Scanning tunneling microscopy imaging of Torpedo acetylcholine receptor.

The synaptic surface of the acetylcholine receptor in membranes from Torpedo californica electric organ has been imaged by scanning tunneling microscopy. The molecule appears pentameric, with one major and four minor protrusions rising above the surface, and these protrusions encompass a large central cavity. The outer diameter of the molecule is 69 +/- 10 A, while the diameter of the cavity, measured at the widest complete contour line delimiting the opening, is 26 +/- 7 A. The images and dimensions obtained are consistent with the structure determined from hybrid density maps obtained by x-ray diffraction and electron microscopy. Thus, scanning tunneling microscopy can be used to obtain overall dimensions and low-resolution structural features of the surface of a membrane-embedded protein.

Effects of ions and pH on the thermal stability of thin and thick filaments of skeletal muscle: high-sensitivity differential scanning calorimetric study.

Differential scanning calorimetry (DSC) is unique for studying conformational changes in supramolecular structures because it is immune to interference by the turbidity and other optical artifacts of a sample solution. We have employed DSC to study thermal stability of myosin and actin in their filamentous forms (i.e., thick and thin filaments). The thermal stability of the myosin monomer, as well as polymers, showed remarkable sensitivities to pH and to the ionic strength of the solution. At pH 7.5, the endotherm of myosin filaments was broad and resembled that of the monomer in solution. Reducing the pH to 6.3 split the endotherm of the filament into two major transitions. The first one, with a Tm of 47 degrees C, a delta Hcal of 805 kcal/mol, and a cooperative ratio (CR) of 0.1, was relatively insensitive to the pH changes whereas the second one which represented approximately 80% of the helical structure was pH sensitive. The second transition released 2.17 H+ per mole at 0.17 M KCl and was defined by a Tm of 53.9 degrees C, a delta Hcal of 917 kcal/mol, and a CR of 0.35. The major fragment contributing to the splitting of the endotherm was interpreted to be S-2 because the Tm of purified S-2 in a similar medium also shifted from 39.5 degrees C at pH 7.3 to 49.6 degrees C at pH 6.0. KCl had similar effects on the shape of the endotherm of the thick filament. A decrease of KCl from 0.2 to 0.1 M enhanced the effect of pH on the second transition.(ABSTRACT TRUNCATED AT 250 WORDS)

Study of effects of pH on the stability of domains in myosin rod by high-resolution differential scanning calorimetry.

Differential scanning calorimetry (DSC) has detected at least six quasi-independent structure domains in myosin rod [Potekhin, S.A., & Privalov, P.L. (1978) Biofizika 23, 219-223]. These domains were found to be remarkably sensitive to pH in the physiological range, i.e., pH 6-8. We compared the thermodynamic characteristics, and studied effects of pH on the stability, of individual domains in rod, light meromyosin (LMM), and subfragment 2 (S-2). In rod, the lowest stability domain (approximately 400 amino acid residues per double strand), with a Tm of 42.4 degrees C, a delta Hcal of 190 kcal/mol, and a delta G of 3.39 kcal/mol, at pH 7.02, destabilized by absorption of protons, is located at the LMM/S-2 junction and split into two parts, one associated with S-2 (approximately 100 residues per double strand) and the other with LMM (300 residues per double strand). The fragment with S-2 is likely a part of the "hinge" suggested by Swenson and Ritchie [(1980) Biochemistry 19, 5371-5375]. All other domains of rod released protons on melting. The domains located in S-2 were the most sensitive to pH and released a total of 0.9 proton on melting. The thermal meltings of all domains in myosin rod, LMM, and S-2 were independent of each other, and enthalpies of melting were additive in the whole pH range studied. Their sensitivities to pH and KCl were also unaffected by the presence or absence of other fragments. For example, domains in an isolated S-2 behaved similarly as they were in the rod, and so were domains in LMM.(ABSTRACT TRUNCATED AT 250 WORDS)

Enthalpic and entropic contributions to actin stability: calorimetry, circular dichroism, and fluorescence study and effects of calcium.

The delta H associated with the thermal unfolding of G-actin has been determined by differential scanning calorimetry (DSC) to be 142 +/- 5 kcal/mol, with the Tm (melting temperature) at 57.2 +/- 0.5 degrees C, at pH 8.0 (heating rate 0.5 K/min). The transition is broad and cannot be treated as a single transition that mimics a two-state process, suggesting the existence of domains. Deconvolution is done to fit it into two quasi-independent two-state transitions. For F-actin, the transition is more cooperative, with a cooperative ratio (the ratio of van't Hoff enthalpy and calorimetric enthalpy) of 1.4, indicating intermonomer interaction. The delta H of the thermal unfolding of F-actin is 162 +/- 10 kcal/mol with a Tm at 67.0 +/- 0.5 degrees C. A state of G-actin similar to that of the heat-denatured form, designated D-actin, is obtained by removing tightly bound Ca2+ with EGTA. The DSC-detectable cooperative transition is completely lost when the free calcium concentration of the medium is 1 x 10(-11) M or lower, using a Ca2+/EGTA buffer system. However, circular dichroism (CD) shows that the helix content of actin, 32% in the G-form, is only partially reduced to 19% in this apo form. The CD spectrum and the helix content of the calcium-depleted actin are almost identical with those of the heat-denatured D form. This loss of 40% of the native helical content is irreversible in both cases. The remaining 60% of the native helical content cannot be further eliminated by heating to 95 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

High-resolution differential scanning calorimetric study of myosin, functional domains, and supramolecular structures.

High-resolution differential scanning calorimetry (DSC) has been employed to study the thermal stability of myosin, its major constitutive fragments (S-1, light chains, and rod), and also reconstituted thick filaments. The thermal denaturation of soluble myosin was complex and was characterized by a multistep endothermic process for the temperature range from 41 to 60 degrees C. The shape of the endotherm was highly dependent on the pH and the ionic strength of the solution, although the delta Hcal (calorimetric enthalpy) of denaturation (1715 +/- 75 kcal/mol) was insensitive to these changes for the solvent conditions used in this study. This value also agrees, within experimental error, with the sum of the denaturation enthalpies obtained for isolated fragments (1724 +/- 79 kcal/mol). In identical conditions of ionic strength, pH, and heating rate, the computer-calculated differential endotherms of domains belonging to S-1 and light chains were superimposable with those of the isolated fragments. Their responses to changes in the solvent condition were also similar. We suggest that the observed functional independence of the major domains in myosin reflects also the independence of their structural stability. The thermal unfolding of the isolated rod was multiphasic and readily reversible (95%). It occurred between 41 and 60 degrees C, with an delta Hcal of 1058 +/- 59 kcal/mol. The melting of S-1 showed a single peak at 46.3 +/- 0.1 degrees C with an delta Hcal of 255 +/- 12 kcal/mol. Light chains melted at 51.0 +/- 0.2 degrees C with an delta Hcal of 85 +/- 15 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of 2-pyrrolidone on the concentration of GABA in rat tissues.

The effects of 2-pyrrolidone, a cyclic lactam of GABA, were studied on blood and organ levels of 2-pyrrolidone, GABA, glutamic acid, glutamate decarboxylase (GAD) and GABA-transaminase (GABA-T). When administered i.p., the only significant effects observed were increases of brain and liver 2-pyrrolidone. In contrast, regular oral administration for 7 months produced significant increases of GABA and glutamic acid in brain and of glutamic acid alone in liver while GAD decreased in brain and increased in liver; GABA-T was unchanged. A new method for the synthesis of radioactive 2-pyrrolidone was set up and the enzymatic conversion of 2-pyrrolidone to GABA was measured by an original procedure. The results obtained in vitro by this method on the conversion of 2-pyrrolidone to GABA catalyzed by tissue slices, together with the observed inhibition of the GABA-dependent oxygen consumption by 2-pyrrolidone, partially explain the effects of the oral administration.

Interaction of low doses of ionizing radiation and carbon tetrachloride on liver superoxide dismutase and glutathione peroxidase in mice.

Male BALB/c mice were treated with intraperitoneal injections of carbon tetrachloride (CCl4) (0.2 g/kg body weight) and/or 50 R of whole-body gamma irradiation, three times per week, for 4 weeks. The effects of the treatments on superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in liver extracts and homogenates, and on alkaline phosphatase (ALP) in serum were investigated. A significant decrease in the SOD and GSH-Px activities in liver extracts and an increase of serum ALP of hepatic origin were found in CCl4-treated animals. In contrast, only an increase in SOD activity was observed in liver homogenates after the combined treatment.

An N-protected gamma-aminobutyric acid dipeptide with anticonvulsant action.

N-Pivaloyl-leucyl-gamma-aminobutyric acid (PLG) is a synthetic dipeptide with a partition coefficient of 1.67 in an ethyl acetate/water system that partially inhibits the synaptosomal uptake and activates the release of [U-14C]gamma-aminobutyric acid [( U-14C]GABA). The displacement of GABA from crude synaptic membranes by PLG occurs with an IC50 of 10(-5) M. The compound has the capacity to cross the blood-brain barrier and increase central GABA levels. Its ED50 on cardiazol -induced convulsions is 60-65 mg/kg. PLG is resistant to hydrolysis by chymotrypsin and partially inhibits the proteolytic activity of trypsin.

Effect of subtoxic amounts of furan, acetylfuran and methylene chloride on some serum enzymes of rat.

Serum transaminases (GOT and GPT) and ornithincarbamyltransferase (OCT) were determined in rats treated with subtoxic doses of furan, acetylfuran, and methylene chloride. Significant increases of all enzymes were observed in methylene chloride treated rats, while only GOT increased in rats treated with acetylfuran and with furan + methylene chloride. Calculation of the GOT/GPT ratios indicated a pattern of toxic hepatitis only for rats treated with acetylfuran and furan + methylene chloride.

Interaction between RNAs from different sources and egg lecithin liposomes.

The interaction of RNAs of molecular weight ranging between 15,000 and 30,000 with egg lecithin large and small unilamellar liposomes has been investigated. Torula and tRNA proved to be incorporated to a similar extent but no linear relationship between molecular weight and percent of incorporation was demonstrated. A relationship between percent of secondary structure of RNA and its ability to be trapped in SUV liposomes is suggested.

Change of catalytic properties of erythrocyte acetylcholinesterase after binding to lecithin liposomes.

Phosphatidylcholine and phosphatidylserine + phosphatidylcholine liposomes were prepared with cholate and erythrocyte acetylcholinesterase (EC 3.1.1.7). Dipalmitoyllecithin- and egg lecithin-acetylcholinesterase complexes exhibit an affinity for acetylthiocholine different from that of the free enzyme. The binding to lecithin apparently abolishes the excess substrate inhibition of acetylcholinesterase; the affinity constants of acetylthiocholine, acetylcholine and acetylcarnitine for the lecithin-bound enzymes are higher than the ones found for the free enzyme. Binding to lecithin decrease the optimum pH value for acetylcholinesterase, increase the resistance of the enzyme to heat denaturation and reduces the extent of activation by Ca2+.

Effect of ACP (pyridoxine-2-oxoglutarate) on hepatic failure after diversion of portal blood flow.

The protective effect of pyridoxine-2-oxoglutarate (ACP) was studied in end-to-side porto-caval shunted rats. A significant improvement of hepatic conditions was shown in ACP-treated rats, while an increased mortality was observed in pyridoxine and 2-oxoglutarate treated rats. Serum glutamic-oxaloacetic transminase, glutamicpyruvic transaminase and lactic dehydrogenase activities, composition of serum proteins, liver mitochondria oxygen consumption rate, plasma ammonia were tested.