红细胞4．1蛋白与血型糖蛋白C／D结合位点研究进展

红细胞膜骨架与脂双层间存在着相互作用,其中带4.1蛋白与血型糖蛋白C/D间的相互作用对维持正常红细胞的形态和机械稳定性起着重要作用,研究表明,带4.1蛋白在血型糖蛋白C、D上的结合位点分别位于血型糖蛋白C的第82～98位氨基酸残基和血型糖蛋白D的第61～77位氨基酸残基．     

Phosphoryl amino acids: Common origin for nucleic acids and protein

A series of compounds (DAP-AA) composed of an amino acid (AA) and a dialkyl phosphoryl group (DAP) is the basic elements of life chemistry. Self-catalysis of DAP-AA gives the self-assembly oligopeptides, even in aqueous medium at 38°C. The oligo-nucleotides could also be assembled from nucleosides' phosphorylation by DAP-AA. DAP-AA acts as the energy source as well as the phosphoryl donor for the synthesis of nuclic Acids and protein. A general expression for the self assembly system is proposed.     

Study of human erythrocyte membrane protein interactions by selective solubilization of Triton-skeletons

Summary— The membrane skeleton, responsible for shape and mechanical properties of the red cell, was purified by the Triton extraction procedure in presence of 5 mM, 150 mM or 600 mM NaCl. The proportion of spectrin, protein 4.1 and actin present in erythrocyte skeletons does not depend on the molarity of NaCl used. In contrast ankyrin, protein band 3 and protein 4.2 are removed from skeletons as the ionic strength increased. Solubilization assays of membrane skeletons were used to study protein interactions inside the skeleton. Solubilization was performed by Tris, a non-selective disruptive reagent, or by p-mercuribenzene sulfonic acid (PMBS), which principally release spectrin and actin. Tris action was assessed by calculation of the percentage of solubilized proteins, which increased proportionally with Tris molarity. PMBS action was kinetically determined as the decrease in skeleton turbidity. With these two reagents, we observed a lower dissociation of skeletons prepared with high ionic strength buffer. Erythrocyte pretreatment with okadaic acid, an inhibitor of serine-threonine phosphatases, revealed a phosphorylation-induced skeleton gelation and a better resistance to Tris-solubilization.     

The basic organization of the Plathelminthes

The basic organization of the Plathelminthes is summarized. Special attention is given to epidermal structures, musculature, extracellular matrices, nervous system and sensory structures, digestive system, totipotent stem cells, protonephridia, reproductive system and life cycle. The latest common ancestor of the Plathelminthes lacked any parenchymal cells and tissues; Plathelminthes do not represent Parenchymia. Discussions concerning the relationships of the Plathelminthes with other Metazoa must be based on the characteristics of the plathelminth stem species.     

Sequence analysis and structural features of the largest known protamine isolated from the sperm of the archaeogastropod Monodonta turbinata

Protamine of the archaeogastropod mollusc Monodonta turbinata has been isolated and characterized. With a mass of 13,476 Da, it is the largest known prolamine. Amino acid sequence of this protamine (106 residues) was established from data provided by automated sequence analysis and mass spectrometry of the protein and of its fragments. The primary structure of the NH₂-terminal region exhibits repetitive sequence motifs Basic-Ser (mainly R-S) and both central and COOH-terminal regions are composed by arginine clusters. The amino acid sequence of Monodonta turbinata protamine shows structural similarities with other protamines from invertebrates and from birds and mammals.     

Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury

To examine whether basic fibroblast growth factor (bFGF) administered to the heart by perfusion can improve cardiac resistance to injury we employed an isolated rat heart model of ischemia-reperfusion injury and determined the extent of functional recovery in bFGF-treated and control hearts. Global ischemia was simulated by interruption of flow for 60 min. Recovery of developed force of contraction (DF), recorded after reestablishment of flow for 30 min, reached 63.8±1.5% and 96.5±3.5% of preischemic levels in control and bFGF-treated hearts (10 g/heart), respectively, indicating that bFGF induced significantly improved recovery of mechanical function. Recoveries of the rates of contraction or relaxation were also significantly improved in bFGF-treated hearts. Extent of myocardial injury, assessed by determination of phosphocreatine kinase in the effluent, was reduced as a result of bFGF treatment. As a first step towards understanding the mechanism and direct cellular target(s) of bFGF-induced cardioprotection, we investigated its fate after perfusion. Perfusion of 10 g bFGF/heart resulted in a 4-fold increase in bFGF associated with the heart compared to control levels, as estimated by biochemical fractionation and immunoblotting. Immunofluorescent staining of the bFGF-perfused hearts revealed intense anti-bFGF staining in association with blood vessels as well as the periphery of cardiomyocytes, suggesting that the latter may be a target for direct bFGF action. In conclusion, our findings of bFGF-induced increases in cardiac resistance to, and improved functional recovery from, ischemia-reperfusion injury indicate that bFGF may have clinical applications in the treatment of ischemic heart disease.     

Redox thermodynamics, acid-base equilibria and salt-induced effects for the cucumber basic protein. General implications for blue-copper proteins

 The reduction potential of the basic blue-copper protein from cucumber peels (CBP) was determined through voltammetric techniques in different conditions of temperature, pH and ionic composition of the medium. The most notable properties of CBP include a positive entropy change upon reduction, a low-pH protonation and detachment of a metal-binding histidine in the reduced protein, and specific binding interactions with a number of anions present in common laboratory buffers, which influence to some extent the redox thermodynamics. The enthalpy and entropy changes accompanying reduction of the Cu(II) center were compared with those for other blue-copper proteins and correlated with spectroscopic data, structural properties and theoretical calculations. This allows some general considerations to be offered regarding the determinants of the reduction potential in this protein class. It emerges, in line with previous studies of the electronic structure of blue-copper sites, that the enthalpic contribution to the reduction potential is mainly modulated by the metal-binding interactions in the trigonal N₂S ligand set, and particularly by the Cu-cysteinate bond, while the entropy term is mainly affected by solvation properties and possibly by the weak axial bond to copper. The role of solvent exposure of the metal site in the active-site protonations in reduced blue-copper proteins is discussed. Finally, it is shown that the Nernst-Debye-Huckel model qualitatively accounts for the ionic strength dependence of the reduction potential. Received: 20 December 1996 / Accepted: 26 March 1997     

Characterization of Myelin Basic Protein Charge Microheterogeneity in Developing Mouse Brain and in the Transgenic Shiverer Mutant

Abstract: Myelin basic protein (MBP) is a highly heterogeneous family of membrane proteins consisting of several isoforms resulting from alternative splicing and charge isomers arising from posttranslational modifications. Although well characterized in the bovine and human species, those in the mouse are not. With the availability of a number of transgenic and knockout mice, the need to understand the chemical nature of the MBPs has become very important. To isolate and characterize the MBP species in murine brain, two methods were adapted for use with the small amounts of MBP available from mice. The first was a scaled-down version of the preparative CM-52 chromatographic system commonly used to isolate MBP charge isomers; the second was an alkaline-urea slab gel technique that required five times less material than the conventional tube gel system and, from these gels, western blots were readily obtained. Murine MBP was resolved into two populations of charge isomers: the 18.5- and 14-kDa isoforms. Isolation and characterization of these charge isomers or components permitted us to assign possible posttranslational modifications to some of them. Component 1 (C-1), the most cationic isomer, had a molecular weight of 14,140.38 ± 0.79. C-2 consisted of two 14-kDa species, 14,136.37 ± 0.74 and 14,204.45 ± 0.70. Two variants, 14,215.57 ± 0.94 and 18,413.57 ± 0.76, constituted C-3. C-4, C-5, and C-8 (the least cationic isomer) each consisted of both 14- and 18.5-kDa isoforms. During myelinogenesis, the 18.5-kDa isoform appeared first (day 4); the 14-kDa isoform appeared at day 16 and subsequently became the dominant isoform. The transgenic shiverer mutant synthesized mainly the 18.5-kDa isoform, but none of the 14-kDa isoform, similar to the 4-day-old mouse. We concluded that the trangenic shiverer was able to initiate myelinogenesis with the 18.5-kDa isoform, but was unable to complete myelinogenesis because of the absence of the 14-kDa isoform.     

Regulation of neuronal nitric oxide synthase by histone,protamine, and myelin basic protein

We examined the effects of endogenous basic proteins rich in the amino acidL-arginine on neuronal NO synthase activity by monitoring cyclic GMP formation in intact neuron-like neuroblastoma N1E-115 cells. Histone, protamine and myelin basic protein significantly stimulated cyclic GMP formation, both in a time- and concentration-dependent manner. These effects were blocked by hemoglobin and NO synthase inhibitors. Removal of the extracellular/intracellular Ca²⁺ gradient by a Ca²⁺ chelator completely abolished the cyclic GMP responses elicited by histone and protamine, suggesting that influex of extracellular Ca²⁺ might be involved in their activation of NO synthase. The effects of myelin basic protein on cyclic GMP formation, however, appeared to be due to Ca²⁺ release from intracellular stores. In cytosolic preparations of rat cerebellum, these basic proteins inhibited the metabolism ofL-arginine intoL-citrulline by NO synthase. We conclude from our findings that endogenous basic proteins might be involved in the regulation of neuronal NO synthase activity. Their effects on the enzyme could be either stimulatory or inhibitory, depending on whether the basic proteins exert their effects extracellularly or intracellularly, respectively.     

Myelin basic protein purified on an ion-exchange continuous polymer bed in the presence of ethylene glycol and salt possesses activity againstp-nitrophenyl acetate

In this paper we describe a fast and mild method based on the use of a unique cation exchanger and buffers containing ethylene glycol and salt for the purification of the myelin basic protein (MBP; MW 18.5 kDa). MBP thus purified hydrolyses catalytically p-nitrophenyl acetate. This esterase activity facilitates not only the purification of MBP but also indicates that probably it is in its native state, i.e. there is a good chance that the purified molecules are structurally and chemically identical. This is a prerequisite to obtain crystals appropriate for x-ray diffraction and other studies.Abbreviations used MBP myelin basic protein - MW molecular weight - kDa kilo Dalton - octyl-POE n-octylpolydisperse oligooxyethylene - CHAPS 3-3-cholamidopropyl dimethylammonio-1-propane-sulfonate - CTAB cetyltrimethylammonium bromide - SDS sodium dodecyl sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS - G 3707 heptaoxyethylene lauryl ether - TWEEN-20 polyoxyethylenesorbitan-monolaurat - EDTA ethylenediaminetetraacetic acid - HEPES N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid)