Somatostatin-detergent interaction

The effect of cationic, anionic and nonionic detergents on the EPR spectrum of spin-labeled somatostatin has been studied. At detergent concentrations well above the critical micelle concentration, nonionic detergents do not alter the EPR spectrum. Sodium dodecyl sulfate markedly alters both the line height ratio and the hyperfine splitting constant, whilst dodecyltrimethylammonium bromide alters only slightly the hyperfine splitting constant and line height ratio. The somatostatin-sodium dodecyl sulfate complex appeared monodisperse by sedimentation equilibrium with about 17 g bound detergent per g peptide. Circular dichroic and difference spectra of the dodecyl sulfate-somatostatin complex show that the tryptophanyl residue is buried in a nonpolar environment and that the secondary and tertiary structure of the peptide is markedly altered. Sedimentation equilibrium studies suggest that two types of dodecyltrimethylammonium-somatostatin complex exist. One type resembles the dodecyl sulfate-peptide complex, whilst the other appears to include several peptide units with only about one gram bound detergent per gram peptide.     

Furosemide-sensitive Na+-K+ cotransport and cellular metabolism in human erythrocytes

Metabolic depletion of human red cells with 2-deoxy-D-glucose in the presence of EGTA decreased ATP to about 4% of the initial value and increased total ouabain- and furosemide-resistant Na+ and K+ effluxes by 20% and 100%, respectively, and furosemide-sensitive Na+ and K+ effluxes by 100% and 60%, respectively. When ATP was restored, all the components of Na+ and K+ fluxes measured returned to baseline levels suggesting a metabolic dependence.     

Plant phosphoglucose isomerase genes lack introns and are expressed in Escherichia coli

Nuclear genes that appear to encode both cytosolic and plastid isozymes of phosphoglucose isomerase (PGI), an essential glycolytic enzyme, have been isolated from three diploid species of the annual wild flower genus Clarkia (Onagraceae). The genes do not contain introns and are expressed to varying degrees in Escherichia coli when cloned in either Charon 35 phage or pUC plasmid vectors. The PGI proteins synthesized in E. coli form dimers, are catalytically active, and their electrophoretic mobilities are similar to those of appropriate Clarkia PGIs. The nucleotide sequence of a gene encoding a plastid isozyme of C. unguiculata is described.     

Denaturation thermodynamics of chicken cardiac metmyoglobin

The unfolding at pH 8 of chicken cardiac aquometmyoglobin was examined as a function of temperature and concentration of guanidinium chloride using the two-state model. The isothermal unfolding data at 25°C were fitted to Tanford's transfer model and the binding model of Aune and Tanford. The estimates obtained for ΔG_D) were virtually identical, viz., 8.3 ±0.3 kcal mol^?1. The chicken metmyoglobin is thus some 5.3 kcal mol^?1 less stable than that of sperm whale metmyoglobin. The unfolding parameters α and Δn were decreased 20% from those of mammalian myoglobins thus far examined, suggesting nonidentity of native conformations. The apparent enthalpy change on unfolding was dependent on both temperature and denaturant concentration. The decreases in the isothermal unfolding parameters from those of sperm whale are principally assigned to three of the 46 sequence changes.     

Calcium uptake by isolated nerve endings: evidence for a rapid component mediated by the breakdown of phosphatidylinositol

Several physiological stimuli, including neuronal depolarization, increase the production of phosphatidate (PA) from phosphatidylinositol (PI) and increase calcium fluxes across cell membranes. To determine if breakdown of PI is required for neuronal calcium uptake, we tested inhibitors of PI-specific phospholipase C on depolarization-dependent uptake of calcium by isolated brain synaptosomes. At a concentration of 0.1 mM these inhibitors reduced calcium uptake produced by depolarization for 1 to 3 sec, but did not affect uptake due to more prolonged depolarization. Exogenous PA also stimulated calcium accumulation by synaptosomes and this uptake was not reduced by the enzyme inhibitors. These results suggest that the rapid calcium influx produced by neuronal depolarization may be mediated by the breakdown of PI.     

Procedure using voltage-sensitive spin-labels to monitor dipole potential changes in phospholipid vesicles: The estimation of phloretin-induced conductance changes in vesicles

Summary Voltage-sensitive membrane potential probes were used to monitor currents resulting from positive or negative charge movement across small and large unilamellar phosphatidylcholine (PC) vesicles. Positive currents were measured for the paramagnetic phosphonium ion or for K⁺-valinomycin. Negative currents were indirectly measured for the anionic proton carriers CCCP and DNP by monitoring transmembrane proton currents. Phloretin, a compound that is believed to decrease dipole fields in planar bilayers, increases positive currents and decreases negative currents when added to egg PC vesicles. In these vesicles, positive currents are increased by phloretin addition to a much larger degree than CCCP currents are reduced. This asymmetry, with respect to the sign of the charge carrier, is apparently not the result of changes in the membrane dielectric constant. It is most easily explained by deeper binding minima at the membrane-solution interface for the CCCP anion, when compared to the phosphonium. The measured asymmetry and the magnitudes of the current changes are consistent with the predictions of a point dipole model. The use of potential-sensitive probes to estimate positive and negative currents, provides a methodology to monitor changes in the membrane dipole potential in vesicle systems.     

The 108-kDA peptidylglycine alpha-amidating monooxygenase precursor contains two separable enzymatic activities involved in peptide amidation

A 43-kDa protein factor that increases the ability of purified bovine peptidylglycine alpha-amidating monooxygenase (PAM)-A and -B to produce alpha-amidated peptides at physiological pH was purified to homogeneity from bovine neurointermediate pituitary. At each step of the purification, the amount of activity correlated with the amount of protein detected on Western blots by antibody to bovine PAM(561-579). In the bovine neurointermediate pituitary the 108-kDa PAM precursor protein is cleaved to form a peptidylglycine alpha-hydroxylating monooxygenase and a peptidyl-alpha-hydroxyglycine alpha-amidating lyase, which function sequentially in the 2-step formation of alpha-amidated peptides.     

Recruitment: the ins and outs of spindle pole formation

Microtubule-organizing centers are a diverse but interrelated set of cellular organelles that appear when needed at specific times in the cell cycle. Diversity is introduced into MTOCs, and thus into microtubule structures, by post-translational modification, by morphological changes, by de novo synthesis, and by recruitment of cytoplasmic proteins. Understanding the mechanisms that lead to such diversity should contribute to a better understanding of the myriad of cellular events which depend on microtubules.