Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. Purification and properties of the enzyme from the electric organ of Electrophorus electricus

An unspecific carboxylesterase was purified 180-fold from acid-precipitated human liver microsomes. The final preparation was homogeneous on disc electrophoresis and polyacrylamide gel electrophoresis in the presence of 6.25 M urea at pH 3.2. A single symmetrical peak was also found on gel filtration and on velocity sedimentation in the analytical ultracentrifuge, whereas slight heterogeneity was observed on isoelectric focusing.The amino acid composition of the purified enzyme is presented. From the results the partial specific volume (0.745 ml × g^?1) and the minimal molecular weight (60,000) could be calculated. Fingerprint maps of tryptic peptides from the carboxymethylated enzyme are shown.The molecular weight as determined by gel filtration, disc electrophoresis, and analytical ultracentrifugation is in the range of 181,000–186,000. For the molecular weight of the subunits a value of 61,500 has been obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The equivalent weight of the enzyme has been estimated to be 62,500 from stoichiometry of its reaction with diethyl-p-nitrophenyl-phosphate. Partial cross-linking of the subunits with dimethyl suberimidate and subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis yielded three bands with molecular weights of 60,000, 120,000, and 180,000.From these results it is concluded that human liver esterase is a trimeric protein. It is composed of three subunits of equal size, and there is one active site per subunit.     

Purification and molecular properties of rabbit liver esterase ES-1A

1. The isolation of esterase ES-1A from rabbit liver microsomes/lysosomes is reported. The purification as measured by methylbutyrate-hydrolysing activity, was about 27-fold with a recovery of 2.4%. 2. The resulting product is apparently homogeneous by polyacrylamide (gradient) gel electrophoresis and sodium dodecyl sulphate/polyacrylamide gradient gel electrophoresis after protein staining. The enzyme exhibits heterogeneity after staining for esterase activity and in isoelectric focusing. 3. The molecular mass of the native protein was found to be about 183 kDa (determined by gel filtration and polyacrylamide gel electrophoresis) with a subunit mass of about 63 kDa, indicating a trimeric structure of the enzyme, with subunits of equal size. 4. ES-1A is a glycoprotein and is classified as a carboxylesterase (EC 3.1.1.1). 5. The high degree of similarity of the properties of rabbit ES-1A with those of mouse ES-6A and rat ES-10 suggests that these three esterases may have a common evolutionary origin.     

Heterogeneity of delta-crystallins of the embryonic mallard lens. Correlation between subunit compositions and isoelectric points.

delta-Crystallins from the lenses of embryonic mallards (Anas platyrhynchos) were analyzed with respect to native and subunit molecular weight, subunit composition, and isoelectric point. NaDodSO4-urea-polyacrylamide gel electrophoresis showed that unfractionated mallard delta-crystallins are composed of approximately equal amounts of subunits with molecular weights near 47 000 and 48 000. Agarose gel chromatography showed that the embryonic mallard delta-crystallins have native molecular weights slightly less than 200 000. Thus, embryonic mallard delta-crystallins appear to be tetramers. Five major and nine minor delta-crystallins were resolved by isoelectric focusing. The five predominant delta-crystallins each cross-reacted with antichick delta-crystallin antiserum, and each had a different proportion of the larger and smaller subunits, indicating a direct relationship between the isoelectric point and the subunit composition. The presence of numerous, minor species of native delta-crystallins with different isoelectric points suggested that the subunits possess charge heteogeneity as well as size heterogeneity.     

Subunit structure of a cortical granule lectin involved in the block to polyspermy in Xenopus laevis eggs

The cortical granule lectin of Xenopus laevis eggs is a large molecular mass glycoprotein involved in the post-fertilization block to polyspermy. We have investigated the subunit structure of this lectin and found that the native molecule contains 10-12 monomers, each of which has considerable charge and size heterogeneity due to glycosylated side chains. In addition, significant amino acid sequence homology is indicated by peptide mapping of subunits separated by isoelectric focusing.     

The third subunit of desulfoviridin-type dissimilatory sulfite reductases.

In addition to the 50-kDa (alpha) and 40-kDa (beta) subunits, an 11-kDa polypeptide has been discovered in highly purified Desulfovibrio vulgaris (Hildenborough) dissimilatory sulfite reductase. This is in contrast with the hitherto generally accepted alpha 2 beta 2 tetrameric subunit composition. Purification, high-ionic-strength gel-filtration, native electrophoresis and isoelectric focussing do not result in dissociation of the 11-kDa polypeptide from the complex. Densitometric scanning of SDS gels and denaturing gel-filtration indicate a stoichiometric occurrence. A similar 11-kDa polypeptide is present in the desulfoviridin of D. vulgaris oxamicus (Monticello), D. gigas and D. desulfuricans ATCC 27774. We attribute an alpha 2 beta 2 gamma 2 subunit structure to desulfoviridin-type sulfite reductases. N-terminal sequences of the alpha, beta and gamma subunits are reported.     

The identification of a glutathione S-transferase isozyme in fetal rat livers which is absent from adult livers

The subunit composition of adult and fetal rat liver glutathione S-transferase was investigated by affinity chromatography followed by polyacrylamide gel electrophoresis in sodium dodecylsulphate. Adult livers contained four major GSH S-T subunits. A previously unidentified subunit was detected in fetal livers. This subunit(s), which differed from that found in rat placenta, had a molecular weight of about 25,500 daltons, gave two bands of pI 8.0 and 8.5 on isoelectric focussing, and reacted on "Western blots" with antibodies raised against the major GSH S-T subunits of adult liver. Densitometric measurements suggest that the newly detected transferase subunit accounted for as much as 26% of GSH S-T in fetal livers.     

Structural differences in ferritins from normal and malignant rat tissues.

Ferritins purified from several normal and malignant rat tissues were examined for amino acid composition, content of tryptic peptides, available sulfhydryl groups and subunit sizes and proportion. Ferritin extracted from adult kidney, neonatal liver and hepatic and renal tumors differed from the ferritin of adult rat liver in migration on electrophoretic gels and in antibody affinity, but did not differ among themselves. Nevertheless, they showed distinctive differences in amino acid composition and tryptic peptide content. All of them and also adult liver ferritin contained two major species of subunits differing in molecular weight. The proportions of subunits, and the available sulfhydryl groups of the intact ferritin molecules, differed among these tissue ferritins. On the basis of amino acid and peptide content, the ferritins of hepatomas and the renal tumor analyzed showec the greatest similarity but not identity. The ferritin of neonatal liver was next most similar. Kidney ferritin differed considerably in composition from tumor and neonatal ferritins, while adult liver ferritin was the most extremely divergent of the series examined. A similar progressive difference was found on examining the proportions of subunits and sulfhydryl groups in these ferritins. However, changes in subunit proportion cannot explain the amino acid and peptide compositional changes.     

Evidence for a multiple subunit composition of plant NAD malic enzyme

Malate dehydrogenase (decarboxylating) (EC 1.1.1.39) was purified to near homogeneity from both a C3 plant, Solanum tuberosum, and a CAM plant, Crassula argentea. Sodium dodecyl sulfate-gel electrophoresis of both enzymes revealed an alpha,beta subunit composition with corresponding molecular mass assignments of 61,000 and 55,000 daltons. Isoelectric focusing under native conditions showed only one constituent malic enzyme form with an isoelectric point of 5.1. No evidence of additional isoenzymes was found. Urea isoelectric focusing showed the alpha subunit to be more acidic than the beta subunit. Peptide mapping by limited proteolysis with Staphylococcus aureus V-8 protease, trypsin, and endoproteinase Arg-C eliminated the possibility that a precursor-product relationship may have existed between the two subunits and demonstrated that they each possess unique primary sequences. Further support for this conclusion was obtained when significant differences in the contents of glutamic acid, isoleucine, and arginine were revealed by amino acid analysis of the isolated subunits. There was no apparent activity associated with the separated subunits (as resolved by urea-DEAE chromatography), but activity could be found in a reconstituted system, thereby indicating an (alpha,beta)n protomeric configuration. This is the first case where malic enzyme has been conclusively shown to be constructed from nonidentical subunits. This phenomenon has been observed only for the NAD malic enzyme isolated from plants.     

Studies on pituitary follitropin. II. Isolation and characterization of the subunits of the ovine hormone.

The α and β subunits of highly potent ovine follitropin have been isolated by dissociation in 8 m urea, pH 7.5, and chromatography on DEAE-Sephadex A25. The isolated subunits display microheterogeneity on polyacrylamide gel electrophoresis and have very low activity in follitropin-specific radioreceptor and radioimmunoassays. The tryptophan fluorescence spectra of native follitropin and the isolated β subunit are different. The recombinant of follitropin α + β subunit had the same activity as the native hormone in the radioimmunoassay, but its activity in the radioreceptor and in vivo bioassay was about 65% of the intact hormone. Substitution of the follitropin α by ovine lutropin α subunit (prepared by a method not involving urea) to form the recombinant restored full activity in all the three assays investigated. The formation of recombined hormone proceeds at a rapid rate and is almost complete by 6 h. The α and β subunits of ovine follitropin differ from each other in amino acid composition. No significant differences were apparent in their carbohydrate composition. The amino acid composition of the ovine follitropin α and lutropin α subunits are very similar. The oxidized α subunit has phenylalanine at its NH₂-terminus while aspartic acid is present at this position in the oxidized β subunit.     

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.     

Isolation and characterization of liver glycogen synthase from diabetic rats

NAD-specific pig heart isocitrate dehydrogenase is composed of three distinct types of subunits: α, β, and γ, which have molecular weights of about 40,000 but differ in amino acid composition and in isoelectric points. When the native enzyme is subjected to polyacrylamide gel electrophoresis under nondenaturing conditions, two major protein bands with M_r values of about 360,000 (band 1) and 100,000 (band 2) and two minor bands (bands 3 and 4) with M_r values of about 40,000 are consistently present. Enzymatic activity, as detected from NADH fluorescence, is distributed throughout the protein-staining region. Analytical isoelectric focusing in urea reveals that band 1 is composed of all three subunits in roughly the normal ratio of 2α:1β:1γ, and is probably an octamer, band 2 of an equal amount of α and β and is probably dimer, while bands 3 and 4 each consist of only the monomeric α subunit. The highest enzymatic specific activity is associated with a region intermediate between octamer and dimer, which includes the 160,000 tetramer. The protein pattern resulting from isoelectric focusing under nondenaturing conditions consists of protein bands comparable in pattern to those in the presence of urea along with bands of intermediate pI values, many of which are associated with enzymatic activity. Analysis of the subunit composition of these bands supports the activity of the α species in isolation and establishes the activity of the separated β component. No activity of the isolated γ subunit species has thus far been demonstrated. However, the highest apparent specific activity is observed when at least two types of subunits are present. These studies indicate that a range of oligomeric species of the enzyme are enzymatically active and that at least three of the four subunit chains comprising the minimum complete enzyme molecule (2α:1β:1γ) possess an active site.     

Characterization of KB cell alkaline phosphatase. Evidence of similarity to placental alkaline phosphatase.

The alkaline phosphatase from KB cells was purified, characterized, and compared to placental alkaline phosphatase, which it resembles immunologically. Two nonidentical nonomeric subunits of the KB phosphatase were found. The two subunits, which have apparent molecular weights of 64,000 and 72,000, can be separated on polyacrylamide gels containing sodium dodecyl sulfate. The Mr = 64,000 KB subunit appears to be identical in protein structure to the monomer of placental alkaline phosphatase. The Mr = 72,000 KB subunit, while differing in the NH2-terminal amino acid, appears also to be very similar to the placental alkaline phosphatase monomer. Both KB phosphatase subunits bind (32P)phosphate, and bind to Sepharose-bound anti-placental alkaline phosphatase. Native KB phosphatase is identical to the placental isozyme in isoelectric point, pH optimum, and inhibition by amino acids, and has a very similar peptide map. The data presented support the hypothesis that the Mr = 64,000 KB phosphatase subunit may the the same gene product as the monomer of placental alkaline phosphatase. This paper strengthens the evidence that the gene for this fetal protein, normally repressed in all cells but placenta, is derepressed in the KB cell line. In addition, this paper presents the first structural evidence that there are two different subunit proteins comprising the placental-like alkaline phosphatase from a human tumor cell line.     

Localization and Characterization of Tubulin-Like Proteins Associated with Brain Mitochondria: The Presence of a Membrane-Specific Isoform

A mitochondrial fraction, purified from pig brain, was found to contain associated polypeptides with the same electrophoretic migration and isoelectric points as the alpha- and beta-tubulin subunits present in brain microtubules. When analyzed by Western blotting these polypeptides reacted specifically with purified tubulin antibodies. The tubulin-like proteins were then visualized in mitochondrial membranes by protein A-gold complexes after the incubation of purified mitochondria with tubulin antibodies. When membrane and microtubule proteins were compared by isoelectric focussing and two-dimensional gel electrophoresis, differences were observed in the patterns of tubulin isoforms. An additional polypeptide, with the electrophoretic migration of beta-tubulin but the isoelectric point of alpha-tubulin, was found to be enriched in the mitochondrial fraction. This peptide had several Staphylococcus aureus V8 protease peptides in common with alpha-tubulin and may result from a posttranslational modification of that subunit.     

Enantiotopic Selectivity of-Pig Liver Esterase Isoenzymes

Pig liver esterase was separated into isoenzyme fractions with known subunit compositions. The fractions showed differences in enantiotopic ester group selectivity in hydrolysis of two substrates of synthetic value, benzylmethylpropanedioic acid dimethyl ester and cis-N-benzyl-2,5-bismethoxy-carbonylpyrrolidine. A difference in aliphatic chain length specificity between the isoenzyme fractions was also observed. The results indicate that pig liver esterase cannot be regarded as homogeneous when used in organic synthesis.     

Tissue-specific and species-specific distribution of -SH groups in cytochrome c oxidase subunits

Cytochrome c oxidase was isolated from pig, bovine, rat and human tissues including liver, heart, diaphragm and kidney. The native and the sodium-dodecyl-sulfate (SDS)-dissociated enzymes were labelled under optimal conditions with N-ethyl-[2,3-14C]maleimide before and after reduction with dithiothreitol, separated into 13 subunits by SDS gel electrophoresis and the radioactive bands were visualized by fluorography. In some cases the radioactive bands were cut out and counted. All isozymes were labelled in subunits I, III, Va and VIIb, and in subunit II after reduction. Labelling of subunit Vb was equivocal, and in no case were subunits IV and VIc labelled. All other subunits were labelled tissue-specifically and/or species-specifically. No differences were found between labelling of the native and SDS-dissociated enzyme. By relating the molar amount of bound N-ethylmaleimide to the known amount of cysteines in subunits of bovine heart cytochrome c oxidase, the percentage of -SH group reactivity was calculated. Only the cysteine of subunit Va was found to be 100% reactive. The distinct and different reactivity of subunit VIIb as compared to subunits VIIa and VIIc clearly establishes this polypeptide as an independent subunit of mammalian cytochrome c oxidase.     

Subunit heterogeneity of acetylcholinesterase

Several different preparations of purified 11 S acetylcholinesterase have been examined for structural heterogeneity. While no contaminant protein was observed in any of the preparations, minor isozymic forms with catalytic activity were observed in addition to the major component both in polyacrylamide gel electrophoresis and in isoelectric focusing. Major differences in the relative composition of the disulfide-reduced polypeptides among the preparations were found by gel electrophoresis in sodium dodecyl sulfate. Several characteristics of these differences strongly suggest that they derive from a proteolytic fragmentation of a single subunit species. In particular, the apparent fragmentation in the crude enzyme solution is inhibited by benzethonium chloride, an inhibitor of proteolysis which also prevents the conversion of 18, 14, and 8 S acetylcholinesterase species to the 11 S form in fresh electric tissue extracts. No significant differences in the enzyme specific activity are observed among the preparations, an observation which indicates that fully active native enzyme molecules are composed of subunits which are heterogeneous with respect to discrete points of polypeptide cleavage.     

Isolation and characterization of the major androgen-dependent glycoprotein of canine prostatic fluid

Canine prostatic fluid, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions, is characterized by the presence of a single diffuse band (Mr approximately 30,000) which accounts for over 90% of the total protein. The biosynthesis of this protein is under androgen control. Castration results in the disappearance of this protein, whereas its presence in the prostate can be maintained in the castrated animal with exogenous androgen. Analysis of the native protein by isoelectric focusing revealed the presence of 10-13 charged variants with pI values in the range of 6.5 to 8.4. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed that each isoform is constructed of two dissimilar polypeptide subunits covalently linked through disulfide bonds. One subunit has a molecular weight of 15,000 (H chain); the second subunit (L chain) has a variable molecular weight in the 12,000-14,000 range. The H and L subunits have been purified by preparative isoelectric focusing and chemically characterized. Based on tryptic peptide mapping, NH2-terminal analysis, amino acid and carbohydrate composition, the H and L subunits are structurally unrelated and consequently appear to be unique gene products. Furthermore, the L subunit is glycosylated which potentially accounts for its size heterogeneity. Quantitative NH2-terminal analysis indicated that the H and L subunits are present in the native molecule in a ratio of 2:1, suggesting that the native molecule is a trimer with an apparent molecular weight of 43,000. Based on electrophoretic data, the glycoprotein also constitutes the major fraction of the soluble protein in canine prostatic tissue; its presence is organ specific. This glycoprotein should prove useful as a marker for prostatic function under varying hormonal and environmental conditions.     

Subunit location and sequences of the cysteinyl peptides of pig heart NAD-dependent isocitrate dehydrogenase

Pig heart NAD-dependent isocitrate dehydrogenase has a subunit structure consisting of alpha 2 beta gamma, with the alpha subunit exhibiting a molecular weight of 39,000 and the beta and gamma each having molecular weights of 41,000. The amino-terminal sequences (33-35 residues) and the cysteinyl peptide sequences have now been determined by using subunits separated by chromatofocusing or isoelectric focusing and electroblotting. Displacement of the N-terminal sequence of the alpha subunit by 11-12 amino acids relative to that of the larger beta and gamma subunits reveals a 17 amino acid region of great similarity in which 10 residues are identical in all three subunits. The complete enzyme has 6.0 free SH groups per average subunit of 40,000 daltons, but yields 15 distinguishable cysteines in isolated tryptic peptides. Six distinct cysteines in sequenced peptides have been located in the alpha subunit. The beta and gamma subunits contain seven and five cysteines, respectively, with tryptic peptides containing three cysteines being common to the beta and gamma subunits. The three subunits appear to be closely related, but beta and gamma are more similar to each other than either is to the alpha subunit. The NAD-specific isocitrate dehydrogenase from pig heart has been shown to have 2 binding sites/enzyme tetramer for isocitrate, manganous ion, NAD+, and the allosteric activator ADP [Colman, R. F. (1983) Pept. Protein Rev. 1, 41-69]. It is proposed that the catalytically active tetrameric enzyme is organized as a dimer of dimers in which the alpha beta and alpha gamma dimers are nonidentical but functionally similar.     

Brain GABAA receptors studied with subunit-specific antibodies

Brain GABA_A/benzodiazepine receptors are highly heterogeneous. This heterogeneity is largely derived from the existence of many pentameric combinations of at least 16 different subunits that are differentially expressed in various brain regions and cell types. This molecular heterogeneity leads to binding differences for various ligands, such as GABA agonists and antagonists, benzodiazepine agonists, antagonists, and inverse agonists, steroids, barbiturates, ethanol, and Cl⁻ channel blockers. Different subunit composition also leads to heterogeneity in the properties of the Cl⁻ channel (such as conductance and open time); the allosteric interactions among subunits; and signal transduction efficacy between ligand binding and Cl⁻ channel opening. The study of recombinant receptors expressed in heterologous systems has been very useful for understanding the functional roles of the different GABA_A receptor subunits and the relationships between subunit composition, ligand binding, and Cl⁻ channel properties. Nevertheless, little is known about the complete subunit composition of the native GABA_A receptors expressed in various brain regions and cell types. Several laboratories, including ours, are using subunit-specific antibodies for dissecting the heterogeneity and subunit composition of native (not reconstituted) brain GABA_A receptors and for revealing the cellular and subcellular distribution of these subunits in the nervous system. These studies are also aimed at understanding the ligand-binding, transduction mechanisms, and channel properties of the various brain GABA_A receptors in relation to synaptic mechanisms and brain function. These studies could be relevant for the discovery and design of new drugs that are selective for some GABA_A receptors and that have fewer side effects.     

Enantiotopic Selectivity of-Pig Liver Esterase Isoenzymes

Pig liver esterase was separated into isoenzyme fractions with known subunit compositions. The fractions showed differences in enantiotopic ester group selectivity in hydrolysis of two substrates of synthetic value, benzylmethylpropanedioic acid dimethyl ester and cis-N-benzyl-2,5-bismethoxy-carbonylpyrrolidine. A difference in aliphatic chain length specificity between the isoenzyme fractions was also observed. The results indicate that pig liver esterase cannot be regarded as homogeneous when used in organic synthesis.