Charge heterogeneity of heat-labile enterotoxins from human enterotoxigenic Escherichia coli

Abstract We purified heat-labile enterotoxins (LThs) from YT3, H-10407 and YT240 strains isolated from human diarrheal patients. These LThs were immunologically identical to each other. The molecular weights of their A and B subunits were also the same by means of SDS-polyacrylamide gel electrophoresis. However, the ionic charges of the molecular surfaces of these LThs were different as shown by polyacrylamide gel isoelectric focusing. Though the p I points of B subunits of the LThs were identical to each other, the p I points of A subunits were found to be different. These data suggest that the ionic charge differences among A subunits cause differences in holo LThs in their charge, and that there is heterogeneity among A subunits produced by strains of human enterotoxigenic Escherichia coli .     

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.     

Purification and characterization of five forms of glutathione transferase from human uterus

Five glutathione transferase (GST) forms were purified from human uterus by glutathione-affinity chromatography followed by chromatofocusing, and their structural, kinetic and immunological properties were investigated. Upon SDS/polyacrylamide slab gel electrophoresis all forms resulted composed of two subunits of identical molecular size. GST V (pI 4.5) is a dimer of 23-kDa subunits. GST I (pI 6.8) and GST IV (pI 4.9) are dimers of 24-kDa subunits whereas GST II (pI 6.1) and GST III (pI 5.5) are dimers of 26.5-kDa subunits. GST V accounts for about 85-90% of the activity whereas the other isoenzymes are present in trace quantities. On the basis of the molecular mass of the subunits, amino acid composition, substrate specificities, sensitivities to inhibitors, CD spectra and immunological studies, GST V appeared very similar to transferase pi. Structural and immunological studies provide evidence that GST IV is closely related to the less 'basic' transferase (GST pI 8.5) of human skin. Extensive similarities have been found between GST II and GST III. The comparison includes amino acid compositions, subunits molecular size and immunological properties. The two enzymes, however, are kinetically distinguishable. The data presented also indicate that GST II and GST III are related to transferase mu and to transferase psi of human liver. Even though GST I has a subunit molecular mass identical to GST IV, several lines of evidence, including catalytic and immunological properties, indicate that they are different from each other. GST I seems not to be related to any of known human transferases, suggesting that it may be specific for the uterus.     

Purification Of Mouse and Human Interferons: Detection Of Subunit Structure

During the purification of mouse and human interferons, multiple active components have been detected. Mouse interferon was purified over 500-fold by differential precipitation, centrifugation, gel chromatography, and isoelectric focusing. On electrofocusing, two molecular forms (A and B) were noted. Form B (pI 7.35) had a molecular weight of about 38,000 and Form A (pI 7.15), which was equally active, a molecular weight of 19,000. Purified Form B was dissociable into Form A, but the reverse reaction occurred to a much lesser extent. Human interferon, purified about 1500-fold, is also composed of multiple molecular forms. Form B (pi 5.60) had a molecular weight of about 24,000 and Form A (pi 5.35), which may contain up to 85% of the total activity, a molecular weight of 12,000. Both forms appear to have equal specific activities. The dissociation of both human and mouse interferons into subunits appeared to take place during dialysis versus low salt (0.01 M tris pH 7.A). The data are consistent with the idea that the native molecule exists as a dimer of similar or identical subunits. Dimer formation, which probably occurs within the cells, does not seem to lead to a measurable cooperative effect between the subunits.     

The subunit structure of a major glutathione S-transferase form, MT, in rat testis. Evidence for a heterodimer consisting of subunits with different isoelectric points

A major glutathione S-transferase form (pI 5.7) in rat testis (MT) purified by S-hexyl-glutathione affinity chromatography, followed by chromatofocusing, showed two polypeptide of pI 6.7 (Yn1) and 6.0 (Yn2), having apparently the same molecular mass of 26 kDa on two-dimensional gel electrophoresis. Rechromatofocusing of the MT preparation after 4 M guanidine hydrochloride treatment revealed two additional protein peaks (pI 6.2 and 5.4). These were identified as the two homodimers consisting of the subunits of MT, Yn1Yn1 and Yn2Yn2, respectively. Furthermore, MT could be reconstituted from Yn1Yn1 and Yn2Yn2. These results indicate that MT is a heterodimer, Yn1Yn2, consisting of subunits with very similar molecular masses but different isoelectric points. The Yn1Yn1 form had glutathione S-transferase activities towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene. However, the Yn2Yn2 form had no activity towards any of the substrates examined. N-terminal amino acid sequences of subunits Yn1 and Yn2 revealed differences at two positions in the first 20 residues; the amino acid compositions of these subunits were also similar but not identical, indicating that these two subunits are different in the primary structure. Subunits Yn1 and Yn2 are immunologically related to each other and also to subunits 3 (Yb1) and 4 (Yb2) but they are not identical. These four subunits also showed a high degree of similarity in N-terminal amino acid sequences. Subunits Yn1 and Yn2 seem to belong to the rat GST 3-4 family or class mu. Subunits Yn1 and 4 can make a heterodimer, which is detectable not only in rat testis, but also in the heart, kidney and lung. The Yn1Yn1 form was not detected in the testis, but is present in rat brain [Tsuchida et al. (1987) Eur. J. Biochem. 170, 159-164]. The Yn2Yn2 form seemed to differ from GST 5-5 and may be a new form of rat glutathione S-transferase.     

Purification and comparison of the structures of human liver acidic alpha-D-mannosidases A and B.

Human liver alpha-D-mannosidases A and B were purified 11 500-fold and 2000-fold respectively. Both showed microheterogeneity when analysed by isoelectric focusing. Alpha-D-Mannosidases A and B are immunologically identical but differ in their range of pI values, molecular masses, uptake into fibroblasts and subunit compositions. Alpha-D-Mannosidase A consists of equimolar proportions of subunits of molecular masses 62 kDa and 26 kDa, which are linked by disulphide bridges in the intact enzyme. Alpha-D-Mannosidase B also contains a small subunit, of molecular mass 26 kDa, and a variable mixture of larger subunits, of molecular masses 58 kDa and 62 kDa. The 62 kDa and 58 kDa subunits, but not the 26 kDa one, contain concanavalin A-recognizing glycans. The 58 kDa subunit has a lower pI, contains less high-mannose glycans but probably contains more mannose 6-phosphate than the 62 kDa subunit. It is postulated that the differences in structure and properties of alpha-D-mannosidases A and B are due to differences in the state of processing of the large subunit. This suggestion is consistent with a single locus on chromosome 19 for lysosomal alpha-D-mannosidase.     

Two immunologically distinct Yc-type subunits are present in rat lung glutathione S-transferases.

Two types of 25 000-Mr subunits are present in rat lung glutathione S-transferase I (pI 8.8). These subunits, designated Yc and Yc', are immunologically and functionally distinct from each other. The homodimers YcYc (pI 10.4) and Yc'Yc' (pI 7.6) obtained by hybridization in vitro of the two subunits of glutathione S-transferase I (pI 8.8) were isolated and characterized. Results of these studies indicate that only the Yc subunits express glutathione peroxidase activity and cross-react with the antibodies raised against glutathione S-transferase B (YaYc) or rat liver. The Yc' subunits do not express glutathione peroxidase activity and do not cross-react with the antibodies raised against glutathione S-transferase B of rat liver. The amino acid compositions of these two subunits are also different. These two subunits can also be separated by the two-dimensional gel electrophoresis of glutathione S-transferase I (pI 8.8) of rat lung.     

Different forms of human liver glutathione S-transferases arise from dimeric combinations of at least four immunologically and functionally distinct subunits.

Four immunologically distinct subunits were characterized in glutathione (GSH) S-transferases of human liver. Five cationic enzymes (pI 8.9, 8.5, 8.3, 8.2 and 8.0) have an apparently similar subunit composition, and are dimers of 26 500-Mr (A) and 24 500-Mr (B) subunits. A neutral enzyme, pI 6.8, is a dimer of B-type subunits. One of the anionic enzymes, pI 5.5, is also a dimer of 26 500-Mr subunits. However, the 26 500-Mr subunits of this anionic enzyme form are immunologically distinct from the A subunits of the cationic enzymes, and have been designated as A'. Immunoabsorption studies with the neutral enzyme, BB, and the antibodies raised against the cationic enzymes (AB) indicate that A and B subunits are immunologically distinct. Hybridization in vitro of the A and B subunits of the cationic enzymes (AB) results in the expected binary combinations of AA, AB and BB. Studies with the hybridized enzyme forms indicate that only the A subunits express GSH peroxidase activity. A' subunits have maximum affinity for p-nitrobenzyl chloride and p-nitrophenyl acetate, and the B subunits have highest activity towards 1-chloro-2,4-dinitrobenzene. The other anionic form, pI 4.5, present in liver is a heterodimer of 22 500-Mr (C) and B subunits. The C subunits of this enzyme are probably the same as the 22 500-Mr subunits present in human lung and placental GSH transferases. The distinct immunological nature of B and C subunits was also demonstrated by immunoaffinity and subunit-hybridization studies. The results of two-dimensional polyacrylamide-gel-electrophoretic analyses indicate that in human liver GSH transferases, three charge isomers of Mr 26 500 (A type), two charge isomers of Mr 24 500 (B type) and two charge isomers of Mr 22 500 (C type) subunits are present.     

Characterization of the Antigenic Subunits of the Envelope Protein of Yersinia pestis

Chemical, physical, and immunological properties of the envelope antigen of Yersinia pestis strains have been investigated. The antigen consists of two components with isoelectric points (pI) of 4.6 and 4.8. One component (pI 4.6) is a protein bound to a small carbohydrate moiety identified as an oligomeric galactan; the other component (pI 4.8) is a simple protein. These two components are antigenically identical. In buffered solution, the antigen exists as aggregates of molecular weights larger than 300,000. The aggregates dissociate into a variety of smaller molecular weight forms depending on the nature of the treatment for dissociation. Each aggregate can be further dissociated into a single antigenic subunit fraction containing protein and glycoprotein species with molecular weights in the range from 15,000 to 17,000. The subunits can be obtained by a dissociation treatment with 0.1% mercaptoethanol in 0.25% sodium dodecyl sulfate at 95 C for 5 min. The subunits will readily reaggregate into a variety of larger molecular weight forms on the removal of dodecyl sulfate.     

Sunflower seed protein: size and charge heterogeneity in subunits of the globulin fraction

The subunit heterogeneity of the globulin fraction of sunflower seeds was investigated by two dimensional electrophoresis, using isoelectric focusing in the first dimension and sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension. Under non reducing conditions, intermediary subunits B, C and D (molecular weight 54 000, 48 000 and 40 000, respectively) were focused within a pI range 5.4-6.0 but intermediary subunits A (molecular weight 60 000) focused within a pI range 6.3-6.8. Under reducing conditions the electrophoretic patterns show that intermediary subunits consist in large "acidic" and small "basic" subunits linked by disulphide bonds. The large subunits of B species are more acidic and less heterogeneous than the corresponding subunits of the A species. These results confirm that helianthinin had a "legumin-type" structure.     

Species Specificity of Brain Cyclic AMP Receptor Proteins

The cyclic AMP binding proteins present in mouse, rat, bovine, and sheep brains were compared. Extracts were isotopically labeled with 8-azido-cyclic [32P]AMP, a photoaffinity analog specific for cyclic AMP binding sites, and then subjected to two-dimensional gel electrophoresis. The resulting autoradiographic patterns were generally similar, but showed consistent species variations. Proteins identified by their size and phosphorylatability as regulatory subunits of Type II protein kinase isozymes were present in all species, but with slight variations in pI. A series of charge variants identified as regulatory subunits of Type I kinase isozymes on the basis of their size was also ubiquitously present, as were several smaller proteins postulated to be proteolytic fragments derived from the regulatory subunits. The major species difference was a series of labeled proteins found only in rodent brains and not in the brains of any ruminant, or in other rodent tissues. These proteins had a molecular weight of 54,000 and a pI range of 5.89--6.26, and could not be endogenously phosphorylated. The identities of these proteins and their relationship to the protein kinase regulatory subunits are unknown.     

Purification and subunit-structural and immunological characterization of five glutathione S-transferases in human liver, and the acidic form as a hepatic tumor marker

Five glutathione S-transferase (GST, EC 2.5.1.18) forms were purified from human liver by S-hexylglutathione affinity chromatography followed by chromatofocusing, and their subunit structures and immunological relationships to rat liver glutathione S-transferase forms were investigated. They were tentatively named GSTs I, II, III, IV and V in order of decreasing apparent isoelectric points (pI) on chromatofocusing. Their subunit molecular weights assessed on SDS-polyacrylamide gel electrophoresis were 27 (Mr X 10(-3)), 27, 27.7,27 and 26, respectively, (26, 26, 27, 26, and 24.5 on the assumption of rat GST subunit Ya, Yb and Yc as 25, 26.5 and 28, respectively), indicating that all forms are composed of two subunits identical in size. However, it was suggested by gel-isoelectric focusing in the presence of urea that GSTs I and IV are different homodimers, consisting of Y1 and Y4 subunits, respectively, which are of identical Mr but different pI, while GST II is a heterodimer composed of Y1 and Y4 subunits. This was confirmed by subunit recombination after guanidine hydrochloride treatment. GST III seemed to be identical with GST-mu with regard to Mr and pI. GST V was immunologically identical with the placental GST-pi. On double immunodiffusion or Western blotting using specific antibodies to rat glutathione S-transferases, GST I, II and IV were related to rat GST 1-1 (ligandin), GST III(mu) to rat GST 4-4 (D), and GST V (pi) to rat GST 7-7 (P), respectively. GST V (pi) was increased in hepatic tumors.     

Isolation of acidic acrosin isoinhibitors (BUSI I A, BUSI IB1 and BUSI I B2) from bull seminal plasma.

Three natural proteinase isoinhibitors with low isoelectric points BUSI I A (pI = 3.9), BUSI I B1 (pI = 3.4 and BUSI I B2 (pI = 3.7) were isolated from bull seminal plasma by gel filtration on Sephadex G-50 and ion exchange chromatography on DEAE-Sephadex and SE-Sephadex. Isoinhibitors Bl and B2 have identical amino acid composition. Isoinhibitor A contains six amino acid residues less than isoinhibitors B1 and B2. Since sugars have been detected in the isoinhibitors, heterogeneity may also be due to the sugar component. The isoinhibitors show the same inhibitory properties; all of them inhibit acrosin, trypsin and chymotrypsin. Glandular kallikrein is also inhibited, but to a very low extent only. The molecular weight (Mr approximately 8 900) was determined by gel filtration.     

格拉姆柱花草(Stylosanthes gracilis H.B.K.cv.Graham)凝集素的纯化与性质研究

为了探讨凝集素在豆科植物与根瘤菌的识别过程中的作用,用ＤＥＡＥ－３２离子交换层析和ＳｅｐｈａｄｅｘＧ－１５０凝胶过滤分离、纯化格拉姆柱花草种子凝集素（ＳＧＬ）,其分子量约为４５ｋＤ,由两个相同的亚基组成,等电点约ｐＨ５．８,它是一种糖蛋白,含糖量约为２．６％．ＳＧＬ的热稳定性强．ＳＧＬ的血凝活性能被甘露糖所抑制．ＳＧＬ对红细胞的凝集作用可能具有种属专一性;ＳＧＬ具有强的促有丝分裂作用;荧光标记实验显示：９株能与格拉姆柱花草植株结瘤的菌株有７株能与ＳＧＬ结合,６株不能与之结瘤的菌株,只有１株能与ＳＧＬ结合,这表明不同根瘤菌菌株对ＳＧＬ的结合能力,和它们在格拉姆柱花草上结瘤能力之间可能具有一定的生物学相关性．     

Staphylococcal L-asparaginase: purification and properties of enzymic protein

Staphylococcal L-asparaginase has been purified 400-fold with 40% recovery. The procedure involves ammonium sulphate precipitation and a column chromatography on Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. protein (pI 4.4) with the approximate molecular weight of 125,000 (estimated by Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. The polyacrylamide-SDS gel electrophoresis indicated two subunits with molecular weight 18,000 and 22,000.     

Two-dimensional gel electrophoresis of ribosomal proteins from streptomycin-sensitive and streptomycin-resistant mutants of Chlamydomonas reinhardi.

Ribosomal proteins from three mutant strains of Chlamydomonas reinhardi were analysed and compared by one-dimensional and two-dimensional gel electrophoresis. One mutant was streptomycin-sensitive the other two were streptomycin-resistant, one with a Mendelian the other with a non-Mendelian pattern of inheritance. In the 30-S subunits of chloroplast ribosomes approximately 25 proteins are found and in the 50-S subunits 34 proteins. The 40-S subunits of cytoplasmic ribosomes contain about 31 proteins and the 60-S subunits 44 proteins. The molecular weights of most proteins in all subunits are in the range of 10 000 to 35 000. However, the 60-S subunits contain in addition a protein of molecular weight 50 000 and the 30-S subunits show 6-7 bands of molecular weights from 50 000 to 83 000. The proteins of the cytoplasmic 80-S ribosomes or of their subunits from all three mutants are electrophoretically identical. The proteins of the 70-S organellar ribosomes and both of their subunits show distinct differences between the three strains. Our results indicate that organellar ribosomal proteins are in part controlled by nuclear DNA and in part by organellar DNA.     

cAMP-dependent protein kinases, their subunits, and cAMP-binding protein from four sources: a comparison of physical characteristics determined by polyacrylamide gel electrophoresis

The physical characteristics of cAMP-dependent protein kinases and their, regulatory subunits from calf uterus, human uterus, human mammary tumor, and rat pituitary and of cAMP-binding protein from calf uterus were determined by quantitative polyacrylamide gel electrophoresis in buffers containing the detergent, Triton X-100. In the four tissues, protein kinases of either type A¹, with molecular weight (M_r) = 200,000, or type B, of M_r = 80,000, or both, previously described were found. Trivial charge isomerism, or size isomerism, exists within each of the two classes, Protein Kinase A and B. The protein kinase recombined from the regulatory and catalytic subunits is not significantly different from the crude or isolated protein kinase. Protein Kinases A and B exist each in either one of the isozyme forms I and II but these are not reflected in polyacrylamide gel electrophoresis at pH 10.2. Protein Kinase B appears to be a product of the partial proteolysis of Protein Kinase A. The regulatory subunits of Protein Kinases A from the four tissues are distinct from those of Protein Kinases B. No physical distinction exists between regulatory subunits derived from isozyme forms I and II. cAMP-Binding Proteins A and B are physically indistinguishable, by polyacrylamide gel electrophoresis at pH 10.2, from the regulatory subunits of Protein Kinases A and B, respectively.     

Purification and characterization of four isolectins of mushroom (Agaricus bisporus)

Four lectins were purified from a mushroom (Agaricus bisporus) by ammonium sulfate fractionation, anion-exchange chromatography, affinity chromatography on bovine submaxillary mucin-Sepharose 4B and preparative isoelectric focusing. They were designated as ABA-I (pI 6.70), II (pI 5.98), III (pI 5.69) and IV (pI 5.53). Polyacrylamide gel electrophoresis of each lectin in the presence of sodium dodecyl sulfate gave a single band with an apparent molecular mass of 16 000 Da. Sedimentation equilibrium analysis suggested that each lectin is a tetramer of subunits. The four lectins were found to have quite similar carbohydrate-binding specificities. The hemagglutination activities of the lectins were effectively inhibited by bovine and porcine submaxillary mucins (BSM and PSM), and NH2-terminal glyco-octapeptides obtained by cyanogen bromide cleavage of human erythrocyte glycophorin A. In addition, desialylated PSM-glycopeptides were more potent inhibitors than untreated PSM-glycopeptides. Among monosaccharides and their glycosides, only methyl N-acetyl-alpha-galactosaminide inhibited lectin binding at a high concentration, but a synthetic oligosaccharide, O-beta-galactopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-alpha-D- galactopyranosyl)-N-tosyl-L-serine, was a strong inhibitor.     

Arylsulfatases of human-lung tumors transplanted into athymic mice. Cancer-associated modification of arylsulfatase B variant

The activities and properties of arylsulfatase A and B from human lung carcinoma transplanted into athymic mice were demonstrated. The activities of arylsulfatase A and B from transplanted carcinomas with four histological types were more than twofold higher as compared to those from surgical tumors, except for arylsulfatase A activity in blastoma. Arylsulfatase B in transplanted tumors was almost completely replaced, except for blastoma, by an anionic B variant (B1) which was a minor component of arylsulfatase B in surgical lung tumor and absent in normal human lung. The properties of arylsulfatases A and B from transplanted tumors were essentially identical, respectively, with those from normal lung or surgical tumors in respect of molecular weight, heat stability, pH optimum, isoelectric point (pI), Km, time course profile and substrate specificity. Arylsulfatase B1 showed the properties similar to B enzyme except for net charge. The cause of the negative charge of tumor B1 enzyme was investigated. By the action of phosphatase, which was added exogenously or had been persistently included in the partially purified enzyme preparation, B1 enzyme (pI 7.5) shifted to about pI 8.2. Treatment of B1 enzyme with neuraminidase, concomitant with the endogenous phosphatase, resulted in marked increase (pI 9.5) of the isoelectric point, identical to that of arylsulfatase B. Thus, it is most probable that tumor B1 enzyme is modified by additional sialic acid and phosphate bound to arylsulfate B.     

Amaranth Globulin Polypeptide Heterogeneity

The polypeptides integrating amaranth globulin-p and 11S-globulin were characterized by two-dimensional electrophoresis, ion-exchange chromatography and RP-HPLC. All polypeptides exhibited charge and hydrophobic heterogeneity. Almost all acid (A, pI 5–7) and basic (B, pI 9–10) polypeptides were present in both globulins, and the same happened with the unprocessed M polypeptides with pI in the range of 7–7.5 which fits well with a sequence containing both the A and B polypeptides. There were other polypeptides only present in 11S-globulin, like some of 41 and 16 kDa, which might come from another precursor or be the products of a different processing of the propolypeptide. These results suggested that, although amaranth subunits from different subfamilies are interchangeable in different oligomers, some structural differences between them might affect the assembly of globulin molecules. Structural differences arising from this behavior could account for the different physicochemical properties of globulin molecules.