l-Alanine uptake in brush border membrane vesicles from the gill of a marine bivalve

Summary Brush border membrane vesicles were prepared from mussel gills using differential and sucrose density gradient centrifugation. These vesicles contained both the maximal Na⁺-dependent alanine transport activity found in the gradient and the maximal activities of -glutamyl transpeptidase and alkaline phosphatase. Electron micrographs showed closed vesicles of approximately 0.1–0.5 m diameter. Transport experiments using these vesicles demonstrated a transient 18-fold overshoot in intravesicular alanine concentration in the presence of an inwardly directed Na⁺ gradient, but not under Na⁺ equilibrium conditions. A reduced overshoot (10-fold) was seen with an inwardly directed K⁺ gradient. Further studies revealed a broad cation selectivity, with preference for Na⁺, which was characteristic of alanine transport but not glucose transport in these membranes. The apparent amino acid specificity of the uptake pathway(s) was similar to that of intact gills and supported the idea of at least four separate pathways for amino acid transport in mussel gill brush border membranes. The apparent Michaelis constant for alanine uptake was approximately 7m, consistent with values forK _t determined with intact tissue.     

Trifluoperazine Activates and Releases Latent ATP-Generating Enzymes Associated with the Synaptic Plasma Membrane

Neurone-specific enolase (NSE) and the brain form of creatine phosphokinase (CPK-BB) were previously found to be present in rat synaptosomal plasma membranes (SPM) using two-dimensional gel (2-D gel) and peptide analysis; enzymatic activities of these and of pyruvate kinase (PK), all involved in ATP generation, were shown to be "cryptic" unless the SPM were treated with Triton X-100. We now show that enzymatic activation also occurs when the SPM are treated with trifluoperazine (TFP). TFP activation occurred even when the enzymes were membrane associated, showing that solubilization was not responsible for "unmasking" the enzyme activities. When TFP treatment was performed at alkaline instead of neutral pH, NSE and CPK-BB were released as well as PK, nonneuronal enolase, and aldolase which were identified by 2-D gel and tryptic peptide analysis. Other proteins released included calmodulin, actin, and the 70-kilodalton heat-shock cognate protein. Tubulin, synapsin I, and a 35-kilodalton basic protein were largely unaffected. The latter was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase on the basis of 2-D gel and peptide analyses and subsequent partial sequencing of a rat brain cDNA coding for the same protein. TFP treatment is thus useful for activating latent enzymes as well as for distinguishing enzymes that have a different disposition on the membrane.     

Calcium-Induced Desensitization of Acetylcholine Release from Synaptosomes or Proteoliposomes Equipped with Mediatophore, a Presynaptic Membrane Protein

A "fatigue" of acetylcholine (ACh) release is described in cholinergic synaptosomes stimulated with the calcium ionophore A23187 or gramicidin. A small conditioning calcium entry, which did not trigger a large ACh release, led to a decrease of transmitter release elicited by a second large calcium influx. This fatigue was half-maximal at approximately 30 microM external calcium and developed in a few minutes. In contrast, activation of release by calcium was very rapid and was half-maximal at approximately 0.5 mM external calcium. Activation and desensitization of release could be attributed to the recently identified presynaptic membrane protein, the "mediatophore." Proteoliposomes equipped with purified mediatophore showed a calcium-dependent activation and "fatigue" of ACh release similar to that of synaptosomes. It was found that the ionophore A23187 rapidly equilibrated internal and external calcium concentrations in proteoliposomes. Thus, the external calcium concentration gave the internal concentration required for activation or desensitization of proteoliposomal ACh release. The mediatophore showed remarkable calcium binding properties (20 sites/molecule) with a KD of 25 microM. The physiological implications of desensitization on the organization of release sites are discussed.     

Purification and Properties of Rubrophilin: A Novel Brain Specific Membrane Polypeptide

Rubrophilin, a unique brain specific polypeptide, was purified to apparent homogeneity from microsomal fractions of bovine brains. The peptide stains pink with Coomassie Brilliant Blue R-250 (C.I. No. 42660) under specific conditions, has an apparent Mr of 53,000, and is acidic with an apparent pI of 4.9. The purification involves initial solubilization of delipidated microsomes in sodium dodecyl sulfate, followed by ammonium sulfate fractionation, reversed ammonium sulfate gradient elution from diatomaceous earth, gel filtration on polyacrylamide (Biogel P-200), gradient elution chromatography from hydroxylapatite, and reverse-phase chromatography from phenyl-Sepharose. A yield of about 5 mg of rubrophilin was obtained from 9 g of microsomal proteins. Amino acid analysis shows that rubrophilin contains only nine amino acids with residues/mol as follows: alanine (102), glutamic acid (97), lysine (65), proline (55), aspartic acid (48), glycine (44), serine (37), threonine (35), and valine (10). Cysteine, methionine, tryptophan, tyrosine, isoleucine, phenylalanine, histidine, and arginine could not be detected. Relative rubrophilin content of vertebrate brains was as follows: mammals greater than birds greater than reptiles greater than fishes. It is present in mouse retina and human neuroblastoma cell cultures but could not be detected in octopus optic lobe or in cultured C-6 rat glioma cells.     

鲫鱼尾部神经分泌系统显微和亚显微结构的季节性变化

鲫鱼尾部神经分泌系统的神经分泌细胞和它的轴突中可观察到各种不同电子密度的颗粒。在性腺各个不同的发育阶段,该系统的分泌物具有累积、充满、释放和恢复这样一种周期性变化,由此说明鲫鱼的尾部神经分泌系统和它的生殖有关。     

The kell system in Senegal: phenotype and gene frequencies

The Kell gene frequencies, determined in Senegal are as follows: Our data enter the limits already known for black African populations.     

Types I and IV collagenolytic and plasminogen activator activities in preovulatory ovarian follicles

During ovulation, enzymatic degradation of the extracellular matrix occurs within and around the graafian follicles. In this study, the activities of several different proteolytic enzymes were measured in the culture media of follicles taken from pregnant mare serum gonadotropin (PMSG)-primed immature rats. At 52 h after PMSG, the follicles were cultured for 2 to 15 h in media with or without human chorionic gonadotropin (hCG). Type I collagenase activity in hCG-stimulated follicles gradually increased within 6 h to 3.3-fold above that of the controls. Relatively pure populations of granulosa cells produced type I collagenase to a similar extent. Likewise, type IV collagenase increased 3.8-fold by 6 h after exposure of the follicles to hCG. In contrast, plasminogen activator activity increased by 3.9-fold at 2 h after hCG, but was negligible at 4, 6, and 15 h after incubation. These results suggest that plasminogen activator may activate both type I and type IV collagenase in hCG-stimulated ovulatory follicles.     

Cationic interactions with Na+-H+ exchange and passive Na+ flux in cardiac sarcolemmal vesicles

Na⁺-H⁺ exchange and passive Na⁺ flux were investigated in cardiac sarcolemmal vesicles as a function of changing the ionic composition of the reaction media. The inclusion of EGTA in the reaction medium resulted in a potent stumulation of Na⁺ uptake by Na⁺-H⁺ exchange. It was found that millimolar concentrations of Mg²⁺ and Li⁺ were capable of inhibiting Na⁺-H⁺ exchange by 80%. One mechanism by which these ions may inhibit intravesicular Na⁺ accumulation by Na⁺-H⁺ exchange is via an increase in Na⁺ efflux. An examination of Na⁺ efflux kinetics from vesicles pre-loaded with Na⁺ revealed that Na⁺, Ca²⁺, Mg²⁺ and Li⁺ could stimulate Na⁺ efflux. Na⁺-H⁺ exchange was potently inhibited by an organic divalent cation, dimenthonium, which screens membrane surface charge. This would suggest that Na⁺-H⁺ exchange occurs in the diffuse double layer region of cardiac sarcolemma and this phenomenon is distinctly different from other Na⁺ transport processes. The results in this study indicate that in addition to a stimulation of Na⁺ efflux, the inhibitory effects of Mg²⁺, Ca²⁺ and Li⁺ on Na⁺-H⁺ exchange may also involve a charge dependent screening of Na⁺ interactions with the membrane.     

A double sequential immunofluorescence method demonstrating the co-localization of urotensins I and II in the caudal neurosecretory system of the teleost,Gillichthys mirabilis

Summary A double immunofluorescence method was devised to localize simultaneously urotensin-I (UI) and -II (UII) immunoreactivities in the caudal neurosecretory system of the goby, Gillichthys mirabilis. In a sequential fashion, sections of the posterior spinal cord and urophysis were treated with antiserum to corticotropin-releasing factor (CRF) that cross-reacts with UI, fluorescein-conjugated sheep anti-rabbit IgG, biotinylated anti-UII and rhodamine-conjugated avidin. UI and UII immunoreactivities appeared to coexist in some neurons and in most fibers and urophysial tissue; the remainder of the fibers and urophysis and the majority of neurons were immunoreactive for CRF/ UI only. No convincing evidence of immunoreactivity for UII only was found. A few nonreactive cells were seen, but these may not be neurosecretory neurons. The two immunoreactive cell types were not segregated topographically, and the intensity of perikaryal immunofluorescence for CRF/UI was variable. To explain these results a hypothesis that all caudal neurosecretory cells may synthesize both UI and UII and that immunoreactive differences may reflect different states of cellular activity, is suggested. This sequential double immunofluorescence method offers several advantages over other techniques and is especially useful for co-localization studies when primary antisera from different species are not available.     

The highly modified membrane of cornified cells in stratified squamous epithelia: A comparison of heterogenous deposits in keratinized and nonkeratinized epithelia

Summary The chemical nature of the thickened plasma membrane of cornified cells in stratified squamous epithelium was investigated in comparison with that in noncornified epithelium. Localizations of transglutaminase, molecular weight 92000 daltons, and detection of epidermal cysteine proteinase inhibitor were effected with a monoclonal antibody and a monospecific rabbit anti-inhibitor immunoglobulin, respectively, directed to the antigens. N-(7-dimethylamino-4-methylcoumarinyl) maleimide was used to demonstrate S-S cross-linking. In all keratinizing epithelia, the enzyme and inhibitor were deposited on membranes of granular cells. S-S bonds were formed in cornification with the appearance of electron-dense material by the inner leaflet. Both enzyme and inhibitors occurred on the corneal epithelium, but S-S linkage and the thickened plasma membrane did not form even at the last stage of maturation. On the other hand, the internal vaginal epithelium in the proestrous stage without keratinization contained the enzyme, but neither inhibitor nor S-S linkage. Both antigens and S-S bonds were detected when keratinization proceeded during estrus. The staining patterns in the epithelium near the vaginal introitus were identical to those in the skin. Cuboidal and simple epithelia exhibited none of those constituents. The findings indicated that heterogenous components contribute to modification of the plasma membrane of cornified cells, but S-S cross-linkages are associated exclusively with formation of the ultrastructurally unique membrane structure. In addition, findings suggested hormonal regulation in the chemical modification of the membrane in estrogen-sensitive internal vaginal epithelium.