A water-lipid interface induces a highly dynamic folded state in apocytochrome c and cytochrome c, which may represent a common folding intermediate.

In this study, we have used CD and NMR techniques to investigate the secondary structure of (apo-) cytochrome c both in solution and when associated with micelles. In aqueous solution, the holoprotein cytochrome c is tightly folded at secondary and tertiary levels and differs strongly from its random-coiled precursor. However, in the presence of 12-PN/12-Pglycol (9:1) micelles, we observed a remarkable resemblance between the CD spectra of these partially helical proteins. The water-lipid interface induces a secondary folding of apocytochrome c, whereas cytochrome c is suggested to partially lose its tertiary structure. The exchange of all amide protons and, using deuterium-labeled proteins, of all amide deuterons with the solvent was monitored by NMR. A rapid exchange rate was observed, indicating that these folding states are highly dynamic. Saturation-transfer NMR of micelle-associated apocytochrome c showed that the exchange takes place at the (sub-) second time scale. The holoprotein in the presence of micelles was found to have two distinct exchange rates: (1) a fast rate, comparable to that found for the micelle-associated precursor and 4.5 times slower than that of the random-coiled apocytochrome c, and (2) a slow rate which is 75 times slower than the precursor in solution. Urea denaturation studies showed the micelle-bound proteins to have a low helix stability, which explains the inability of the lipid-induced secondary structure to prevent its labile protons from rapid exchange. The uniqueness of this lipid-induced highly dynamic folding state of (apo-) cytochrome c is demonstrated by comparison with amphiphilic polypeptides like melittin, and its implications for membrane translocation and functioning are discussed.     

Reserpine-induced post-receptor reduction in muscarinic-mediated airway smooth muscle contraction

Radioligand binding was conducted on airways of the rat and human, surgically subdivided into trachea, lung airways, and parenchyma. 3H-QNB bound uniformly to receptors in separate sections of the rat and human airway. Receptor densities generally were ranked: lung airways greater than trachea greater than parenchyma. Receptor subtypes were identified mostly by pirenzepine displacement of bound 3H-QNB. The rat trachea, and rat and human lung airways had a uniformly low affinity for pirenzepine while rat and human parenchyma demonstrated both high and low affinity pirenzepine binding. Inhibition of methacholine-stimulated smooth muscle contraction by the M1 receptor antagonist, pirenzepine, and M2 receptor antagonist, gallamine, was studied in rat trachea and bronchus in vitro. Schild plot pA2 values were compatible with low potency antagonism, thereby favoring the presence of M3 receptors at these smooth muscle sites. Reserpine treatment of rats (0.5 mg kg-1 day-1 for 7 days) produced a decrease in peak tension in response to methacholine without changing the muscarinic receptor character (Kd 3H-QNB), population density (Bmax in fmol mg-1 protein), or function (methacholine EC50). These results indicate that muscarinic receptor heterogeneity exists in the airway of both laboratory rat and man. While the muscarinic receptor subserving airway smooth muscle contraction appears to be the M3 subtype, decreased contractile responses to methacholine by trachea and bronchus from reserpine-treated rats were receptor independent.     

Two-dimensional electrophoresis of proteins in principal cells, spermatozoa, and fluid associated with the rat epididymis

Spermatozoa, fluids, and principal cells from different regions of the epididymis were characterized by two-dimensional electrophoresis. Rete testis fluid was collected after 36-h efferent duct ligation, and cauda epididymal fluid was collected by retrograde perfusion through the vas deferens. Spermatozoa were collected after their exudation from minced caput and corpus epididymal tissue. Principal cells were recovered after enzymatic disaggregation and centrifugal elutriation of epididymides. Two-dimensional polyacrylamide gel electrophoresis was used to prepare protein profiles of all samples. Comparison of the proteins found in rete testis fluid versus those found in cauda epididymal fluid revealed a dramatic change in composition, including the loss, addition, or retention of specific proteins as well as changes in the relative concentrations of certain proteins. Prominent cauda epididymal fluid proteins, possibly contributed by the epididymal epithelium, were detected at 16, 23, and 34 kDa. After epididymal transit, a considerable decrease was observed in the number of aqueous-soluble sperm proteins. Differences in the protein composition of epididymal epithelial principal cells from the caput versus corpus epididymidis were also noted, suggesting that functional differences exist for these epididymal regions. Of particular interest was the occurrence of a prominent protein of approximately 20-23 kDa found in all sperm samples, in fluids, and in caput and corpus principal cells. However, this protein was absent in cauda epididymal sperm after 36-h efferent duct ligation. The rapid loss of this protein from sperm after efferent duct ligation suggests that this surgical intervention may affect spermatozoa residing within the epididymis.     

The membrane as an environment of minimal interconversion. A circular dichroism study on the solvent dependence of the conformational behavior of gramicidin in diacylphosphatidylcholine model membranes

The conformation of gramicidin in diacylphosphatidylcholine model membranes was investigated as a function of the solvent in which peptide and lipid are initially codissolved. By use of circular dichroism it is demonstrated that, upon removal of the solvent and hydration of the mixed gramicidin/lipid film, it is the conformational behavior of the peptide in the organic solvent that determines its final conformation in dimyristoylphosphatidylcholine model membranes. As a consequence, parameters that influence the conformation of the peptide in the solvent also play an essential role, such as the gramicidin concentration and the rate of interconversion between different conformations. Of the various solvents investigated, only with trifluoroethanol is it possible directly to incorporate gramicidin entirely in the beta 6.3-helical (channel) configuration. It is also shown that the conformation of gramicidin in the membrane varies with the peptide/lipid ratio, most likely as a result of intermolecular gramicidin-gramicidin interactions at higher peptide/lipid ratios, and that heat incubation leads to a conformational change in the direction of the beta 6.3-helical conformation. Using lipids with an acyl chain length varying from 12 carbon atoms in dilauroylphosphatidylcholine to 22 carbon atoms in dierucoylphosphatidylcholine, it was possible to investigate the acyl chain length dependence of the gramicidin conformation in model membranes prepared from these lipids with the use of different solvent systems. It is demonstrated for each solvent system that the distribution between different conformations is relatively independent of the acyl chain length but that the rate at which the conformation converts toward the beta 6.3-helical configuration upon heating of the samples is affected by the length of the acyl chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solid-state 15N-NMR evidence that gramicidin A can adopt two different backbone conformations in dimyristoylphosphatidylcholine model membrane preparations

Using [15N-Val7]gramicidin A it is shown by solid state 15N-NMR that in dimyristoylphosphatidylcholine model membrane preparations evidence is obtained for two different backbone conformations of gramicidin. One of these conformations is the familiar channel state while a second conformation possesses very different dynamic and structural characteristics. The relative amounts of the conformations depend upon the solvent used to initially codissolve peptide and lipid. Furthermore, by incubation of the samples at modestly elevated temperatures a conversion can be induced from the non-channel to the channel state in a lipid environment.     

Hemodynamic effects of synchronous high-frequency jet ventilation in mitral regurgitation

We tested the hypothesis that increases in intrathoracic pressure (ITP), by decreasing the pressure gradient for anterograde left ventricular (LV) ejection, should augment cardiac output in acute mitral regurgitation (MR). In a pentobarbital-anesthetized closed-chest canine model, LV stroke volume (SLLV) was measured by integration from an aortic flow probe signal. MR was induced by a regurgitant ring. ITP was elevated over apnea by means of intermittent positive-pressure ventilation (IPPV), asynchronous (asynch) high-frequency jet ventilation (HFJV), and cardiac cycle-specific (synch) HFJV. IPPV resulted in the greatest increase in ITP. MR caused a fall in SVLV and a rise in LV filling pressure that were not altered by IPPV. Compared with IPPV or apnea, both asynch and synch HFJV increased SVLV and reduced LV filling pressures (P less than 0.05). Systolic synch HFJV induced a greater increase in SVLV (32%) than diastolic synch HFJV (26%) despite similar ventilatory settings. Our data suggest that when LV contractility is normal but MR impairs forward flow, cardiac cycle-specific increases in ITP will augment forward flow.     

Interaction mode specific reorganization of gel phase monoglyceride bilayers by beta-lactoglobulin.

The interaction between beta-lactoglobulin and sonicated aqueous dispersions of the gel phase forming monoglyceride monostearoylglycerol were studied using isothermal titration calorimetry, direct binding experiments, differential scanning calorimetry, leakage of a fluorescent dye and solid-state (31)P- and (2)H-NMR. In the absence of a charged amphiphile, monostearoylglycerol forms a precipitate. Under these conditions, no interaction with beta-lactoglobulin was observed. In the presence of the negatively charged amphiphile dicetylphosphate, the gel phase monostearoylglycerol formed stable and closed, probably unilamellar, vesicles with an average diameter of 465 nm. beta-Lactoglobulin interacts with these bilayer structures at pH 4, where the protein is positively charged, as well as at pH 7 where the protein is negatively charged. Under both conditions of pH, the binding affinity of beta-lactoglobulin is in the micromolar range as observed with ITC and the direct binding assay. At pH 4, two binding modes were found, one of which is determined with ITC while the direct binding assay determines the net result of both. The first binding mode is observed with ITC and is characterized by a large binding enthalpy, a decreased enthalpy of the MSG L(beta) to L(alpha) phase transition and leakage of a fluorescent dye. These characteristics are explained by a beta-lactoglobulin induced partial L(beta) to coagel phase transition that results from a specific electrostatic interaction between the protein and the charged amphiphile. This explanation is confirmed by solid-state (2)H-NMR using 1-monostearoylglycerol with a fully deuterated acyl chain. Upon interaction with beta-lactoglobulin, the isotropic signal in the (2)H-NMR spectrum of the monostearoylglycerol-dicetylphosphate mixture partially transforms into a broad anisotropic signal which could be assigned to coagel formation. The second binding mode probably results from an aspecific electrostatic attraction between the negatively charged bilayer and the positively charged protein and causes the precipitation of the dispersion. At pH 7, only the first binding mode is observed.     

The membrane interaction of amphiphilic model peptides affects phosphatidylserine headgroup and acyl chain order and dynamics. Application of the "phospholipid headgroup electrometer" concept to phosphatidylserine

Deuterium nuclear magnetic resonance (2H NMR) was used to study the interaction of amphiphilic model peptides with model membranes consisting of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine deuterated either at the beta-position of the serine moiety ([2-2H]DOPS) or at the 11-position of the acyl chains ([11,11-2H2]DOPS). The peptides are derived from the sequences H-Ala-Met-Leu-Trp-Ala-OH (AX, one-letter code with X = MLWA) and H-Arg-Met-Leu-Trp-Ala-OH (RX+) and contain a positive charge of +1 (AXme+) or +2 (RXme2+) at the amino terminus or one positive charge at each end of the molecule (AXetN2+). Upon titration of dispersions of DOPS with the peptides, the divalent peptides show a similar extent of binding to the DOPS bilayers, which is larger than that of the single charged peptide. Under these conditions the values of the quadrupolar splitting (delta vq) of both [2-2H]DOPS and [11,11-2H2]DOPS are decreased, indicating that the peptides reduce the order of both the DOPS headgroup and the acyl chains. The extent of the decrease depends on the amount of peptide bound and on the position of the charged moieties in the peptide molecule. The effects exerted by the peptides on the delta vq value of [2-2H]DOPS are consistent with the PS headgroup responding as a molecular electrometer to the surface charge resulting from the presence of the peptides in the lipid-water interface. The effects on the acyl chain deuterons are in agreement with a localization of the peptides intercalated in between the lipid headgrouops.(ABSTRACT TRUNCATED AT 250 WORDS)     

A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation

The sea urchin egg has a rich history of contributions to our understanding of fundamental questions of egg activation at fertilization. Within seconds of sperm-egg interaction, calcium is released from the egg endoplasmic reticulum, launching the zygote into the mitotic cell cycle and the developmental program. The sequence of the Strongylocentrotus purpuratus genome offers unique opportunities to apply functional genomic and proteomic approaches to investigate the repertoire and regulation of Ca(2+) signaling and homeostasis modules present in the egg and zygote. The sea urchin "calcium toolkit" as predicted by the genome is described. Emphasis is on the Ca(2+) signaling modules operating during egg activation, but the Ca(2+) signaling repertoire has ramifications for later developmental events and adult physiology as well. Presented here are the mechanisms that control the initial release of Ca(2+) at fertilization and additional signaling components predicted by the genome and found to be expressed and operating in eggs at fertilization. The initial release of Ca(2+) serves to coordinate egg activation, which is largely a phenomenon of post-translational modifications, especially dynamic protein phosphorylation. Functional proteomics can now be used to identify the phosphoproteome in general and specific kinase targets in particular. This approach is described along with findings to date. Key outstanding questions regarding the activation of the developmental program are framed in the context of what has been learned from the genome and how this knowledge can be applied to functional studies.