Characterization of the sex steroid binding protein of human pregnancy serum. Improvements in the purification procedure

The sex steroid binding protein (SBP) of human pregnancy serum was purified to homogeneity by the sequential use of ammonium sulfate precipitation, affinity chromatography on 5alpha-dihydrotestosterone-17beta-succinyldiaminoethyl-(1,4-butanediol diglycidyl ether)-agarose, and preparative polyacrylamide gel electrophoresis. The yield of pure SBP was improved from 5% as originally reported [Mickelson, K. E., and Petra, P. H. (1975), Biochemistry 14, 957] to 34%. Homogeneity of SBP was shown by equilibrium sedimentation ultracentrifugation in 6 M guanidine hydrochloride containing 0.1 M mercaptoethanol which yields a minimum molecular weight of 36 335 +/- 525. The protein is also homogeneous when examined by gel electrophoresis in the presence of sodium dodecyl sulfate. A value of 52 000 for the molecular weight is obtained by this method. SBP partially purified from Cohn fraction IV has also a molecular weight of 52 000 by gel electrophoresis in the presence of sodium dodecyl sulfate; that fraction is contaminated with another protein of molecular weight 90 000 which must be removed to obtain homogeneous SBP. The amino acid composition of SBP isolated from pregnancy serum is presented.     

M-protein in chicken cardiac muscle.

Chicken cardiac muscle myofibrils lack a visible M-line. Antibodies against chicken breast muscle M-protein, an M-line component with M_r = 165 000, were used to demonstrate the presence of a similar protein in chicken heart muscle. The immunoreplica technique showed the heart protein to have about the same molecular weight as the breast muscle M-protein on polyacrylamide slab gels in the presence of sodium dodecyl sulfate (SDS). Positive staining within the H-zone was observed when the indirect immunofluorescence technique was used to localize the M-protein in isolated heart myofibrils. This result was confirmed by electron microscopic investigations on longitudinal sections of antibody-incubated heart muscle fiber bundles showing the antibody against M-protein to be bound within a region corresponding to the M-line region of breast muscle myofibrils.     

Subunits of the extracellular hemoglobin of Tubifex tubifex.

The molecular weight of the extracellular hemoglobin of Tubifex tubifex determined by equilibrium sedimentation is 3.0 +/- 0.2 . 10(6). Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that the hemoglobin dissociated into four subunits: 13 000 (subunit 1), 21 000 (subunit 2), 23 000 (subunit 3) and 47 000 (subunit 4); in the presence of mercaptoethanol two subunits were observed, 13 000 +/- 1000 (subunit I) accounting for 70--80% of the whole molecule, and 26 000 (subunit II). Electrophoresis of the subunits obtained in the absence of mercaptoethanol showed that subunit I originated from subunits 1 and 4, while subunit II originated from subunits 2 and 3. These relationships were supported by N-terminal group determinations. Gel filtration in 6 M guanidine hydrochloride showed that the molecular weight of subunit I is 17 500 and that of subunit II, 36 000. Tubifex hemoglobin appears to consist of at least seven polypeptide chains.     

Mammalian high molecular weight and monomeric forms of valyl-tRNA synthetase

Valyl-tRNA synthetase from rat liver sediments at 15.5 S with a Stokes radius of 90 A, corresponding to a native molecular weight of 585,000. Purification of valyl-tRNA synthetase to homogeneity by a combination of conventional and affinity column chromatography yields a fully active monomeric form of valyl-tRNA synthetase with a sedimentation coefficient of 7.7 S and a Stokes radius of 45 A. The subunit molecular weight of the monomeric valyl-tRNA synthetase is 140,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In the presence of 400 mM KCl, the purified monomeric valyl-tRNA synthetase associates to a high molecular weight form. The high molecular weight valyl-tRNA synthetase in the homogenate can be readily converted to the monomeric form by controlled trypsinization. The kinetic parameters of the two forms are nearly identical. The results suggest that the high molecular weight valyl-tRNA synthetase is a homotypic tetramer and converts to the monomeric valyl-tRNA synthetase after the cleavage of a small peptide.     

Photoreaction center of photosynthetic bacteria. 2. Size and quaternary structure of the photoreaction centers from Rhodospirillum rubrum strain G9 and from Rhodopseudomonas sphaeroides strain 2.4.1.

The photoreaction center from Rhodospirillum rubrum strain G9 binds about 6 times as much sodium dodecyl sulfate as certain proteins commonly used as molecular weight markers for sodium dodecyl sulfate--polyacrylamide gel electrophoresis. This presumably explains the apparent discrepancy between the molecular weight of the photoreaction center determined by electrophoresis (76 000) and its minimal molecular weight (87 000). The molecular weight of the photoreaction center solubilized with Triton X-100 was determined by three different methods: conventional sedimentation equilibrium, a combination of sedimentation velocity and gel filtration measurements, and sedimentation equilibrium in H2O and in D2O. Each technique required a determination of the amount of bound detergent. All three methods gave molecular weight values close to 60 000. A similar molecular weight was found for the photoactive beta gamma dimer obtained from the photoreaction center of Rhodopseudomonas sphaeroides strain 2.4.1 which, as a whole, had a molecular weight of 87 000. These results indicate that the photoreaction center from Rp. sphaeroides is an oligomer of the type alpha 1 beta 1 gamma 1. In contrast, the photoreaction center from Rs. rubrum appears to be dissociated, in solution, into a photoactive beta gamma dimer and a free polypeptide alpha.     

Pallidin. Purification and characterization of a carbohydrate-binding protein from Polysphondylium pallidum implicated in intercellular adhesion.

A carbohydrate-binding protein from Polysphondylium pallidum, a species of cellular slime mold, was purified to homogeneity by adsorption to formalinized erythrocytes and elution with D-galactose. The protein, for which we propose the name PALLIDIN, is assayed by its activity as an agglutinin of erythrocytes. It was previously shown to have different carbohydrate-binding specificities than discoidin, a carbohydrate-binding protein from Dictyostelium discoideum, another species of slime mold. Evidence has been presented previously that each of these proteins is detectable on the cell surface. In the present report we show that the physico-chemical properties of pallidin are different from discoidin. Pallidin has a subunit molecular weight of 24 800 +/- 1100 determined by polyacrylamide electrophoresis in the presence of dodecyl sulfate and 2-mercaptoethanol, compared to 26 100 +/- 1000 for discoidin. The weight-average molecular weight of pallidin is 250 000 +/- 50 000 determined by equilibrium sedimentation in the presence of D-galactose compared to 100 000 +/- 2000 for discoidin. In equilibrium sedimentation studies, pallidin exhibited some heterogeneity at equilibrium while discoidin was homogeneous. The amino acid composition of pallidin is generally similar but clearly different from the composition of discoidin. The isoelectric point of pallidin is 7.0 compared to 6.1 for discoidin. Like discoidin, pallidin contains no detectable hexosamine or neutral sugar. These results establish that agglutinins from two species of cellular slime molds are distinct. The different properties of the cell-surface agglutinins, pallidin and discoidin, are consistent with their suggested role in species-specific cellular recognition and adhesion in the species of slime mold from which they are derived.     

Purification and characterization of Novikoff ascites tumor protein kinase.

A protein kinase, designed KII, has been purified 5000-fold from Novikoff ascites tumor cells. The purification procedure also allows for the purification of a second major protein kinase, designated KI, as well as RNA polymerase I and II. Purified KII has a sedimentation constant of 7.6 S and a Stokes radius of 39 A, suggesting a molecular weight of about 122000. Polyacrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate suggests the enzyme is composed of subunits of molecular weights 44 000, 40 000, and 26 000 present in a molar ratio of 1:1:2. Incubation of the enzyme alone in the presence of [gamma-32P]ATP results in the phosphorylation of the 26 000-dalton subunit. Protein kinase II actively phosphorylates phosvitin, casein, and nonhistone chromosomal proteins but does not phosphorylate basic proteins such as histones or protamine to an appreciable extent. Km values of 3.6 micron for ATP and 6.5 micronM for GTP were determined in the presence of 4mM Mg2+. The enzyme is neither stimulated by cyclic adenosine 3',5'-monophosphate or cyclic guanosine 3', 5'-monophosphate nor inhibited by the regulatory subunit of rabbit muscle protein kinase. Its activity is stimulated by KCl at concentrations below 0.2 M and inhibited by higher concentrations.     

Isolation and characterization of mitochondrial alanine aminotransferase from porcine tissue.

Mitochondrial alanine aminotransferase L-alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2) has been isolated in homogeneous form from both porcine liver and kidney cortex, but in low yield. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate or 8 M urea gave a single band. An isoelectric point of 8.5 +/- 0.5 and a molecular weight of 75--80 000 were obtained. The enzyme is specific for L-alanine and is inhibited by D-alanine, aminooxyacetate and cyclosterine. The Km for pyruvate and glutamate is 0.4 mM and 32 mM, respectively. These values are similar to those determined for the cytoplasmic enzyme; however, at high concentrations, both compounds strongly inhibit the mitochondrial enzyme, an inhibition not observed with cytosolic alanine aminotransferase. These characteristics and the fact that the mitochondrial alanine aminotransferase was inactivated by procedures effective in the preparation of the cytosolic enzyme, clearly differentiate the two proteins and further support different roles for the two alanine aminotransferases in vivo.     

Erythrocruorin from the water-flea Daphnia magna. Quaternary structure and arrangement of subunits.

The subunit structure of erythrocruorin from the cladoceran Daphnia magna was studied. The native protein was found to have a sedimentation coefficient (S2(20), w) of 17.9 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 494 000 +/- 33 000. Iron and haem determinations gave 0.312 +/- 0.011% and 3.84 +/- 0.04%, corresponding to minimal molecular weights of 17900 +/- 600 and 16 100 +/- 200 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a molecular weight of 31 000 +/- 1 500. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol is 31 100 +/- 1300. On a molecular-weight basis, Daphnia erythrocruorin is composed of 16 identical polypeptide chains carrying two haem groups each. The native structure is stable between pH5 and 8.5. At alkaline and acidic pH, a gradual decrease in the sedimentation coefficient down to 9.8S occurs. Above pH 10 and below pH4, a slow component with S20, w between 2.7S and 4.0S is observed. The 2.7S, 4.0S and 9.8S species are identified as single-chain subunits, subunit dimers and half-molecules respectively. We propose a model for the molecule composed of 16 2.7S subunits grouped in two layers stacked in an eclipsed orientation, the eight subunits of each layer occupying the vertices of a regular eight-sided polygon. Support for this arrangement is provided from electron microscopy and from analysis of the pH-dissociation pattern.     

Nature of the phosphorylated 26 000 molecular weight protein extracted from dog thyroid with contractile proteins.

Dog thyroid contractile proteins are characterized by their ATPase activity at high KCl concentration. In the presence of Ca(2+), 80 nmol ATP are hydrolyzed per min per mg protein. This Ca(2+) -ATPase activity is inhibited by Mg(2+) but not influenced by sodium azide. The 26 000 molecular weight protein which is present in thyroid contractile protein preparations and the phosphorylation of which is stimulated by thyroid stimulating hormone (TSH) is suggested to be identical to the lysine-rich histones (H1). Indeed, radioactive thyroid H1 histones added to unlabelled thyroid slices copurify with the contractile proteins and migrate at the same level as the 26 000 molecular weight when submitted to electrophoresis in polyacrylamide sodium dodecyl sulfate gels of different acrylamide concentrations.     

Purification and properties of debranching enzyme from dogfish muscle.

Glycogen debranching enzyme (4-alpha-glucanotransferase amylo-1,6-glucosidase, EC 2.4.1.25 + 3.2.1.33) was purified 140-fold from dogfish muscle in a rapid, high-yield procedure that takes advantage of a strong binding of the enzyme to glycogen, and its quantitative adsorption to concanavalin A-Sepharose only when the polysaccharide is present. The final product was hrophoresis in the presence and absence of dodecyl sulfate. A molecular weight of 162,000 +/- 5000 was determined by sedimentation equilibrium analysis in good agreement with the value of 160,000 estimated by gel electrophoresis, but a low-sedimentation constant of 6.5 S suggests that the enzyme is asymmetric. The molecule appears to be made up of a single polypeptide chain with no evidence for multiple repeating sequences: it could not be dissociated into smaller fragments by dodecyl sulfate even after complete carboxymethylation; tryptic cleavage of the native protein yielded only two fragments of molecular weight 20,000 and 140,000 without loss of enzymatic activity. The amino acid composition of the enzyme is reported; no covalently bound phosphate or carbohydrate could be detected. All 32 sulfhydryl groups present were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions; eight reacted readily in the native enzyme without loss of catalytic activity, while substitution of eight additional ones lowered the activity by 50%. Inactivation was greatly reduced by glycogen; the polysaccharide also influenced markedly the electrophoretic behavior of the enzyme and large filamentous aggregates were formed when solutions of both were mixed. Purified debranching enzyme releases 3 mumol of glucose min-1 mg-1 at 19 degrees C, pH 6.0, from a glycogen limit dextrin and one-tenth this amount when the native polysaccharide is used as substrate; glycogen is quantitatively degraded in the presence of phosphorylase. None of the usual sugar phosphates or nucleotide effectors of glycolysis affected enzymatic activity. No phosphorylation by either dogfish or rabbit skeletal muscle protein kinase or phosphorylase kinase could be demonstrated, nor any direct interaction with phosphorylase as measured by SH-group reactivity, enzymatic activity, or rate of phosphorylase b to a conversion. Purification of the 160,000 molecular weight M-line protein of skeletal muscle resulted in the quantitative removal of debranching enzyme, indicating that the two proteins are different.     

Pseudocatalase from Lactobacillus plantarum: evidence for a homopentameric structure containing two atoms of manganese per subunit

An improved procedure for the isolation of the pseudocatalase of Lactobacillus plantarum has been devised, and the quaternary structure and manganese content of this enzyme have been reexamined. Sedimentation equilibrium of the native enzyme at several salt concentrations gave a molecular weight of 172 000. The subunit weight, obtained by sedimentation equilibrium in 6.4 M guanidinium chloride, with or without prior reduction and carboxymethylation, was 34 kilodaltons. The amino acid composition indicated 150 Arg + Lys, and after exhaustive tryptic digestion, 32 peptides were resolved. These data suggest that the pseudocatalase is a homopentamer. Cross-linking with dimethyl suberimidate, followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, yielded five major bands, another indication of pentameric structure. The manganese content was found to be 1.8-2.4 per subunit. S20,W was found to be 9.6 S and f/f0 = 1.2, suggesting a globular structure of Stokes radius 44 A.     

Isolation of an actin polymerization stimulator from bovine thyroid plasma membranes

An actin polymerization stimulator was purified from bovine thyroid plasma membranes by DNase I affinity column chromatography. Although the molecular weight of the protein was about 42,000 (42K) by sodium dodecyl sulfate polyacrylamide gel electrophoresis, it did not comigrate with actin. In the presence of 30 mM KCl, the 42K protein facilitated formation of actin filaments when analyzed by a centrifugation method, accelerated the initial phase of actin polymerization as measured in an Ostwald viscometer and increased the length of filaments as shown by electron microscopy. The 42K protein also accelerated the initial phase of actin polymerization in the presence of 100 mM KCl and 2 mM MgCl₂ but did not affect the final viscosity. The effect of the 42K protein was diminished by 5 uM cytochalasin B or 1 uM cytochalasin D. This 42K protein may anchor actin filaments onto the thyroid plasma membrane.     

Purification and some properties of the histidyl-tRNA synthetase from the cytosol of rabbit reticulocytes.

The histidyl-tRNA synthetase of rabbit reticulocyte cytosol has been purified 84 000-fold to apparent homogeneity with a specific activity of 687 nmol of histidyl-tRNA formed per min per mg of protein. Ten to 15% of the enzyme activity is sedimented with the ribosomes while the remainder is in the cytosol. The purified enzyme has a molecular weight of 122 000 as determined by sucrose density gradient centrifugation. Gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate suggests that it is composed of two similar subunits with a molecular weight of approximately 64 000. The enzyme has a magnesium optimum of 45 mM; however, this is reduced to 5 mM in the presence of an intracellular potassium concentration (160 nM). The enzyme acylates the two histidine tRNA isoacceptors of rabbit reticulocytes with similar Km values and at similar rates.     

Physical properties and subunits of DNA dubia erythrocruorin.

The erythrocruorin of the freshwater leech Dina dubia possessed an S20,w of 61 S and exhibited a slightly sigmoid oxygenation curve with n congruent to 1.6 and P50 = 2.4 mm at pH 7.4. A minimum mol. wt of 23 000 +/- 2100 per heme group was determined from the iron and heme contents, 0.22 +/- 0.02 and 2.92 +/- 0.35 weight %. The subunit composition of this erythrocruorin was investigated using polyacrylamide gel electrophoresis and gel filtration in sodium dodecyl sulfate at neutral pH and gel filtration at pH 9. Sodium dodecyl sulfate electrophoresis of Dina erythrocruorin revealed the presence of five subunits (1-5) with mol. wts of about 13 000, 21 000, 23 000, 25 000 and 31 000, respectively. When the erythrocruorin was reduced with mercaptoethanol prior to dodecyl sulfate electrophoresis, three subunits (I-III) were observed, two possessing molecular weights in the range 12 000-14 000 (I and II) and one possessing a molecular weight of about 28 000. One of the subunits I, II, was provided by the dissociation of the 31 000 subunit. Subunit III (28 000) consisted of subunits 2, 3, and 4. It is likely that not all of the polypeptide chains constituting Dina erythrocruorin are associated each with a heme group.     

Purification and properties of an NADP-specific 6-phosphogluconate dehydrogenase from Streptococcus faecalis.

A procedure is described for the purification of 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP oxidoreductase (decarboxylating) EC 1.1.1.44) from cell extracts of Streptococcus gaecalis. A 180-fold purification was achieved with an over-all yield of about 12% and an average specific activity of 14. The enzyme was homogeneous as determined by polyacrylamide gel electrophoresis, immunoelectrophoresis, and sedimentation equilibrium, studies. Its weight average molecular weight, as measured by sedimentation equilibrium, was 108,000 +/- 3,600. Other methods employed for molecular weight determinations gave values that ranged between 106,000 and 115,000. An analysis of the enzyme by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed it to be a dimer composed of subunits having equal molecular weight. The amino acid composition of the streptococcal enzyme is reported. The apparent Km values for NADP and 6-phosphogluconate were calculated from kinetic data and found to be 0.015 mM and 0.024 mM, respectively. Kinetic studies also indicated that the binding of one substrate did not affect the apparent affinity of the enzyme for the other substrate.     

Molecular weights of apoprotein B obtained from human low-density lipoprotein (apoprotein B-PI) and from rat very low density lipoprotein (apoprotein B-PIII)

Human low-density lipoproteins (LDL) were isolated from single donors by differential centrifugation between densities of 1.020 and 1.050 g/mL. The LDL were reduced and alkylated in 7 M guanidine hydrochloride, and the lipid was removed by multiple extractions in the cold with a mixture of diethyl ether and ethanol. Sedimentation studies on the resultant human apoprotein B (apoprotein B-PI) at low concentrations in 6.00 M guanidine hydrochloride showed a single sharp boundary with a sedimentation coefficient of 2.15 +/- 0.04 S at 25 degrees C, uncorrected for viscosity or density. Diffusion experiments performed in the same solvent at low speeds in the analytical ultracentrifuge gave a D25 = 0.694 +/- 0.043 Fick. Combining these values with an apparent specific volume of 0.703 mL/g yielded a molecular weight of 387 000, indistinguishable from that obtained by sedimentation equilibrium analysis in 7 M guanidine hydrochloride. Similar values were also obtained by calibrated sedimentation analysis, by Sepharose 2B chromatography in guanidine hydrochloride, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rat very low density lipoproteins (VLDL), isolated from sera of Triton WR1339 treated animals, were used as the source of rat apoprotein B-PIII. The delipidated VLDL were solubilized in sodium dodecyl sulfate, and apoprotein B-PIII was isolated by Sepharose 4B chromatography. With appropriate corrections for density and viscosity, the behavior of rat apoprotein B-PIII was identical, upon analytical ultracentrifugation, in 6 and 7.7 M guanidine hydrochloride, corresponding to sedimentation and diffusion coefficients of 1.47 S and 0.92 Fick, respectively, in 6 M guanidine hydrochloride. These data may be combined to yield a molecular weight of 210 000. Similar values were obtained by calibrated sedimentation analysis, by Sepharose 2B chromatography in guanidine hydrochloride, and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate.     

Isolation and characterization of bovine factor VII.

Factor VII (proconvertin) has been purified approximately 5 x 10(5)-fold from bovine plasma with an overall yield of 30%. The isolation procedure involves barium sulfate adsorption and elution, DEAE-Sephadex batchwise adsorption and elution, benzamidine-agarose column chromatography, heparin-agarose column chromatography, and preparative polyacrylamide gel disc electrophoresis. The final product was homogeneous when examined by gel electrophoresis in the presence of sodium dodecyl sulfate. A minimal molecular weight of 45,500 was determined by sedimentation equilibrium. The molecular weight estimated by sodium dodecyl sulfate gel electrophoresis was 54,000. Factor VII is composed of a single polypeptide chain possessing an amino-terminal sequence of Ala-Asn-Gly-Phe-Leu-. The amino acid and carbohydrate compositions of factor VII are also reported.     

Beta-Adrenergic receptors in brown-adipose tissue. Characterization and alterations during acclimation of rats to cold.

Aldehyde dehydrogenase from bovine liver has been purified to homogeneity. Amino acid composition showed a high content of cysteine of 32 mol/mol enzyme. The enzyme is composed of four identical subunits as judged by sodium dodecyl sulfate gel electrophoresis and end-group analysis. The molecular weight was determined to be 220 000 +/- 10 000 by sedimentation equilibrium analysis in an analytical ultracentrifuge. The Michaelis constants for NAD+, glyceraldehyde and acetaldehyde were found to be 47 micron, 170 micron and 130 micron, respectively.     

Purification to homogeneity and some properties of L-phenylalanine ammonia-lyase of irradiated mustard (Sinapis alba L.) cotyledons.

1. Lyase (L-Phenylalanine ammonia-lyase, EC 4.3.1.5) from far-red light-irradiated mustard cotyledons was purified to a single protein using ammonium sulphate fractionation, column chromatography on L-phenylalanyl-Sepharose 4B and on Sephadex G-200, isoelectric focusing and polyacryalmide gel electrophoresis. 2. The enzyme constituted 0.01% of total cellular protein, did not catalyse the deamination of L-tyrosine, had a pH optimum of pH 8.6 and an isoelectric point of pH 5.6. 3. The sedimentation coefficient was estimated as 11.3 S, the Stokes' radius 4.25 nm, and the molecular weight 240 000 +/- 9000 (S.E.). 4. Electrophoresis on denaturing polyacrylamide gels gave a single stained protein band corresponding to a subunit molecular weight of 55 000 indicating a tetrameric structure of equal (or near-equal) size subunits. 5. Maximum velocity (V) for the purified lyase at 25 degrees C was 3.83--4.10 nkat. 1(-1) enzyme and Km value 0.151--0.154 mM. Negative cooperativity (Hill coefficient, n = 1.08) was not detected over the substrate concentration range tested. 6. A putative non-diffusible inhibitor isolated from dark-grown gherkin hypocotyls inhibited the homogeneously purified mustard lyase.