籽瓜种子蛋白组分和生化特性的研究(简报)

籽瓜是西瓜的变种,其种子板大、味香,是人们喜爱的食品。籽瓜种子蛋白质含量约为38%,其中78%是球蛋白。Blagrove(1980)报道。南瓜、西瓜、甜瓜及黄瓜等瓜类种子球蛋     

Purification and characterization of mojave (Crotalus scutulatus scutulatus) toxin and its subunits

An acidic lethal protein, Mojave toxin, has been isolated from the venom of Crotalus scutulatus scutulatus. The purified toxin had an i.v. LD₅₀ of 0.056 μg/g in white mice. Disc polycrylamide gel electrophoresis at pH values of 9.6 and 3.8 and isoelectric focusing in polyacrylamide gels with a pH 3.5–10 Ampholyte gradient were used to establish the presence of one major protein band. The pI of the most abundant form of the toxin was determined to be 5.5 by polyacrylamide gel isoelectric focusing experiments. The molecular weight was established to be 24,310 daltons from amino acid composition data. Mojave toxin was shown to consist of two subunits, one acidic and one basic with isoelectric point (pI) values of 3.6 and 9.6, respectively. Amino acid analyses established molecular weights of 9593 for the acidic component and 14,673 for the basic component. The acidic subunit consisted of three peptide chains intermolecularly linked by cystine residues. The basic subunit was a single polypeptide chain with six intramolecular disulfide bonds. The basic subunit was lethal to test animals with an intravenous LD₅₀ of 0.58 μg/g. Following recombination of the subunits a recombinant toxin was isolated which was identical to the native toxin by comparisons of electrophoretic mobility and toxicities. Comparisons of circular dichroism spectra also indicated reassociation to the native toxin structure. Phospholytic activity was associated with Mojave toxin and the basic subunit was responsible for this enzymic activity. Phospholipase activity of the basic subunit was inhibited by addition of the acidic subunit.     

A BRAIN-SPECIFIC PROTEIN WITH AFFINITY FOR DNA1

Abstract— A procedure has been developed which allows the isolation from rat brain cytosol of a soluble acidic protein, designated DNA-110 protein, having two basic properties: selective affinity for single-stranded DNA and immunological specificity to the nervous system. Only two major purification steps, DNA-cellulose chromatography and affinity chromatography on immunoadsorbents are needed to give apparently pure protein. The purification steps of the DNA-110 protein have been followed by immunological assay. DNA-110 has a molecular weight of 68,000 and an isoelectric point of 5.9. It accounts for 1.95% of the total soluble protein and its concentration is 216 μg per g wet weight of rat brain. DNA-110 is immunologically unrelated to other soluble acidic brain-specific proteins and glycoproteins.     

Ribosomes of Physarum polycephalum. Subunits and protein composition.

Ribosomes from Physarum polycephalum were purified. Optimal conditions for preparation and stability of subunits were determined. KCl concentration above 200 mM induced protein dissociation from the subunits. It was observed that dissociated ribosomes were more stable in a low ionic strength buffer than in 200 mM KCl, where the 40 S was preferentially degraded by ribonucleases. Ribosomal proteins were analyzed by two-dimensional gel electrophoresis. The first dimension was carried out at pH 8.6 while the second was run at pH 4.6. The monosome contained sixty seven proteins, of which six were acidic. Two proteins were lost after subunit dissociation. Twenty six basic and two acidic proteins were observed in the 40 S subunit while the largest subunit gave thirty nine spots on the basic part of the gel and three additional spots on the acidic side. Five proteins were shared by 40 S and 60 S.     

Stoichiometric analysis of the flagellar hook-(basal-body) complex of Salmonella typhimurium

The stoichiometries of components within the flagellar hook-(basal-body) complex of Salmonella typhimurium have been determined. The hook protein (FlgE), the most abundant protein in the complex, is present at approximately 130 subunits. Hook-associated protein 1 (FlgK) is present at approximately 12 subunits. The distal rod protein (FlgG) is present at approximately 26 subunits, while the proximal rod proteins (FlgB, FlgC and FlgF) are present at only approximately six subunits each. The stoichiometries of the proximal rod proteins and hook-associated protein 1 are, within experimental error, consistent with values of 5 or 6, and 11, respectively. Such values would correspond to either one or two turns of a helical structure with a basic helix of approximately 5.5 subunits per turn, which is the geometry of both the hook and the filament and, one supposes, the rod and hook-associated proteins. These stoichiometries may derive from rules for the heterologous interactions that occur when a helical structure consists of successive segments constructed from different proteins; the stoichiometries within the hook and the distal portion of the rod must, however, be set by different mechanisms. The stoichiometries for the ring proteins are approximately 26 subunits each for the M-ring protein (FliF), the P-ring protein (FlgI), and the L-ring protein (FlgH); the protein responsible for the S-ring feature is not known. The rings presumably have rotational rather than helical symmetry, in which case the stoichiometries would be directly constrained by the intersubunit bonding angle. The ring stoichiometries are discussed in light of other information concerning flagellar structure and function.     

Studies on subunit assembly of broad bean legumin in a reconstitution system

The constituent subunits of 11S globulin of broad bean, legumin, were separated into basic subunits (BS, a mixture of BSI, BSII, and BSIII) and acidic subunits (ASI, ASII, and ASIII). The 11S components were formed in reconstitution reactions from combinations of BS and one or two each of the acidic subunits. The reconstituted 11S components were similar to the native legumin; they all consisted of acidic (A) and basic (B) subunits linked by disulfide bridges in the ratio of 1:1 and had the 6 (AB) structure. In the reconstitution of 11S components, ASI preferentially selected BSI from among the three kinds of BS, and ASII and ASIII exhibited selectivities for BSII and BSIII, respectively. The same selectivities were observed in the reconstitution reaction containing all subunits and in the renaturation reaction from the reduced-denatured state. The selectivity of each acidic subunit for basic subunits coincides with the combination of acidic and basic subunits in the native legumin. The 11S component was reconstituted from any combination of the intermediary subunits examined. This may be one of the reasons for the occurrence of heterogeneity of legumin molecular species.     

Analysis of the protein composition of yeast ribosomal subunits by two-dimensional polyacrylamide gel electrophoresis

The number of proteins in yeast ribosomal subunits was determined by two-dimensional polyacrylamide gel electrophoresis. The 40S subunit obtained after dissociation of ribosomes at high ionic strength contains 30 different protein species (including six acidic proteins). The 60S subunit, obtained in the same way contains 39 different species (including 1 acidic protein). While the total number of protein species found in yeast ribosomes, thus, is in close agreement with those reported for other eukaryotic organisms, the distribution between acidic and basic proteins is quite different. When the ribosomes were dissociated at low ionic strength, four extra protein spots appeared in the electropherograms of both 40S and 60S subunits. We consider these proteins to be nonribosomal.     

Identification of initiation factors and ribosome-associated phosphoproteins by two-dimensional polyacrylamide gel electrophoresis.

A two-dimensional polyacrylamide gel electrophoresis procedure has been used to identify initiation factors rapidly in the high-salt-wash fraction from reticulocyte ribosomes. Initiation factors are identified by relative mobility and by co-electrophoresis with purified factors. A creatine phosphate/ATP/GTP/Pi exchange system is described which has been used to maintain [gamma-32P]ATP and [gamma-32P]GTP at constant specific activity in the cell-free protein-synthesizing system. Phosphorylated proteins associated with the protein-synthesizing complex have been identified using a combination of the two procedures. The salt-wash fraction contains eight major phosphorylated proteins and a number of minor ones. Two phosphorylated proteins are observed to comigrate with two of the three subunits of eukaryotic initiation factor 2 (eIF-2), the initiation factor involved in binding Met-tRNAf onto the 40-S subunit and promoting dissociation of 80-S ribosomes. eIF-4B, one of the proteins involved in binding mRNA to 40-S subunits is also phosphorylated. The remainder of phosphorylated proteins in the high-salt-wash fraction are not previously characterized initiation factors and have not been identified further. Two of the six phosphoproteins associated with the salt-washed ribosomes comigrate with ribosomal proteins; one is the major phosphorylated protein in 40-S ribosomal subunits, the other is an acidic protein.     

Multiple molecular forms of mouse liver arginase

Hepatic arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is an oligomer composed of three or four subunits. The present studies indicate heterogeneity in the size and charge of arginase subunits in mouse liver. Two types of arginase subunits with molecular weights of approximately 35,000 and 38,000 have been found. These two subunits are detected in liver cytosol or in purified preparations of arginase after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Two dimensional SDS-PAGE revealed multiple ionic forms of arginase for both the 35,000 and 38,000 subunits; the subunits contain basic proteins (pI range 7.8-9.1) and acidic proteins (pI range 5.8-6.4). Limited proteolysis by trypsin eliminated the molecular weight differences between the subunits without substantially affecting either their isoelectric points or activity. Comparative peptide maps and amino acid analyses of the 35,000- and 38,000-Da subunits showed that they were very similar. The data indicate that a neutral peptide (approx 3000 Da) is responsible for the differences in subunit molecular weight and that the multiple sized and charged forms are variants of the same protein.     

Class III human liver alcohol dehydrogenase: a novel structural type equidistantly related to the class I and class II enzymes

The primary structure of class III alcohol dehydrogenase (dimeric with chi subunits) from human liver has been determined by peptide analyses. The protein chain is a clearly distinct type of subunit distantly related to those of both human class I and class II alcohol dehydrogenases (with alpha, beta, gamma, and pi subunits, respectively). Disregarding a few gaps, residue differences in the chi protein chain with respect to beta 1 and pi occur at 139 and 140 positions, respectively. Compared to class I, the 373-residue chi structure has an extra residue, Cys after position 60, and two missing ones, the first two residues relative to class I, although the N-terminus is acetylated like that for those enzymes. The chi subunit contains two more tryptophan residues than the class I subunits, accounting for the increased absorbance at 280 nm. There are also four additional acidic and two fewer basic side chains than in the class I beta structure, compatible with the markedly different electrophoretic mobility of the class III enzyme. Residue differences between class III and the other classes occur with nearly equal frequency in the coenzyme-binding and catalytic domains. The similarity in the number of exchanges relative to that of the enzymes of the other two classes supports conclusions that the three classes of alcohol dehydrogenase reflect stages in the development of separate enzymes with distinct functional roles. In spite of the many exchanges, the residues critical to basic functional properties are either completely unchanged--all zinc ligands and space-restricted Gly residues--or partly unchanged--residues at the coenzyme-binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polymerization of Soybean 11S Globulin Due to Reactions with Peroxidizing Linoleic Acid

Soybean 11S globulin was polymerized by incubating with peroxidizing linoleic acid. The molar ratio of the acidic subunits to the basic subunits of 11S globulin decreased with the elapse of the incubation time. The acidic subunits were lost faster and formed polymers more easily than the basic subunits. The acidic and basic subunits in 11S globulin were fractionated by DEAE-Sephadex gel chromatography. Each of the acidic and basic subunits was allowed to react with peroxidizing linoleic acid individually. The results also showed that the acidic subunits formed polymers faster than the basic subunits. Both succinylated and acetylated 11S globulins were also submitted to the incubation with peroxidizing linoleic acid. The polymerization of the modified protein was suppressed by masking ε-amino groups.     

Isolation and Partial Characterization of Heat-denatured Products of Soybean 11S Globulin and Their Analysis by Electrophoresis

Heat denaturation of soybean 11S globulin was examined at 70° and 100°C in phosphate buffer (pH 7.6), at 0.01 and 0.5 ionic strength. Gel electrophoresis (Davis system) indicated that heat-denatured soybean 11S globulin contained two major components (buffer-soluble form). But they were not identified at 70°C-0.5 ionic strength. Gel filtration followed by SDS-gel electrophoresis showed that the major components were composed of a monomer and at least three of kinds of oligomers containing only an acidic subunit. Gel filtration of the precipitate formed at 100°C at 0.5 ionic strength gave two peaks. SDS-gel electrophoresis indicated that the first peak contained aggregates of highly polymerized subunits, and the second peak contained a monomer of basic subunit and seven kinds of oligomers with various proportions of basic subunits to an acidic subunit.     

Purification and characterization of protein phosphatase 1I activating kinase from bovine brain cytosolic and particulate fractions

The activating kinase of protein phosphatase 1I is distributed in approximately equal amounts between the cytosolic and particulate fractions of bovine brain homogenates. Both species of this protein kinase have been purified to near homogeneity. The cytosolic form, purified about 7,000-fold, has an apparent Mr = approximately 75,000, as estimated by gel filtration chromatography on Sephacryl S-300. The enzyme contains two subunits, with apparent Mr = 52,000 and 46,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both subunits undergo phosphorylation when the enzyme is incubated with Mg2+ and [gamma-32P]ATP. Peptide maps of the two subunits are different, and rabbit antibodies to the 52-kDa subunit show only very minor cross-reactivity to the 46-kDa subunit. These observations indicate that the two subunits are different. The species of protein phosphatase 1I activating kinase that is associated with the membrane fraction has an apparent Mr = approximately 105,000 as estimated by gel filtration. This species also contains two subunits, with apparent Mr = 52,000 and 46,000, the properties of which are very similar, if not identical, to those of the two subunits comprising the cytosolic form of the protein kinase.     

Variation and inheritance of the arachin polypeptides of groundnut (Arachis hypogaea L.)

Summary Variation in the arachin polypeptides of groundnut genotypes was observed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Three regions could be observed on the electropherogram. Region 1, corresponding to conarachin, did not show any variation; region 2, consisting of arachin acidic subunits, showed variation; region 3, containing the arachin basic subunits, did not show any variation. There are four varietal classes of arachin polypeptide patterns: class A comprised three acidic subunits of arachin of molecular weights 47.5, 45.1 and 42.6 kd and a basic subunit of 21.4 kd; class B, with three acidic subunits of molecular weights 47.5, 45.1 and 41.2 kd and a basic subunit of 21.4 kd; class C of an additive pattern of class A and class B; class D, of two acidic polypeptides of 47.5, 45.1 kd and the basic 21.4 kd subunit. Of the 90 genotypes studied, 73% belong to class A, 15% to class B and 6% each to class C and D. Analysis of F₂ seeds from a cross between class A and class B genotypes showed that the two polypeptides (42.6 kd and 41.2 kd) are coded by nonallelic genes and also revealed that class C and class D patterns arose as a result of hybridisation between class A and class B. A. monticola, the progenitor of A. hypogaea, showed a pattern similar to the additive pattern of class A and class B while some diploid Arachis species had the 41.2 kd polypeptide. Based on arachin polypeptide patterns the probable origin of A. hypogaea has been suggested.     

Identification of novel subunits of the TOM complex from Arabidopsis thaliana

The preprotein translocase of the outer mitochondrial membrane (also called TOM complex) from Arabidopsis thaliana was characterized by Blue-native gel electrophoresis (BN-PAGE) and Electrospray Tandem Mass Spectrometry (ESI-MS/MS). BN-PAGE allows to prepare a very stable 390 kDa complex that includes six different protein types: the 34 kDa translocation pore TOM40, the 21/23 kDa preprotein receptor TOM20, the small TOM component TOM7 and three further subunits of 10, 6.3 and 6.0 kDa. Primary structures of all TOM subunits were elucidated. The 10 kDa subunit represents a truncated version of the TOM22 preprotein receptor and the two 6 kDa proteins represent subunits possibly homologous to fungal TOM6 and TOM5, although sequence conservation is at the borderline of significance. TOM40, TOM7 and one or both of the 6 kDa subunits form a subcomplex of about 100 kDa. The six TOM proteins from Arabidopsis are encoded by 12 genes, at least 11 of which are expressed. While the subunit composition of the TOM complex from fungi, animals and plants is remarkably conserved, the domain structure of individual TOM proteins differs, e.g. acidic domains in TOM22 and the 6 kDa TOM subunits from Arabidopsis are absent. The domain structure of the Arabidopsis TOM complex does not support the so-called ‘acid chain hypothesis’, which explains the translocation of proteins across the outer mitochondrial membrane of mitochondria by the binding of preproteins to acidic protein domains within the TOM complex. Functional implications are discussed.     

Isolation and partial characterization of a 40 S ribosomal subunit-transfer ribonucleic acid binding factor from rabbit reticulocytes.

A factor that catalytically promotes the codon-directed, GTP-independent binding of tRNA to 40 S ribosomal subunits has been isolated from the postribosomal supernatant and the ribosomal wash of rabbit reticulocytes. The factor is a heat labile, sulfhydryl reagent-sensitive protein of a molecular weight of approximately 50,000. It consists of two non-identical subunits of Mr equals approximately 30,000 and 20,000. Its basic character has been confirmed by the high ratio of basic amino acids to nonamidic aspartic and glutamic acid present in the purified protein. Formation of a factor promoted 40 S-poly(U)-phenylalanyl-tRNA initiation complex causes a shift in the Mg-2+ concentration optimum for polyphenylalanine synthesis from 8 mM to 4mM.     

Reevaluation of the Subunit Molecular Weights of Soybean 11S Globulin

The acidic and basic subunits are the main constituents of soybean 11S globulin. Each of these two subunits consists of three major polypeptides of similar size. The molecular weights of the acidic and basic subunits have been previously estimated to be 37,000 and 22,000, respectively, by SDS-polyacrylamide gel electrophoresis (Catsimpoolas et al, J. Set Food. Agric., 22, 448 (1971)). Reevaluation of the molecular weights by 6 m guanidine gel chromatography gave the values of 28,000 and 18,000, respectively. These are supported by results of equilibrium sedimentation in the same solvent. The previously reported values seem to have been overestimated, especially for the acidic subunits. The overestimations seem to be related to the high percentage of acidic amino acids, which causes the conformation of the SDS-protein polypeptide complexes of these subunits to deviate from those of proteins usually employed as standards for molecular weight estimations.     

Protein subunits and amino acid composition of wild lentil

Three wild species of lentil, Lens orientalis, L. ervoides and L. nigricans were investigated for protein subunits of the albumin protein fraction (APF), globulin protein fraction (GPF) and for protein and free amino acid composition. The APF and GPF formed 12.7–16.8 % and 34.7–49.0 %, respectively, of the meal nitrogen. SDS-PAGE showed APF to contain 15 to 20 major and a similar number of minor protein subunits ranging in M_r at least from 14 400 to 94 000. The GPF was also heterogenous and contained some subunits having M_r similar to APF subunits but none < 15 000. The three wild lentil species were distinguishable by their protein subunit composition. The protein amino acid composition of the wild species was identical and similar to that of the cultivated lentil. The wild species, like the cultivated species (L. culinaris), contained major amounts of free arginine, glutamic and aspartic acids, serine and a number of unidentified amino acids. L. orientalis, L. nigricans and the cultivated lentil contained two acidic and two basic unidentified amino acids. However, L. ervoides was distinctly different in that it contained only the two acidic plus one neutral unidentified amino acid, but none of the two basic unidentified amino acids.     

Cytidine deaminase from Escherichia coli B. Purification and enzymatic and molecular properties

Cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5) from Escherichia coli has been purified to homogeneity through a rapid and efficient two-step procedure consisting of anion-exchange chromatography followed by preparative electrophoresis. The final preparation is homogeneous, as judged by a single band obtained by disc gel electrophoresis performed in the absence and presence of denaturing agents. The native protein molecular weight determined by gel filtration is 56 000. Sodium dodecyl sulfate disc gel electrophoresis experiments conducted upon previous incubation of the enzyme with dimethyl suberimidate suggest an oligomeric structure of two identical subunits of 33 000 molecular weight. The absorption spectrum of the protein reveals a maximum at 277 nm and a minimum at 255 nm. The isoelectric point is at pH 4.35. Amino acid analysis indicates an excess of acidic amino acid residues as well as six half-cystine residues. No interchain disulfide groups have been evidenced. According to Cleland's nomenclature, kinetic analysis shows a rapid-equilibrium random Uni-Bi mechanism. Cytidine deaminase is competitively inhibited by various nucleosides. Km values for cytidine, deoxycytidine, and 5-methylcytidine are 1.8 X 10(-4), 0.9 X 10(-4), and 12.5 X 10(-4) M, respectively.