9, 1986)     

Molecular motions and thermotropic phase behavior of cholesteryl esters with triolein

The phase behavior of cholesteryl esters with triglyceride has been characterized by differential scanning calorimetry (DSC), light microscopy, and polarizing light microscopy (PLM). Temperature-dependent molecular motions determined by 13C NMR spectroscopy were correlated with thermotropic phase behavior. Two systems, cholesteryl oleate (CO) and a 3/1 w/w mixture of cholesteryl linoleate (CL) and CO, were examined in the presence of small amounts of triolein (TO). Both systems exhibited metastable cholesteric and smectic (or only smectic) phases. Increasing amounts of TO progressively lowered the liquid-crystalline phase transition temperatures and eventually abolished the cholesteric phase, but at differing amounts of TO for the two systems (between 4% and 5% with CL/CO and between 7% and 10% with CO). DSC and PLM showed a progressive broadening of the phase transitions as well as an overlapping of the temperature ranges of the cholesteric and smectic phases. At greater than or equal to 4% TO, a separate isotropic liquid phase coexisted with liquid-crystalline phases. 13C NMR spectroscopy was used to monitor the molecular motions of the cholesteryl ester steroid ring and acyl chain in liquid and liquid-crystalline phases. In the liquid phase, no significant changes in fatty acyl motions, as reflected in spin-lattice relaxation time (T1) and nuclear Overhauser enhancement (NOE) values, were found on addition of TO. The line width (v 1/2) of the steroid ring resonances increased markedly near (1-5 degrees C above) the isotropic liquid----liquid-crystal phase transition temperature (TLC). However, the C3/C6 v 1/2 ratio at 1 degree C above TLC was greater for mixtures exhibiting an isotropic----cholesteric transition than for mixtures exhibiting an isotropic----smectic transition. Rotational correlation times calculated for motions about the long molecular axis and the nonunique axis showed (i) that the ring motions became more anisotropic as TLC was approached and (ii) that the motions were more anisotropic at TLC + 1 degree C for systems exhibiting a cholesteric phase than for systems exhibiting only a smectic phase. 13C line widths in spectra of the cholesteryl ester liquid-crystalline phases suggested that TO perturbed the cholesteryl ester intermolecular interactions and increased the rates of cholesteryl ester molecular motions relative to neat esters.     

Structure and polymorphism of 1,2-dioleoyl-3-acyl-sn-glycerols. Three- and six-layered structures

Triacylglycerols, which usually contain at least one unsaturated fatty acid, are the most important forms of stored biological lipids in teleosts, mammals, and most plants. Since the physical properties of such mixed-chain triacylglycerols are poorly understood, a systematic study of such compounds has been initiated. Stereospecific 1,2-dioleoyl-3-acyl-sn-glycerols were synthesized with even carbon saturated fatty acyl chains of 14-24 carbons in length. Their polymorphic behavior was examined by differential scanning calorimetry and X-ray powder diffraction. The thermal behavior revealed from one to four major polymorphic transitions depending upon saturated chain length. Plots of enthalpy of fusion and entropy vs. carbon number for melting of the most stable polymorph were linear throughout the series with slopes of 1.0 kcal/mol per carbon atom and 2.6 cal/(mol K) per carbon atom, respectively. These slopes indicate that the saturated chains are packed in a well-ordered tightly packed lattice. When the compounds were rapidly cooled to 5 degrees C, X-ray powder diffraction revealed strong beta' (ca. 3.8 and 4.2 A) reflections and weak beta (ca. 4.6 A) reflections. The beta subcell reflections intensified when the compounds were heated to within 5 degrees C of the melting temperature of the highest melting polymorph. Evidence of an alpha phase was not seen on 30-min X-ray exposures for any of the compounds. In the proposed packing arrangement the saturated and unsaturated chains are segregated into layers. The stable form of all compounds exhibits a triple layer packing mode in which a bilayer of oleoyl chains is segregated from an interdigitated layer of saturated chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a photoreactivation-deficient mutant of Chlamydomonas

A mutant deficient in photoreactivation has been isolated following mutagenesis of Chlamydomonas reinhardi with N-methyl-N'-nitro-N'-nitrosoguanidine. The mutant is deficient in the photorepair of pyrimidine dimers from nuclear DNA but appears to be normal in the rate of photorepair of dimers from chloroplast DNA. Cell-free extracts prepared from the photoreactivation-deficient mutant have about 17% of the DNA photolyase activity of wild-type cells. These results are consistent with the hypothesis that nuclear and chloroplast DNA photolyases are controlled by two separate genes.     

Binding of [3H]Serotonin to Skeletal Muscle Actin

We previously observed that the neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) binds with high- and low-affinity interactions to an actin-like protein prepared from rat brain synaptosomes. In this study, we examined its binding to highly purified actin obtained from rabbit skeletal muscle. Monomeric G-actin bound serotonin with high and low affinities, exhibiting equilibrium dissociation constants (KD values) of 5 X 10(-5) M and 4 X 10(-3) M, respectively. The serotonin binding site on actin was distinct from those sites previously characterized for divalent cations, nucleotides, and cytochalasin alkaloids. The binding of serotonin (1 microM) to G-actin was increased as much as 26-fold by divalent cations. Potassium iodine (KI) increased the affinity of G-actin for serotonin, KD values for this binding being 3 X 10(-7) M and X 10(-5) M. Serotonin bound with even higher affinity to polymerized F-actin, with KD values of 2 X 10(-8) M and 2 X 10(-5) M. However, the total number of binding sites on F-actin was only about 4% of the number of G-actin. The binding of serotonin (0.1 microM) to G-actin could be inhibited by phenothiazines (1 microM) or reserpine (10 microM), but not by classical antagonists of serotonin receptors or by drugs that release serotonin or inhibit its uptake. The binding of serotonin to actin in vivo may participate in a contractile process related to neurotransmitter release.     

Association of Serotonin, Dopamine, or Noradrenaline with an Actin-Like Component in Pheochromocytoma (PC12) Cells

A rat pheochromocytoma (PC12) cell line was used to examine the possibility that 5-hydroxytryptamine (serotonin), 3,4-dihydroxyphenylethylamine (dopamine), or noradrenaline may be associated with cytoplasmic actin, as was suggested by previous in vitro binding studies on an actin-like protein from rat brain synaptosomes. When PC12 cells were incubated with [3H]serotonin. [3H]dopamine, or [3H]noradrenaline for 30 min at 37 degrees C, approximately 2-4% of the radioactivity present in the cells was found to be associated with a high-molecular-weight (actin-like) component in supernatant fractions. Evidence relating this monoamine binding component to actin filaments includes: (a) its strong absorption by myosin filaments at low ionic strength: (b) a decrease in its affinity for myosin in the presence of 1 mM ATP, which lowers the affinity of authentic actin for myosin: (c) displacement of bound [3H]serotonin from it by DNase I, which binds strongly to actin and which inhibits [3H]serotonin binding to actin in vitro; (d) an increase in its binding of each monoamine (by 25-40%) after PC12 cells were preincubated with 10 microM cytochalasin B (a drug that induces depolymerization of F-actin). These findings suggest that serotonin, dopamine, or noradrenaline may associate with actin filaments in vivo.     

The effects of crude oil on the growth of Spartinaalterniflora Loisel. and Spartina cynosuroides (L.) Roth

Greenhouse studies were conducted to examine the effects of crude oil on the growth of Spartinaalterniflora Loisel. and S. cynosuroides (L.) Roth from North Carolina. The way in which crude oil came into contact with the plant tissue and/or substratum was an important factor in determining the responses of both species to oil pollution. Plants recovered from a single application of oil to aerial tissue with relatively little impact on productivity. The presence of an oil layer on the surface of an overlying layer of water had little impact on existing aerial portions of S. alterniflora plants; however, regrowth following harvest was completely inhibited. Incorporation of oil into the substratum significantly reduced aerial productivity and regrowth of S. alternflora and S. cynosuroides. Observations suggest that decreased productivity and regrowth may have been caused by decreased root and rhizome growth. Regrowth potential of S. alterniflora grown in oiled substratum was greater in fine-textured marsh substratum than in sand substratum.     

Column cellulose hydrolysis reactor: Cellulase adsorption profile

Summary A column cellulose hydrolysis reactor was set up using a single passage of cellulase enzyme which was followed with a continuous percolation of buffer. Hydrolysis rates were found to decline precipitously upon the removal of the non-adsorbed cellulase components. By comparing specific activities of the cellulase before and after adsorption on the cellulose column, it was concluded that the adsorption efficiencies for the cellulase components decreased from exoglucanase (1,4--d-glucan cellobiohydrolase EC 3.2.1.91) to endoglucanase [1,4-(1,3;1,4)--d-glucan 4-glucanohydrolase, EC 3.2.1.4] to -glucosidase (-d-glucoside glucohydrolase, EC 3.2.1.21). Of the adsorbed cellulase components, the rate of endoglucanase leaching from the cellulose column was 20 times that for the exoglucanase despite the greater adsorption efficiency of the latter. By analysing the cellulase components which were bound and not bound by the cellulose column and comparing them with a purified exoglucanase enzyme on sodium dodecyl sulfate polyacrylamide gels, it was confirmed that the major cellulase component adsorbed to the cellulose column was an exoglucanase component. The resultant loss of other cellulase components from the reactor was probably the cause for the much reduced rate of cellulose hydrolysis when these components were flushed out of the column.