Conversion of Des-tyrosine-γ-endorphin by brain synaptic membrane associated peptidases: Identification of generated peptide fragments

Des-tyrosine-γ-endorphin, a β-endorphin fragment with neuroleptic-like properties, was digested with a cSPM fraction of rat brain. A profile of metabolites and a time course of conversion were obtained by HPLC analysis of the digests. Quantitative amino acid analysis and a second HPLC fractionation step which was designed to separate and to identify very similar des-tyrosine-γ-endorphin fragments, combined with dansyl end group determination allowed the characterization of β-LPH 65–77, β-LPH 66–77 and β-LPH 62–73 as main conversion products. In the digests the C-terminal leucyl peptides β-LPH 67–77 and β-LPH 68–77 as well as the N-terminal glycyl peptides β-LPH 62–74 and β-LPH 62–76 were minor components. The data indicate the involvement of several types of peptidase activities in the conversion process. It is suggested that these peptidases have a role in mediating in vivo des-tyrosine-γ-endorphin effects. In addition, this study points to the capacity of the brain to gene-rate small peptides with neuroleptic-like properties.     

Identification of the mRNA coding for the ACTH-beta-lipotropin precursor in a human ectopic ACTH-producing tumor

The mRNA coding for the ACTH-β-lipotropin precursor from a human ectopic ACTH-producing thymic carcinoid was identified by blot hybridization analysis with the bovine cDNA as a probe. The mRNA from the tumor had the same length (approximately 1,100 nucleotides) as that from the human pituitary. An additional hybridization-positive RNA species of a larger size was found in the tumor. Cell-free translation of the mRNA from the tumor as well as from the pituitary yielded a product with an apparent molecular weight of 38,000 that was reactive both with antibody to ACTH and with antibody to β-endorphin.     

Characterization of a plasma factor having opiate and immunoactivity like beta-endorphin.

A factor from human plasma having opiate-like activity was characterized in the present study. In addition to its ability to inhibit the binding of [³H]-methionine-enkephalin to opiate receptors, it also cross-reacted with two β-endorphin specific anti-sera. Compared with β-endorphin, the plasma factor had a shorter action on inhibiting the contraction of the guinea pig ileum. By gel-filtration chromatography, the size of this factor was intermediate between that of β-endorphin and methionine-enkephalin.     

Isolation and amino acid composition of two somatostatin-like peptides from ovine hypothalamus: somatostatin-28 and somatostatin-25

Two $\text{b}$-type cytochrome components, differing in their mid-point redox potentials as well as their reactivities toward CO, are demonstrated in the membrane particle of $\text{Azotobacter}\text{vinelandii}$ by potentiometric titration. The low redox component is revealed to be CO-reactive while the higher redox component is not. Accordingly, the later cytochrome is believed not to be directly involved in the reduction of oxygen while the former component is the cytochrome $\text{o}$ of $\text{Azotobacter}$.     

Calmodulin selectively modulates the guanylate cyclase activity by repressing the lipid phase separation temperature in the inner half of the bilayer of rat brain synaptosomal plasma membranes

The association of [¹²⁵I-]calmodulin with rat brain synaptosomal plasma membranes, when incubated for 1 h at 25° in the presence or in absence of 20 M Ca²⁺, follows a sigmoid path with a Hill coefficient h=1.79±0.12 and h=1.72±0.11, respectively. The total association of calmodulin with the membrane increased approx. 60%–80% at all the range of calmodulin concentrations used in the presence of 20 M Ca²⁺. A three fold increase of guanylate cyclase activity was shown in the presence of low concentrations of calmodulin (up to 10 mM); higher concentrations (up to 40 mM) however, led to a progressive inhibition of the enzyme activity with respect to maximal stimulation. Calmodulin increased the lipid fluidity of synaptosomal plasma membranes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(r_o/r)-1]^–1. Arrhenius-type plots of [(r_o/r)-1]^–1 indicated that the lipid separation of the membrane at 22.7±1.2° was perturbed by calmodulin such that the temperature was reduced to 16.3±0.9° and 15.5±0.8° in the absence or in the presence of 20 M Ca²⁺. Arrhenius plots of guanylate cyclase and acetylcholinesterase activities exhibited brak points at 25.7±1.4° and 22.3±1.0° in control synaptosomal plasma membranes, respectively. The break point for the guanylate cyclase was reduced to 16.3±0.9° in calmodulin treated synaptosomal plasma membranes whereas that of acetylcholinesterase remained unaffected (21.1±0.9°). The allosteric properties of guanylate cyclase by Mn-GTP (as reflected by changes in the Hill coefficient) were modulated by calmodulin while those of acetylcholinesterase by fluoride (F^–) were not altered. We propose that calmodulin achieves these effects through asymmetric perturbations of the membrane lipid structure and that increase in membrane fluidity of the inner leaflet of the membrane induced by calmodulin may be an early key event to the process of neurotransmitter release.     

Oxytocin biotransformation in the rat limbic brain: chemical characterization of two oxytocin fragments and proposed pathway for oxytocin conversion

Two peptide fragments of oxytocin were isolated by high-pressure liquid chromatography from digests of oxytocin obtained after exposure to a SPM preparation of the rat limbic brain. The structures of these peptides, being Gln-Asn-Cys(O)_x-Pro-Leu-GlyNH₂ and Gln-Asn-Cys(-S-S-Cys)-Pro-Leu-GlyNH₂, were assessed by quantitative amino acid analysis, combined with the determination of N-terminal end groups and cysteic acid residues after performic acid treatment. The fragments comprised the 4–9 and 1,4–9 sequences of oxytocin, respectively. The types of proteolytic enzymes involved in their formation are discussed and a pathway for the conversion of oxytocin by SPM is proposed.     

Monoclonal antibodies identify common and differentiation-specific antigens on the plasma membrane of guard cells of Vicia faba L.

Monoclonal antibodies were raised against the plasma membrane of Vicia faba L. guard cells by immunizing either with total membranes from purified guard-cell protoplasts or with sealed, predominantly right-side-out plasma-membrane vesicles prepared from abaxial epidermes of V. faba by aqueous two-phase partitioning. Hybridoma screening was performed by enzyme-linked immunosorbent assay using polystyrene-adsorbed plasma-membrane vesicles as solid phase and by indirect immunofluorescence analysis using unfixed, immobilized protoplasts in a microvolume Terasaki assay. A range of monoclonal antibodies was characterized and is reported here. One monoclonal antibody, G26-6-B2, is guard-cell-specific and does not react with mesophyll-cell protoplasts of the same species. It binds to a periodate-resistant but trypsin-labile epitope, probably a differentiation-specific plasma-membrane protein.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - GCP guard cell protoplast(s) - Ig immunoglobulin - MAB monoclonal antibody - MCP mesophyll-cell protoplast(s) - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

A novel plasma membrane quinone reductase and NAD(P)H:quinone oxidoreductase 1 are upregulated by serum withdrawal in human promyelocytic HL-60 cells

We have studied changes in plasma membrane NAD(P)H:quinone oxidoreductases of HL-60 cells under serum withdrawal conditions, as a model to analyze cell responses to oxidative stress. Highly enriched plasma membrane fractions were obtained from cell homogenates. A major part of NADH-quinone oxidoreductase in the plasma membrane was insensitive to micromolar concentrations of dicumarol, a specific inhibitor of the NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase), and only a minor portion was characterized as DT-diaphorase. An enzyme with properties of a cytochrome b ₅ reductase accounted for most dicumarol-resistant quinone reductase activity in HL-60 plasma membranes. The enzyme used mainly NADH as donor, it reduced coenzyme Q₀ through a one-electron mechanism with generation of superoxide, and its inhibition profile by p-hydroxymercuribenzoate was similar to that of authentic cytochrome b ₅ reductase. Both NQO1 and a novel dicumarol-insensitive quinone reductase that was not accounted by a cytochrome b ₅ reductase were significantly increased in plasma membranes after serum deprivation, showing a peak at 32 h of treatment. The reductase was specific for NADH, did not generate superoxide during quinone reduction, and was significantly resistant to p-hydroxymercuribenzoate. The function of this novel quinone reductase remains to be elucidated whereas dicumarol inhibition of NQO1 strongly potentiated growth arrest and decreased viability of HL-60 cells in the absence of serum. Our results demonstrate that upregulation of two-electron quinone reductases at the plasma membrane is a mechanism evoked by cells for defense against oxidative stress caused by serum withdrawal.     

Stimulation of phosphoinositide degradation and phosphatidylinositol-4-phosphate phosphorylation by GTP exclusively in plasma membrane of rat brain

The effect of GTP on the hydrolysis of [³H]phosphatidyinositol (PI), [³H]phosphatidylinositol-4-phosphate (PIP) and [³H]phosphatidylinositol-4,5-bisphosphate (PIP₂) by phospholipase C of rat brain plasma membrane, microsomes and cytosol was determined. Moreover the regulation of PI and PIP phosphorylation by GTP in brain plasma membrane was investigated.In the presence of EGTA PIP₂ was actively degradted, opposite to PI and PIP which require Ca²⁺ for their hydrolysis. Addition of calcium ions in each case caused stimulation of inositide phosphodiesterase(s). GTP independently of calcium ions activates by about 3 times phospholipase C acting on PIP and PIP₂ exclusively in the plasma membrane. PI degradation was unaffected by GTP. In the presence of Ca²⁺ guanine nucleotides have synergistic stimulatory effect on plasma membrane bound phospholipase C acting on PIP₂. PIP kinase of brain plasma membrane was stimulated by GTP by about 20–100% in the presence of exogenous and endogenous substrate respectively. PI kinase was negligible activated by about 20% exclusively in the presence of endogenous substrate. These results indicated that guanine nucleotide modulates the level of second messengers as diacylglycerol and IP₃ through the activation of phospholipase C acting on PIP₂ exclusively in brain plasma membrane. The stimulation of phospholipase C by GTP may occur directly or through the enhancement of substrate level PIP₂ due to stimulation of PIP kinase.     

Binding and degredation of insulin by plasma membranes from bovine liver isolated by a large scale preparation

With the large-scale preparation described, as much as 1 kg of bovine liver can be processed, giving a yield of more than 1 g plasma membrane protein. From analytical and morphological criteria the plasma membrane fraction isolated mainly derives from bile-canalicular and contiguous areas of the hepatocytes.The insulin binding activity is quite similar to insulin receptors in otherr cell systems and membrane preparations. Insulin-degrading activity is very low in the isolated plasma fraction. Most of degrading activity is located in a microsomal membrane fraction. Neverthless the K_m and the pH dependence of the insulin-degrading activity in both fractions are nearly identical.From these studies we conclude that binding and degradation of insulin are two independent processes located on different cell organelles.     

Glycans of synaptosomal plasma membrane glycoproteins from adult rat forebrain: Characterization after fractionation by concanavalin A-Sepharose affinity chromatography

Glycopeptides obtained by exhaustive proteolytic digestion of synaptosomal plasma membranes from adult rat forebraini were separated by affinity chromatography on concanavalin A-Sepharoe. Concanavalin A-binding glycopeptides are essentially made up of mannose and N-acetylglucosamine in a molar ration of 3.45:1, whereas glycopeptides not bound to concanavalin A have a complex monosaccharide composition. By gel filtration on Bio-Gel P-30, concanavalin A-binding glycopeptides appear as low-molecular-weight glycopeptides (migrating like ovalbumin glycopeptides), whereas glycopeptides not bound to concanavalin A behave as high-molecular-weight glycopeptides (migrating like fetuin glycopeptides). Comparison of concanavalin A-binding glycopeptides from rat brain synaptosomal plasma membranes with concanavalin A-binding glycoproteins isolated from the same membrane fraction shows clear differences in monosccharide composition. We demonstrate here that this discrepancy is due to the presence on most concanavalin A-binding glycoprotein subunits of at least two different types of glycan: in addition to the concanavalin A-binding glycans, these glycoprotein subunits carry other glycans which do not interact with concanavalin A. Biological implications of the presence of two (or more) types of glycan on the same polypeptide are discussed.     

In situ localization of P-glycoprotein (ABCB1) in human and rat brain.

Transport of several xenobiotics including pharmacological agents into or out of the central nervous system (CNS) involves the expression of ATP-dependent, membrane-bound efflux transport proteins such as P-glycoprotein (P-gp) at the blood-brain barrier (BBB). Previous studies have documented gene and protein expression of P-gp in brain microvessel endothelial cells. However, the exact localization of P-gp, particularly at the abluminal side of the BBB, remains controversial. In the present study we examined the cellular/subcellular distribution of P-gp in situ in rat and human brain tissues using immunogold cytochemistry at the electron microscope level. P-gp localizes to both the luminal and abluminal membranes of capillary endothelial cells as well as to adjacent pericytes and astrocytes. Subcellularly, P-gp is distributed along the nuclear envelope, in caveolae, cytoplasmic vesicles, Golgi complex, and rough endoplasmic reticulum (RER). These results provide evidence for the expression of P-gp in human and rodent brain capillary along their plasma membranes as well as at sites of protein synthesis, glycosylation, and membrane trafficking. In addition, its presence at the luminal and abluminal poles of the BBB, including pericytes and astrocyte plasma membranes, suggests that this glycoprotein may regulate drug transport processes in the entire CNS BBB at both the cellular and subcellular level.     

Determination of ajulemic acid and its glucuronide in human plasma by gas chromatography-mass spectrometry

A method using gas chromatography-mass spectrometry (GC-MS) and solid-phase extraction (SPE) was developed for the determination of ajulemic acid (AJA), a non-psychoactive synthetic cannabinoid with interesting therapeutic potential, in human plasma. When using two calibration graphs, the assay linearity ranged from 10 to 750 ng/ml, and 750 to 3000 ng/ml AJA. The intra- and inter-day precision (R.S.D., %), assessed across the linear ranges of the assay, was between 1.5 and 7.0, and 3.6 and 7.9, respectively. The limit of quantitation (LOQ) was 10 ng/ml. The amount of AJA glucuronide was determined by calculating the difference in the AJA concentration before ("free AJA") and after enzymatic hydrolysis ("total AJA"). The present method was used within a clinical study on 21 patients suffering from neuropathic pain with hyperalgesia and allodynia. For example, plasma levels of 599.4+/-37.2 ng/ml (mean+/-R.S.D., n=9) AJA were obtained for samples taken 2 h after the administration of an oral dose of 20 mg AJA. The mean AJA glucuronide concentration at 2h was 63.8+/-127.9 ng/ml.     

Modulation of the organization of erythrocyte membrane phospholipids by cytoplasmic ATP. The susceptibility of isoionic human erythrocyte ghosts to attack by detergents and phospholipase C

Human erythrocyte ghosts were prepared in media of physiological ionic composition, and these “isoionic” ghosts were then lysed and resealed in media of varying Ca²⁺, Mg²⁺ and ATP concentrations. The susceptibilities of these ghosts to limited attack by various detergents and by phospholipases C were then compared with the susceptibilities of intact cells to similar attack: attack was assessed by measurements of lysis and of phospholipid hydrolysis. Ghosts were more readily attacked than cells by anionic detergents (cholate, glycocholate, dodecyl sulphate) and by phospholipases C, but Triton X-100 and cetyltrimethylammonium attacked cells and ghosts to the same extent. Mg · ATP^2? partially protected ghosts from attack by the anionic detergents and by the phospholipases C of Bacillus cereus and of Clostridium perfringens. Protection by Mg · ATP^2? occurred only if Mg · ATP^2? had access to the cytoplasmic surface of the membrane. Adenylyl(β-γ-methylene)diphosphonate, a non-hydrolysable ATP analogue, protected as effectively as did Mg · ATP^2?. Internal Mg · ATP^2? caused a marked reduction in the hydrolysis by phospholipases of phosphatidylethanolamine and sphingomyelin, but had no appreciable effect upon the simultaneous hydrolysis of phosphatidylcholine. It therefore seems that interaction of ATP with sites on the cytoplasmic surface of the erythrocyte membrane can, without ATP hydrolysis, cause changes in the organization of the outer surface of the membrane that specifically render phosphatidylethanolamine and sphingomyelin less accessible to attack by extracellular phospholipases.     

灌胃和皮下注射脂肪膜蛋白卵黄抗体对大鼠生长和脂肪沉积的影响

目的：研究脂肪膜卵黄抗体不同处理对大鼠生长和脂肪沉积的影响。方法：选用140 g左右雌性SD大鼠96只,随机分成4组,分别灌胃阴性卵黄和含脂肪细胞膜蛋白抗体的阳性卵黄;皮下注射阴性卵黄和含脂肪细胞膜蛋白抗体的阳性卵黄。灌胃每3 d给予1 ml卵黄,皮下注射连续4 d经背部皮下多点注射1 ml卵黄,1月后同方式加强1次。75 d后屠宰并采集血样测定。结果：阳性卵黄处理后大鼠体重和摄食量无显著差异。灌胃阳性卵黄降低肠系膜脂指数、子宫周脂指数和肾脂肪囊指数（P〈0.05）;降低血清甘油三酯（P〈0.05）,升高血清游离脂肪酸（P〈0.01）;降低血清Leptin、胰岛素和TNF-α水平（P〈0.01或P〈0.05）,但对腓肠肌生长、血清总胆固醇无显著影响。皮下注射阳性卵黄提高腓肠肌指数（P〈0.05）;降低血清甘油三脂（P〈0.01）;降低血清Leptin（P〈0.01）,升高血清TNF-α（P〈0.05）;而对脂肪沉积、血清游离脂肪酸、总胆固醇和胰岛素无显著影响。结论：脂肪细胞膜蛋白卵黄抗体能有效改善机体组成,灌胃的效果优于皮下注射。     

Rates of cholesterol exchange between human erythrocytes and plasma lipoproteins

Rates of exchange of labelled cholesterol between human erythrocytes and three plasma lipoprotein species, LDL, HDL₂ and HDL₃, were measured over a range of lipoprotein concentrations and temperatures. The exchange rates reached limiting, concentration-independent values, which were the same for the three lipoproteins. The temperature dependencies correspond to activation energies of 12 kcal in the limiting rate region, and at lower lipoprotein concentrations to activation energies of 11 to 22 kcal. Calculations based on simple collision theory indicate that energetic barriers to the exchange are far smaller than indicated by these activation energies and that no particular orientation of lipoprotein molecules is required for the exchange. The occurrence of a limiting rate may be a result of the adsorption of lipoprotein molecules onto a limited number of sites on the cell surface, or of a slow process occurring in the membrane, possibly the diffusion of cholesterol. Present data do not permit a choice between these models.     

Quantitation of plasma membrane fatty acid-binding protein by enzyme dilution and monoclonal antibody based immunoassay

Summary A plasma membrane fatty acid-binding protein (h-FABPPm) has been isolated from rat hepatocytes. Analogous proteins have also been identified in adipocytes, jejunal enterocytes and cardiac myocytes, all cells with high transmembrane fluxes of fatty acids. These 43 kDa, highly basic (pl = 9.1) FABP_pm 's appear unrelated to the smaller, cytosolic FABP's (designated FABP's) identified previously in the same tissues. h-FABP_pm appears closely related to the mitochondrial isoform of glutamic-oxaloacetic transaminase (mGOT), and both the purified protein and liver cell plasma membranes (LPM) possess GOT enzymatic activity. From their relative GOT specific activities it is estimated that h-FABP_pm constitutes approximately 2% of LPM protein, or about 0.7 × 10⁷ sites per cell. A monoclonal antibody-based competitive inhibition enzyme immunoassay (CIEIA) for h-FABP_pm is described; it yields an estimate of 3.4 x 10⁷ h-FABP_pm sites per hepatocyte. Quantitated by either method, h-FABPPm appears to be a highly abundant protein constituent of LPM.     

Degradation of cholecystokinin-like peptides by a crude rat brain synaptosomal fraction: a study by high pressure liquid chromatography

Degradation of CCK-8, CCK-4, and related peptides by a crude synaptosomal fraction of rat brain was investigated by monitoring the tryptophan fluorescence of reaction products after HPLC fractionation. At 20 degrees C, the half disappearance time was 52 min for CCK-8, 35 min for unsulphated CCK-8, 20 min for unsulphated CCK-7, 6 min for Tyr(SO3H)-Trp-Met-Asp-Phe-NH2, and 3 min only for CCK-4. Caerulein was much more resistant than CCK-8, and Boc-CCK-4 and Aoc-CCK-4 remained stable for at least 3 h. The apparent Km for CCK-8 and CCK-4 was 40 microM and maximal activity on CCK-8 was observed at pH 7.0. Zn2+ was strongly inhibitory. The protease inhibitors puromycin and bacitracin, the metal chelator 1,10-phenanthroline, and the sulphydryl blocking agents N-ethylmaleimide and p-chloromercuribenzoate greatly reduced the release of tryptophan from CCK-8. Puromycin inhibition of CCK-8 degradation provoked the accumulation of a CCK-7-like peptide, and that of CCK-4 degradation was of a competitive type (Ki = 2 microM). The CCK-8 degrading activity of brain synaptosomes was present in the cytosol as well as in synaptic membranes.     

Different changes of plasma membrane beta-adrenoceptors in rat heart after chronic administration of propranolol, atenolol and bevantolol

Recently, tissue segment binding method with a hydrophilic radioligand [(3)H]-CGP12177 was developed to detect plasma membrane beta-adrenoceptors in rat heart (Horinouchi et al., 2006). In the present study, propranolol (40 mg kg(-1) day(-1)), atenolol (40 mg kg(-1) day(-1)) and bevantolol (200 mg kg(-1) day(-1)) were administered to rats for 6 weeks, and the changes of plasma membrane beta-adrenoceptors and their mRNA expression in rat ventricle were examined. Chronic administration of propranolol increased the beta(1)-adrenoceptors but decreased the beta(2)-adrenoceptors without changing total amount of plasma membrane beta-adrenoceptors. Atenolol increased both plasma membrane beta(1)- and beta(2)-adrenoceptors, whereas bevantolol had no effect on the beta-adrenoceptor density and their subtype proportions. In contrast, the density of beta-adrenoceptors detected in conventional homogenate binding study was extremely low (approximately 60% of plasma membrane beta-adrenoceptors detected with the tissue segment binding method) and the effects of chronic administration of beta-adrenoceptor antagonists were not necessarily in accord with those at the plasma membrane beta-adrenoceptors. The mRNA levels of beta(1)- and beta(2)-adrenoceptors were not altered by propranolol treatment, while beta(1)-adrenoceptor mRNA significantly decreased after administration of atenolol or bevantolol without changing the level of beta(2)-adrenoceptor mRNA. The present binding study with intact tissue segments clearly shows that the plasma membrane beta(1)- and beta(2)-adrenoceptors of rat heart, in contrast to the homogenate binding sites and the mRNA levels, are differently affected by chronic treatment with three beta-adrenoceptor antagonists; up- and down-regulations of beta(1)- and beta(2)-adrenoceptors, respectively, by propranolol, and up-regulation of both the subtypes by atenolol, but no significant change in both the subtypes by bevantolol.