Membrane biochemistry最新文献

The main purpose of this work was to identify the macromolecules carrying the surface charge of endothelial cells. This was done by measuring changes in cell electrophoretic mobility caused by enzymatic removal of glycocalyx components. Endothelial cells were removed from the bovine pulmonary artery using nonenzymatic procedures, plated, and identified by immunocytochemical methods and electron microscopy. Cultured cells were suspended in saline and placed in the lumen of a capillary in a Rank Brothers electrophoresis instrument. Voltage was applied between the ends of the capillary, and the velocity acquired by the cells was measured with a microscope. Preincubating the cells in protein-free saline for 1 h reduced the mobility by 25%. This reflects the loss of proteoheparan sulfate from the cell surface. Cell mobility was totally suppressed by exposing the entire cell surface to chondroitin sulfate lyase, but it was only slightly diminished when the enzyme was applied only to the cell side facing the culture medium. A partial decrease in mobility was obtained after enzymatic removal of either heparin, heparan sulfate, or collagen. The results indicate that sulfated glycosaminoglycans are the main carriers of the surface change in vascular endothelial cells. The asymmetrical effect of chondroitinase on the two sides of the cell indicates a distribution polarization for glycosaminoglycans in endothelial cells.

The 7F0----5D0 excitation spectrum of Eu3+ bound to the high-affinity calcium sites of SR (Ca2+ + Mg2+)-ATPase diminishes upon occlusion of the Eu3+ into the interior of the enzyme. This "quenching" was found to be caused by the enzyme itself because trypsin digestion could relieve it. The level of digestion needed to relieve the quenching is beyond the level needed to eliminate occlusion; thus, the two processes are not related. Ca2+ is required during digestion to preserve the quenching, indicating close proximity between the Ca2+ site(s) and the quenching segment. Synthetic peptides were found that could mimic the native enzyme's ability to quench the Eu3+ fluorescence, although no native sequence has yet been identified that could emulate the enzyme.

Like other neurotransmitter receptors, muscarinic acetylcholine receptors are subject to regulation by the state of receptor activation. Prolonged increases in the concentration of muscarinic agonists result in a decrease in receptor density and loss of receptor sensitivity, both in vivo and in vitro. On the other hand, when the receptor is deprived of acetylcholine for a long duration in vivo, the receptor becomes more sensitive in responding to muscarinic agonists. However, it has been more difficult to demonstrate increases in receptor concentration that accompany this supersensitive state. The purpose of this review is to provide current information related to the characteristics of muscarinic receptor regulation and the molecular mechanisms underlying this phenomenon, regarding both the density of receptors and their transduction mechanisms. Furthermore, possible feedback regulatory roles of different second messenger signals are discussed. Particular emphasis is dedicated to molecular mechanisms of regulation of neuronal muscarinic receptors.

Gastric glands isolated from rabbit stomach were incubated in isosmotic medium or media made hyposmotic by 50-100 mOsm/kg. As indicated by radiolabeled aminopyrine accumulation, acid secretion was nearly 3 times greater in 200 mOsm/kg hyposmotic than in isosmotic medium after a 30-min incubation. The hyposmotic stimulation appeared within 2 min, peaked at 10-15 min and declined almost to the isosmotic control by 45 min. As estimated by the wet weight corrected for inulin extracellular space, the intracellular water of the glands also peaked at 15 min and returned to the isosmotic norm by 45 min. Hyposmotic stimulation of acid secretion directly involved the parietal cell, since parietal cells obtained from gastric glands were also stimulated. That the hyposmotic response was direct was indicated by omeprazole inhibition of aminopyrine accumulation in hyposmotic medium.

Nitrosoguanidine mutagenesis was employed to isolate an Escherichia coli mutant conditionally altered in respiratory chain components. Mutant R25 was able to grow on glucose, fructose, and glycerol but failed to grow on succinate and acetate (suc-). Also, R25 exhibited leaky growth on DL-lactate, fumarate, and malate (lct*). The lct* mutation pleiotropically affected a number of respiratory chain components and its expression was conditional with the growth substrate. Glucose-grown R25 resting cell suspensions oxidized DL-lactate and formate; however, these two substrates were not oxidized by fructose- or glycerol-grown cell suspensions. The same conditional pattern was observed for the concentration of cytochrome components, the membrane-associated oxidation of NADH and formate, and formate phenazine methosulfate (PMS) reductase activity; succinate oxidase and PMS reductase activities were not exhibited by membranes under any growth condition due to the suc- mutation. R25 membrane-associated H(+)-translocating ATPase activity was not conditional with the growth substrate. R25PC, a spontaneous lct+ suc- partial revertant of R25, did not exhibit the conditional pattern of R25. The lct* mutation was found to map in the 27-30-min region and the suc- mutation in the 15-17-min region of the E. coli genome. Two distinct classes of R25 P1kc transductants were isolated that differed in both their growth response on succinate and DL-lactate and their oxidase activities.

The level of biosynthesis and the composition of polyglycerophosphatides (phosphatidylglycerol, phosphatidyglycerolphosphate, and diphosphatidylglycerol or cardiolipin) and phosphatidylinositols were examined in mitochondria and microsomes, respectively, isolated from neonatal and adult rat heart and liver. Biosynthesis of [3H]polyglycerophosphatides [( 3H]phosphatidylglycerol and [3H]phosphatidylglycerolphosphate) was 4.5 times higher in neonatal than in adult heart mitochondria, whereas in the respective liver mitochondria this synthesis was only 15% higher in neonatal mitochondria. The biosynthesis of [3H]phosphatidylinositol was twice as high in neonatal as in adult heart microsomes, but very similar in the respective liver microsomes. The major biosynthesized polyglycerophosphatide was [3H]phosphatidylglycerol. The accumulation of [3H]phosphatidylglycerolphosphate depended on the origin of the mitochondria. Under our experimental conditions [3H]phosphatidylinositol was the only synthesized phosphoinositide in all microsomes. The biosynthesis of cardiolipin depended on the origin of the mitochondria and was highest in adult rat liver mitochondria and lowest in adult heart mitochondria. In all cases the biosynthesized [14C,3H] cardiolipin from [14C]phosphatidylglycerol and [3H]CDP-diglycerides had a ratio of 14C/3H around unity. The biosynthesis of [3H]CDP-diglycerides, the key precursor for the biosynthesis of phosphatidylglycerol, phosphatidylinositol, and cardiolipin, was 30% higher in neonatal than in adult heart microsomes and very similar in the respective liver microsomes. The subcellular localization of the enzymes required for the biosynthesis of the lipids and liponucleotides examined was found to be the same in membranes isolated from neonatal and adult rat heart and liver.

Renal brush border membrane vesicles (BBMV) of the dog possess at least two ATPase activities. In the present study, we have examined the effect of pH, ions, and inhibitors on the activity of ATPase in BBMV. Two different sets of conditions were identified that produced stimulation of ATPase activity. A unique stimulation of BBMV ATPase activity occurred at acidic pH in the presence of 1 mM ZnCl2. In the absence of Zn2+, a second ATPase activity was stimulated by alkaline pH values with peak stimulation occurring between pH 8.5 and 9.0. The results suggest that the alkaline pH-stimulated hydrolysis of ATP probably represents the activity of BBMV alkaline phosphatase. The unique acidic pH + Zn2(+)-stimulated ATPase activity must represent the activity of a second protein other than the alkaline phosphatase, since purified alkaline phosphatase did not show this activity. The biochemical identity and physiological function of this renal BBMV ATPase activity remain to be determined, but it may be an ecto-ATPase.

Gastric glands incubated in hyposmotic medium (200 mOsm) accumulated aminopyrine, a measure of acid secretion, to the same extent as that of paired glands in isomotic medium containing histamine (10(-4) M). These maximal responses to hyposmolality and histamine were not additive. The hyposmotic response peaked earlier than the histamine response. Hyposmotic stimulation was nearly abolished by preincubation of the glands with metiamide and cimetidine, H-2 histamine antagonists. In the presence of histaminase, no hyposmotic stimulation occurred. The response to forskolin, a stimulant of adenylate cyclase, was equivalent in hyposmotic and isosmotic media. These results indicate that hyposmolality releases histamine from a paracrine cell in the gastric gland and that histamine binds to H-2 receptors on the parietal cell to initiate a cyclic AMP-mediated stimulation of acid secretion.

In this paper, we describe a simple and reproducible method for purifying large quantities of ryanodine receptor from skeletal muscle membranes. The procedure involves the use of ion exchange chromatography and sucrose gradient centrifugation to purify the protein which has been identified as the calcium release protein of the sarcoplasmic reticulum (Imagawa, T., Smith, J., Coronado, R. and Campbell, K. (1987) J. Biol. Chem. 262:16,636-16,643). Addition of micromolar quantities of unlabeled ryanodine prior to solubilization and throughout the isolation procedure appears to stabilize the tetrameric structure of the ryanodine receptor. The purified receptor, consisting predominantly of a 400K polypeptide on SDS-PAGE, binds [3H]ryanodine with a binding affinity similar to that in membranes. Overall recovery of ryanodine binding activity was 21% of the initial activity with a 30-fold purification of the receptor.