Molecular weights of nitrogenase components from Azotobacter vinelandii.

Meniscus depletion sedimentation equilibrium ultracentrifuge experiments were performed on purified MoFe and Fe proteins of $\text{Azotobacter vinelandii}$. The MoFe protein was found to have a molecular weight of 245,000, using an experimentally confirmed partial specific volume of 0.73. The MoFe protein formed one band on sodium dodecyl sulfate gel electrophoresis and had a subunit molecular weight of 56,000. The subunit molecular weight from ultracentrifuge experiments in 8 M urea was 61,000. The molecular weight of the Fe protein was calculated to be 60,500 in meniscus depletion experiments. Similar experiments in 8 M urea solvent indicated a subunit molecular weight of 30,000. A subunit molecular weight of 33,000 was obtained from sodium dodecyl sulfate gel electrophoresis experiments.     

Variability in the apparent molecular weight of inhibin

An in vitro bioassay based on suppression of GnRH-stimulated FSH secretion by pituitary cells in culture was used to monitor inhibin activity after dialysis, gel filtration or polyacrylamide gel electrophoresis of protein preparations from a variety of gonadal secretions and extracts under native and dissociating conditions. The suggestion that inhibin is a peptide of molecular weight less than 5000 was not confirmed. Although some fractions of low molecular weight suppressed FSH secretion, the amount of activity was low and the dose response curves were not parallel with a standard preparation of inhibin. Under most conditions, inhibin eluted with an apparent molecular weight of about 90 000. However, gel filtration of rete testis fluid protein in 1 M acetic acid resulted in elution of inhibin activity with a lower apparent molecular weight and with polyacrylamide gel electrophoresis in 0.1% (w/v) sodium dodecylsulfate, the apparent molecular weight was 30 000. It is concluded that inhibin is a protein which tends to aggregate and coelute with larger molecules.     

Studies on structural proteins of the rat liver ribosomes. I. Molecular wights of the proteins of large and small subunits.

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'three-dimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.     

Purification and subunit composition of a GTP-binding protein from maize root plasma membranes

When frozen plasma membranes isolated from maize seedling roots are thawed, a significant portion of GTP-binding activity goes into solution. The GTP-binding protein was purified by ion exchange chromatography on Mono-Q and gel filtration on Superose 6. Its molecular weight was estimated at 61 kDa by gel filtration. The same molecular weight was obtained upon solubilization of the GTP-binding protein with cholic acid followed by gel filtration in the presence of this detergent. SDS-PAGE demonstrated that the isolated GTP-binding protein consists of two types of subunit of molecular weights 27 kDa and 34 kDa.     

Evidence for naturally occurring hyaluronic acid binding protein in rat liver

Hyaluronic acid binding protein (HBP) was purified homogeneously from normal adult rat liver by hyaluronate-sepharose affinity chromatography. The molecular weight of this protein as determined by gel filtration was found to be 64,000 daltons. This protein HBP appeared as a single band in non-dissociating gel electrophoresis and has a subunit of molecular weight approximately 12,000 as determined by SDS-gel electrophoresis.     

Purification of a bovine counterpart to human prealbumin and its interaction with a bovine retinol-binding protein

A bovine counterpart to human prealbumin was purified from bovine serum by thiol-disulfide exchange chromatography on thiol-Sepharose 4B and affinity chromatography on human retinol-binding protein linked to Sepharose 4B. The bovine prealbumin had alpha1-mobility on agarose gel electrophoresis at pH 8.6. It has the same molecular weight as human prealbumin on gel filtration and consisted of subunits with a molecular weight of 12 500. This is compatible with a tetrameric structure for the bovine protein. Antiserum against human prealbumin cross-reacted with bovine prealbumin and vice versa. The bovine prealbumin formed at high ionic strength complexes with another bovine serum protein which were dissociated at low ionic strength. This property was used to isolate a protein from bovine serum, by chromatography on bovine prealbumin linked to Sepharose which cross-reacted with antiserum against human retinol-binding protein; had a molecular weight of 21 000 and alpha 2-mobility on agarose gel electrophoresis. It was concluded that the latter protein was a bovine retinol-binding protein.     

火菇素的分离纯化及其分子量的测定*

火菇素是一种从金针菇FlammulinaVelutipes中分离纯化出来的具有抗癌活性的简单蛋白。用SDS-PAGE系统,通过与已知分子量的标准参照蛋白比较,确定火菇素的分子量为24kDa,这一结果与其氨基酸组成分析相比偏大,进一步通过电喷雾离子化质谱法(ESIMS)分析,精确测定火菇素的分子量为19891.13Da。推测由于火菇素蛋白与SDS的非正常结合是造成蛋白质在SDS-PAGE中迁移变慢导致偏差的原因。     

Organization of glycosaminoglycan chains in a chondroitin sulfate-dermatan sulfate proteoglycan from bovine aorta

A chondroitin sulfate-dermatan sulfate proteoglycan was isolated from bovine aorta intima by extraction of the tissue by 4 M guanidine hydrochloride. The proteoglycan was purified by CsCl isopycnic centrifugation followed by gel filtration and ion-exchange chromatography. The proteoglycan had 21.9% protein, 22.1% uronate, 21.4% hexosamine and 10.8% sulfate. Glycosaminoglycan chains obtained from the proteoglycan by beta-elimination were resolved by gel filtration into two fractions, one containing chondroitin 6-sulfate with an approximate molecular weight of 49 000 and the other containing chondroitin 4-sulfate and dermatan sulfate in a proportion of 2:1 with an approximate molecular weight of 37 000. Digestion of the proteoglycan by chondroitinase ABC or AC yielded a protein core with similar composition and behavior in gel filtration and SDS-polyacrylamide gel electrophoresis. An approximate molecular weight of 180 000 was estimated for the core protein. Dermatan sulfate chains with an approximate molecular weight of 10 000 were observed only in the digest of chondroitinase AC. Limited trypsin hydrolysis of the proteoglycan yielded three peptide fragments containing chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in varied proportions. A tentative structure for the proteoglycan was suggested.     

Molecular weight hetergeneity of proteins of the ribosomes and ribosomal subunits from dry pea seeds]

The molecular weight distribution of the total protein of ribosomes and ribosomal subunits isolated from dry pea seeds was studied by electrophoresis in polyacrylamide gel, containing sodium dodecyl sulfate. It was demonstrated that overall protein of 80 S ribosomes is separated into a number of fractions with molecular weights of 10000-64000. Treatment of ribosomes with 0.5 per cent tritone, 0.5 per cent and 1 per cent deoxycholate does not change the general pattern of the molecular weight distribution of ribosomal proteins. The large subunit reveals 19 protein zones (14 major and 5 minor zones), their molecular weights are varying from 10000 to 54000. The majority of proteins of the large subunit have molecular weights of 14000--32000. The molecular weights of 17 protein zones of the small subunit (7 major and 10 minor zones) vary from 10000 to 64000. The majority of proteins of both large and small subunits have molecular weights of 14000--32000. Electrophoretic separation of proteins in the split gel confirmed the fact that the proteins of large subunit differ in molecular weights from those of the small subunit. Thus, ribosomal proteins of pea seeds are shown to produce a typical (for 80S ribosomes) pattern of molecular weight distribution under polyacrylamide gel electrophoresis in the presence of sodium dodecul sulphate.     

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.     

Purification and characterization of an ubiquitin-immuno-reactive protein localized in the cap of young basidiocarp in the basidiomycete Coprinus cinereus.

The ubiquitin-immuno-reactive protein with a molecular weight of 27,800 daltons, which is mainly present in the cap of young basidiocarp, was purified from the basidiomycete Coprinus cinereus. The molecular weight of the native protein was approximately 55,000 as determined by gel filtration chromatography. The isoelectric point of the protein was 4.4. The amino-terminal sequence of the protein was also determined.     

Organization of glycosaminoglycan chains in a chondroitin sulfate - dermatan sulfate proteoglycan from bovine aorta

A chondroitin sulfate - dermatan sulfate proteoglycan was isolated from bovine aorta intima by extraction of the tissue by 4 M guanidine hydrochloride. The proteoglycan was purified by CsCl isopycnic centrifugation followed by gel filtration and ion-exchange chromatography. The proteoglycan had 21.9% protein, 22.1% uronate, 21.4% hexosamine and 10.8% sulfate. Glycosaminoglycan chains obtained from the proteoglycan by β-elimination were resolved by gel filtration into two fractions, one containing chondroitin 6-sulfate with an approximate molecular weight of 49 000 and the other containing chondroitin 4-sulfate and dermatan sulfate in a proportion of 2:1 with an approximate molecular weight of 37 000. Digestion of the proteoglycan by chondroitinase ABC or AC yielded a protein core with similar composition and behavior in gel filtration and SDS-polyacrylamide gel electrophoresis. An approximate molecular weight of 180 000 was estimated for the core protein. Dermatan sulfate chains with an approximate molecular weight of 10 000 were observed only in the digest of chondroitinase AC. Limited trypsin hydrolysis of the proteoglycan yielded three peptide fragments containing chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in varied proportions. A tentative structure for the proteoglycan was suggested.     

Characteristics of human milk glutathione peroxidase

Human milk glutathione peroxidase (GPx) was purified 4500-fold using acetone precipitation and purification by repetitive ion-exchange and gel filtration chromatography with an overall yield of 34%. Homogeneity was established by gel electrophoresis. Using gel filtration, the molecular weight (mol wt) of the enzyme was estimated to be 92 kdalton (kD). The monomeric molecular weight was estimated to b 23 kD from polyacrylamide gel electrophoresis, indicating that the native enzyme consists of four identical subunits. The molecular weight of each subunit was supported by amino acid analysis. Selenium (Se) content of the purified enzyme was 0.31%, in a stoichiometry of 3.7 g-atoms/mol. Data from these studies reveal that GPx provided approximately 22% of total milk Se, but only 0.025% of the total protein.     

Purification and characterization of 33 kilodalton protein of spinach chloroplasts

A protein was prepared from spinach chloroplasts in a highly purified form. The isoelectric point of the protein was 5.2. The apparent molecular weight was estimated to be 33 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea, and 34 000 by gel filtration column chromatography with Sephadex G-100. The protein was provisionally named '33 kilodalton protein' according to the molecular weight. The absorption spectrum of the protein did not show any absorption band in the visible region. No histidine was found in the amino acid analysis of the protein. The 33 kilodalton protein was released from the thylakoid membrane by EDTA-treatment and also by sonic oscillation. The protein was bound to System II particles, but not to System I particles.     

Number and Molecular Weights of Foot-and-Mouth Disease Virus Capsid Proteins and the Effects of Maleylation

Evidence was obtained by gel electrophoresis that foot-and-mouth disease virus (FMDV) type A(12) protein migrates mainly in a zone corresponding to polypeptide(s) approximately 25,000 daltons in molecular weight. Additional minor components were observed, four with molecular weights ranging from 10,000 to 22,500 daltons and one with a molecular weight of 37,500 daltons. The minor components comprised about 10% of the total protein and were present in variable amounts. The 75S empty capsids contained primarily 25,000-, 37,500- and 50,000-dalton zones. These molecular weights were estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate versus proteins of known molecular weight, including poliovirus and vesicular stomatitis virus proteins. Maleylation of the amino residues of FMDV protein solubilized it to about 5 to 10 mg/ml in aqueous, nondenaturing solvents. This permitted molecular weights to be estimated also by gel filtration. Maleylation of 70% of the available amino groups of the FMDV protein produced heat and sodium dodecyl sulfate-stable polymeric aggregates of 10 to 20% of the 25,000-dalton zone. It also resulted in an increase in the molecular weight of this zone by an amount equivalent (ca. 1,000) to that expected from the added maleyl residues.     

Isolation of collagen glucosyltransferase as a homogeneous protein from chick embryos

Collagen glucosyltransferase was isolated as a homogeneous protein from chick embryos by a procedure consisting of ammonium sulphate fractionation, two affinity chromatographies and two gel filtrations. The specific activity of the purified enzyme was 32,000 times that of the 15,000 x g supernatant of the embryo homogenate, and the enzyme was pure when examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis using three different gel compositions. The molecular weight of the enzyme was about 72,000-78,000 by sodium dodecyl sulphate polyacrylamide gel electrophoresis, the value being dependent on the gel composition. The apparent molecular weight by gel filtration was dependent on the purity and protein concentration. The sedimentation coefficient S20,w was 4.7. The data suggest that the enzyme molecule consists of one polypeptide chain.     

Purification of soluble acetylcholinesterase from Japanese quail brain by affinity chromatography.

The purification of a soluble acetylcholinesterase from Japanese quail brain using affinity chromatography on concanavalin A-Sepharose and edrophonium-Sepharose is described. The affinity matrix was synthesized by coupling an inhibitor edrophonium to epoxy-activated Sepharose. Acetylcholinesterase was purified 10,416-fold with a specific activity of 2500 U/mg protein. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol gave only one band with a molecular weight of 62.5 kDa. The molecular weight of the purified acetylcholinesterase was estimated to be 245.5 kDa by gel chromatography on Sephacryl S-200 under nondenaturing conditions. Based on the molecular weight obtained by both SDS-PAGE and gel filtration the purified acetylcholinesterase was assumed to be a tetrameric form.     

Spinach nitrate reductase: purification, molecular weight, and subunit composition

Nitrate reductase was purified about 3,000-fold from spinach leaves by chromatography on butyl Toyopearl 650-M, hydroxyapatite-brushite, and blue Sepharose CL-6B columns. The purified enzyme yielded a single protein band upon polyacrylamide gel electrophoresis under nondenaturing conditions. This band also gave a positive stain for reduced methylviologen-nitrate reductase activity. The specific NADH-nitrate reductase activities of the purified preparations varied from 80 to 130 units per milligram protein. Sucrose density gradient centrifugation and gel filtration experiments gave a sedimentation coefficient of 10.5 S and a Stokes radius of 6.3 nanometers, respectively. From these values, a molecular weight of 270,000 ± 40,000 was estimated for the native reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured enzyme yielded a subunit band having a molecular weight of 114,000 together with a very faint band possessing a somewhat smaller molecular weight. It is concluded that spinach nitrate reductase is composed of two identical subunits possessing a molecular weight of 110,000 to 120,000.     

Lack of interaction between hormonal proteins and synthetic carrier ampholytes (Ampholine)

Using ¹⁴C-labeled Ampholine, radioiodinated and unlabeled human growth hormone, and ovine prolactin, no significant degree of interaction could be detected by polyacrylamide gel electrophoresis and gel chromatography. Protein binding to excess Ampholine was negligible. Ampholine binding to excess protein was below the detection limit of 0.2 moles of Ampholine per mole of protein. Commercial Ampholine contains large molecular weight components that cannot be separated by gel chromatography from proteins in the 20–30,000-molecular weight range.     

The 'hollow cylinder' protein of erythrocyte membranes.

The 'hollow cylinder' protein (Harris, J.R. (1968) Biochim. Biophys. Acta 150, 534-537) has been purified from human erythrocyte membranes. The molecular weight of the native protein, as determined by analytical ultracentrifugation, was found to be 747,000. By means of sodium dodecyl sulphate gel electrophoresis, the purified protein was shown to be composed of three different low molecular weight polypeptides of average molecular weight 25,000. This study provides convincing evidence that the spectrin tetramer is not responsible for the characteristic electron microscopic appearance of the hollow cylinder protein.