Highly acidic proteins from human brain: purification and properties of Glu-50 protein

Three extremely acidic proteins were isolated from human brain and purified to apparent homogeneity. One of them, Glu-50 protein, contained much glutamic acid (about 50% of the total amino acids). Its purification involved ammonium sulfate fractionation, DEAE-Sephadex A-50 chromatography, and gel filtration on Sephadex G-100 and G-75. Its molecular weight was determined to be 11,000 by SDS polyacrylamide gel electrophoresis and 34,000-36,000 by gel filtration on Sephadex G-75, suggesting that it consists of three identical polypeptide chains. Its isoelectric point was pH 3.9. Its N-terminal amino acid sequence was NH2-Asp-Glu-Pro-Pro-Asp-Glu and its C-terminal amino acid was Lys. It contained no detectable carbohydrate.     

Isolation, properties, and the complete amino acid sequence of a second form of 60-kDa glycoprotein esterase. Orientation of the 60-kDa proteins in the microsomal membrane

A second form (form 2) of glycosylated esterase was isolated from liver microsomal membranes and characterized. The subunit molecular weight of form 2 is identical to that of the 60-kDa protein previously reported (Ozols, J. (1987) J. Biol. Chem. 262, 15316-15321). The NH2 terminus of the form 2 enzyme is blocked. Digestion of form 2 with pyroglutamyl aminopeptidase, followed by electroblotting and sequence analysis of the blotted protein, indicated that a pyroglutamyl residue was located at the NH2 terminus. Sequence analysis of the deblocked protein as well as characterization of the peptides obtained from enzymatic and chemical cleavages of the intact protein led to the elucidation of its complete amino acid sequence. The protein is a single polypeptide consisting of 532 residues. Carbohydrate is attached at asparaginyl residue 249. The sequence of form 2 esterase is 50% identical to the sequence of form 1 enzyme. The amino acid sequence of the first 26 residues of form 1 enzyme from human liver microsomes shows that 23 residues are identical to that of rabbit form 1, but only 8 residues that are identical to form 2. Treatment of the forms 1 and 2 isozymes with N-glycosidase F or endo-N-acetylglucosaminidase H resulted in a decrease of their subunit molecular weights, indicating that the carbohydrate attached is of the high mannose type. To determine whether the 60-kDa proteins are located on the cytoplasmic or luminal side of the endoplasmic membrane, microsomes were treated with proteolytic enzymes and the two 60-kDa isozymes were isolated and characterized. Sequence analysis of both proteins indicated that their NH2 termini were unaffected by proteolysis. Form 1 isozyme isolated from trypsin-treated microsomes, however, lacked the COOH-terminal heptapeptide (residues 533-539). These results, in addition to the finding of an N-linked carbohydrate, suggest that the two 60-kDa proteins are oriented on the luminal side of the endoplasmic membrane.     

Two-dimensional electrophoretic analysis of secretory-granule,granule-membrane,and plasma-membrane proteins of rat parotid cells

Secretory granules and plasma membranes were isolated from rat parotid cells and characterized enzymatically and by electron microscopy. The proteins of the secretory granule membranes, the secretory granules and the plasma membranes were characterized by two-dimensional polyacrylamide gel electrophoresis and visualized by silver staining. The granule membrane contains 166 polypeptides of which only 26 are also present in the granule contents. The membrane proteins have isoelectric points between 4.75 and 6.45 and apparent molecular weights of 17 000 to 190 000 Daltons. The granule content proteins are surprisingly complex and contain 122 polypeptides with molecular weights of 11 000 to 138 000 and isoelectric points of 4.8 to 6.55. Thirteen of these peptides are present as major species. The plasma membrane contains 172 polypeptide species with molecular weights from 17 000 to 200 000 Daltons and isoelectric points of 5.0 to 6.8. Thirty-five of the plasma membrane proteins are also present in the secretory granule membranes indicating that the two membranes have some enzymatic or structural properties in common. Thus, secretory granule membranes and plasma membranes from parotid cells have a more complex polypeptide composition than has previously been shown for membranes of this type. The systems developed are suitable for the analysis of regulatory events such as protein phosphorylation, proteolytic processing, and other types of post-translational modifications that may be important to the secretory mechanism.     

Studies on thyroid hormone-binding proteins. I. The subunit structure of human thyroxine-binding globulin and its interaction with ligands.

Thyroxine-binding globulin was isolated from human plasma by ammonium sulfate fractionation, chromatographic separations on diethylaminoethyl-Sephadex, gel chromatography, and two different electrophoretic procedures. The highly purified was homogeneous when subjected to polyacrylamide gel electrophoresis, ultracentrifugation analyses, and immunochemical determinations. The weight average molecular weight as determined by sedimentation equilibrium ultracentrifugations was 54,000 and by sedimentation diffusion data 55,000. Amino acid analyses indicated a minimum of 110 amino acid residues per molecule. By determination of the minimum in the curve for the fraction of maximum deviation from the amino acid analyses it was found that the minimum molecular weight for the polypeptide was 12,200. Carbohydrate analyses demonstrated the presence f equimolar amounts of amnnose, galactose, and glucosamine, and the carbohydrate portion constituted 7.5% of the total weight. The amino acid analyses suggested that thyroxine-binding globulin is composed of 4 subunits. Molecular weight determinations by gel chromatography in 6 M guanidine hydrochloride indicated the presence of three species of globulin with apparent molecular weights 52,000, 25,000, and 13,500, respectively. Prolonged storage in guanidine hydrochloride promoted a more than 60% yield of the monomeric species. Moreover, a half-molecule of thyroxine-binding globulin was isolated and shown to consist of two polypeptide chains of similar molecular weight...     

Investigations on the outer membrane proteins of Salmonella typhimurium.

The number, nature and organization of the outer membrane proteins of Salmonella typhimurium have not yet been resolved. Therefore these proteins were isolated using a concentrated solution of guanidine hydrochloride and studied using different analytical techniques. Upon chromatography on Sephadex G-200 four fractions were obtained. Only the fraction containing a protein of molecular weight 13,000 produced immunoprecipitation reactions with the antisera raised against the whole bacteria. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, 7 major proteins were found, with molecular weights between 13,000 and 43,000. Isoelectric focusing on 4.6% polyacrylamide gels resolved the outer membrane proteins into 10 bands with apparent isoelectric points between 5.0 and 8.4. Finally these proteins could be further resolved into as many as 50 spots where a two-dimensional electrophoresis was carried out with isoelectric focusing in the first dimension, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate in the second dimension. These results demonstrated that the outer membrane proteins of S. typhimurium are extremely heterogeneous. To investigate the mode of organization of lipopolysaccharides in the outer membrane, the membrane proteins were separated by the liquid isoelectric focusing technique. Lipopolysaccharides were primarily found to be associated with a protein of isoelectric point 7.8.     

Isolation of human alpha-fetoprotein in two fractionation steps and demonstration of homogeneity.

Human alpha-fetoprotein (hAFP) has been isolated from cord serum in 40% yield using an isolation procedure consisting of only two major steps: affinity chromatography followed by preparative polyacrylamide gel electrophoresis (PAGE). The final product appeared homogeneous on the basis of five independent criteria for purity. Sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) demonstrated a single polypeptide chain with molecular weight of 71,000. The protein exhibited an apparent isoelectric point (pI') of 4.85, molecular radius of 3.0 nm and a valence (net H+/molecule) of 21.9 derived from computation of analytical PAGE data. The two-step isolation procedure made it possible for a single operator to isolate milligram amounts of hAFP in a matter of weeks.     

Characterization of the membrane matrix derived from the microsomal fraction of rat hepatocytes

A highly purified membrane preparation derived from the microsomal fraction of rat hepatocytes has been chemically characterized and fractionated by means of gel filtration. The preparation has been freed of ribosomes and intravesicular protein and has a composition on a w/w basis of 52.1% protein, 45.0% phospholipid, 2.9% carbohydrate and no RNA. $\text{97 ± 2\%}$ of the total membrane phosphorus is accounted for as phospholipid phosphorus.Determination of the molecular weight distribution of the constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave values ranging from 171 000 to 16 000 for the major classes of proteins. Although several membrane glycoproteins have been identified, the most prominent species has an apparent molecular weight of 171 000, 40% of the total microsomal protein is present' in the 49 000–60 000 molecular weight region. Examination of the intrinsic polypeptide composition of membranes obtained from smooth and degranulated rough endoplasmic reticulum revealed no detectable qualitative differences.Sodium dodecyl sulfate-solubilized microsomal membrane proteins were separated by gel filtration into much simplified molecular weight classes, some of which showed predominantly a single electrophoretic component. Amino acid analysis of individual fractions showed a noticeable trend toward a decreasing ratio of acidic to basic residues with decreasing molecular weight.Membrane phosphorus was distributed between two chromatographic fractions: one containing the membrane phospholipid (97% of the total) as well as essentially all the cholesterol, the other, at the inclusion volume of the gel filtration system, containing small molecular weight species (3% of the total phosphorus). The absence of a ribonuclease-resistant RNA component eluting near the void volume clearly distinguishes the microsomal membrane from the nuclear envelope.     

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.     

Isolation of Human α-Fetoprote in Two Fractionation Steps and Demonstration of Homogeneity

Human α-fetoprotein (hAFP) has been isolated from cord serum in 40% yield using an isolation procedure consisting of only two major steps: affinity chromatography followed by preparative polyacrylamide gel electrophoresis (PAGE). The final product appeared homogeneous on the basis of five independent criteria for purity. Sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) demonstrated a single polypeptide chain with molecular weight of 71,000. The protein exhibited an apparent isoelectric point (pÍ) of 4.85, molecular radius of 3.0 nm and a valence (net H⁺/molecule) of 21.9 derived from computation of analytical PAGE data. The two-step isolation procedure made it possible for a single operator to isolate milligram amounts of hAFP in a matter of weeks.     

Low molecular weight plasma proteins isolated from preparations of human immunoglobulin

Low molecular weight proteins co-purified with IgG constitute 0.22% of the total protein purified from human plasma by ion-exchange chromatography on DEAE-cellulose. We have found that these low molecular weight proteins were obtained free of immunoglobulin by ultrafiltration in 5 M guanidinium chloride. Electrophoresis and isoelectric focusing in polyacrylamide gels demonstrated that this fraction of low molecular weight proteins is remarkably heterogeneous. Chromatography of an Mr 6000 to 12 000 fraction on hydroxyapatite resolved fourteen discrete protein peaks. Three of the peaks contained proteins which appeared to be homogeneous on acid-urea polyacrylamide gels. Two of these proteins were similar in composition to B2 globulin and may represent degradation products of some larger protein. The third protein was found to have an amino-terminal sequence identical to C3a. This population of low molecular weight plasma proteins has previously been shown to contain the cystic fibrosis mucociliary inhibitor and is here shown to contain two proteins similar to B2 globulin, C3a and many proteins remaining to be characterized. The presence of these low molecular weight proteins in measurable concentrations may be insufficiently appreciated in studies using 'purified' immunoglobulins as biological or chemical probes.     

Chemical properties and amino acid composition of beta1-bungarotoxin from the venom of Bungarus multicinctus (Formosan banded krait)

beta-Bungarotoxin purified from the venom of Bungarus multicinctus (Formosan banded krait) contained no carbohydrate and behaved as a homogeneous protein on polyacrylamide gel electrophoresis at pH 4.1 and SDS-polyacrylamide gel electrophoresis without 2-mercaptoethanol treatment. Its molecular weight and isoelectric point were estimated to be about 21,000 by gel filtration and about 9.5 by isoelectric focusing, respectively. The toxin treated with the reducing agent was split into two polypeptide chains as revealed by SDS-polyacrylamide gel electrophoresis and their molecular weights were calculated to be about 13,000 and 7,000. The two polypeptide chains (the large one named the A chain and the small one the B chain) were isolated by gel filtration after reduction of disulfide bonds in the toxin followed by alkylation. The A chain contained 120 amino acid residues including 13 half-cystines and the B chain 60 residues including 7 half-cystines. The two chains were supposed to link by disulfide bond(s) in the intact toxin which contained no free sulfhydryl groups. The N-terminal residues of the A and B chains were asparagine and arginine and the C-terminal ones were glutamine and proline, respectively, in accordance with the results of the terminal analyses of the intact toxin.     

Identification of Dictyostelium discoideum plasma membrane proteins by cell surface labeling and quantitative two-dimensional gel electrophoresis

Plasma membrane proteins of the cellular slime mold Dictyostelium discoideum were characterized by two-dimensional polyacrylamide gel electrophoresis using a variety of labeling techniques and a microcomputer-based videodensitometer. Algorithms for the determination of molecular weights and isoelectric points were developed to aid in the comparison of polypeptides from different autoradiographs, Coomassie blue-stained gels, and Western blots. Cell homogenates were compared to plasma membranes isolated by a silica density perturbation technique and to cytoskeletons obtained by nonionic detergent extraction. Plasma membrane proteins were distinguished from subcellular contaminants by lactoperoxidase-catalyzed radioiodination, by selective labeling with N-hydroxysuccinimidyl-2-iminobiotin, and by quantitatively determining the enrichments of individual polypeptides from gels of plasma membrane proteins relative to their counterparts in gels of total cell lysate proteins. In contrast to defining plasma membrane purity by measuring a representative marker enzyme activity, the quantitative two-dimensional gel analysis strategy presented allowed for a rigorous evaluation of the enrichments of all detectable polypeptides in the subcellular fraction. Quantitative two-dimensional gel analysis avoided problems encountered with marker enzyme activation or inhibition during subcellular fractionation as enrichments were based solely on polypeptide amounts. It was also capable of identifying a wider spectrum of plasma membrane proteins than any of the labeling techniques employed in this study. A high resolution two-dimensional gel catalog was generated containing information about plasma membrane protein orientation in the bilayer, association with the cytoskeleton, phosphorylation state, glycosylation state, copy number, isoelectric point, and molecular weight.     

Purification and properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase was purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polyacrylamide gel electrofocusing, about 710,000-fold with a 35% yield from the liver cytosol of thioacetamide-treated rats. The final specific activity was approximately 24,400 nmol/min/mg of protein. The apparent molecular weight of the enzyme determined by gel filtration analyses on Sephacryl S-200 was 55,000 in the presence of 0.25 M NaCl and 145,000 in its absence. The minimum molecular weight of the enzyme was determined to be 54,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was estimated as 5.7 in the presence of 8 M urea. Some catalytic properties of the enzyme were also studied.     

Purification and characterization of B-protein from human serum

B-Protein, present in the serum of individuals with cancer, has been purified to electrophoretic homogeneity. The purification procedure consisted of chromatography on Sephacryl S-200, Affi-Gel Blue, Con A--Sepharose 4B, wheat germ lectin--Sepharose and preparative polyacrylamide gel electrophoresis. The molecular weight of B-Protein is estimated to be 100 000 to 120 000. It is a glycoprotein which appears to be composed of two subunits, each with a molecular weight of approximately 52 000. Analytical polyacrylamide gel electrophoresis and analytical ultracentrifugation data indicate that purified B-Protein is homogeneous. Isoelectric focusing studies also show the purified B-Protein to be homogeneous in composition consisting of a single band of pI = 4.8. Amino acid analysis is consistent with this acidic isoelectric point. Other analyses indicate that B-Protein contains 7% carbohydrate and 7% lipid in the form of triglycerides.     

Purification and characterization of a low molecular weight 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414

A low molecular weight 1,4-beta-glucan glucanohydrolase (endoglucanase) (1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) has been isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by a two-step procedure of gel filtration and ion-exchange chromatography. The isolated enzyme appeared homogeneous upon polyacrylamide gel electrophoresis at pH 2.9, isoelectric focusing in a polyacrylamide gel slab, sedimentation equilibrium analysis and chromatography of the reduced and alkylated enzyme on a column of Sepharose 6B in 6 M guanidine - HCl. A molecular weight was calculated at approx. 20 000 and the isoelectric point was determined at pH 7.52. The purified enzyme was not a carbohydrate-containing protein.     

Calmodulin-activated protein kinase activity of adipocyte microsomes

A calmodulin-activated phosphorylation activity was identified in microsomal (endoplasmic reticulum) preparations from rat adipocytes. Activity was not detected in mitochondrial or plasma membrane fractions. Although the phosphorylation of several proteins was enhanced by addition of calmodulin, the major calmodulin-sensitive protein had a molecular weight of 54,000. A series of experiments were conducted to determine if the microsomal phosphorylation was either calmodulin-containing phosphorylase kinase or calmodulindependent myosin light chain kinase. The phosphorylation of the 54,000 Dalton band in microsomal preparations was 1) not significantly reduced by potential competing protein substrates, e.g. actomyosin or phosphorylase b, 2) nearly equally well phosphorlyated at pH 8.6 or pH 7.0, unlike actomyosin or phosphorylase b, and 3) not increased by addition of phosphorylase kinase or myosin light chain kinase. The results demonstrate that this microsomal calmodulinactivated phosphorylation is catalysed by a protein kinase distinct from phosphorylase kinase or myosin light chain kinase.     

Influence of thyroid status on the electrophoretic pattern of rat liver mitochondrial proteins]

Liver mitochondria were isolated from normal and thyroidectomized rats and their protein components analyzed by polyacrylamide gel electrophoresis. In whole mitochondria 35 protein fractions with MW ranging from 10,000 to 135,000 were characterized. In the absence of thyroid hormone secretion, the amount of a MW 54,000 fraction was always decreased. Injection of small doses of 3,5,3'-triiodo-L-thyronine to the thyroidectomized animal restored the quantity of that protein fraction to normal. Isolated outer mitochondrial membranes showed the presence of 20 protein fractions. These fractions revealed no change after thyroidectomy. The mitoplast, which contained 35 fractions, exhibited a decrease of the MW 54,000 component in thyroidectomized rats. The mitoplast was separated into several fractions. Water soluble matrix proteins presented molecular weights ranging between 40,000 and 55,000. Proteins, which were slightly bound to the inner mitochondrial membrane and could be extracted by KCl, presented molecular weights between 25,000 and 45,000. Structural proteins showed a principal specific component of MW equals 23,000. Electrophoretic patterns obtained with these submitochondrial fractions were similar in normal and thyroidectomized animals. The mitoplast fraction which contained the insoluble cytochromes (a, a3, b, c1) was isolated ; its principal constituent, of MW 54,000 was significantly decreased after thyroidectomy. Thus, the lack of thyroid hormone secretion lowered the level of a protein constituent bound to the inner membrane of liver mitochondria. The synthesis of this constituent could be controlled by mitochondrial nucleic acids.     

Purification and physicochemical and immunological analysis of chicken alpha-fetoprotein

Chicken alpha-fetoprotein was isolated from 12 to 13-day-old embryonic chicken serum by column chromatography on CM-Sephadex C-50. Hydroxyapatite and DEAE-Sephadex A-25. The purified protein was homogeneous based on polyacrylamide gel electrophoresis, immunoelectrophoresis and isoelectric focusing. The purified protein had the following physicochemical and immunological properties. (1) It was a glycoprotein with a single polypeptide chain. (2) The molecular weight of the protein was estimated at 71,000 by SDS-polyacrylamide gel electrophoresis. (3) The isoelectric point of the protein was 4.90. (4) The amino acid composition of the protein was similar to those of mammalian alpha-fetoproteins. (5) The protein showed no steroid-binding capacity. (6) It was immunologically distinct from mammalian alpha-fetoprotein. (7) No immunological cross-reaction was observed between the protein and chicken albumin.     

Effect of Inhibition of Glycosylation on the Appearance of a 60 kD Membrane Glycopolypeptide in Endomembrane Fractions of Soybean Root

Endomembrane (endoplasmic reticulum, Golgi apparatus, plasma membrane) proteins of soybean (Glycine max) root cells are highly glycosylated. We investigated whether N-linked oligosaccharide moieties are essential for the correct intracellular transport of plant endomembrane glycoproteins. Excised roots were incubated with tunicamycin, to block cotranslational glycosylation of proteins, and dual labeled with [³H]glucosamine and [³⁵S] (methionine, cysteine). In the presence of tunicamycin, the incorporation of glucosamine into membrane proteins was inhibited by 60 to 90% while amino acid incorporation was only slightly affected. Autoradiograms of two-dimensionally separated polypeptides from each endomembrane fraction revealed the presence of at least one new polypeptide in tunicamycin-treated tissue. The new polypeptide was of the same isoelectric point but lower molecular weight than a preexisting polypeptide. The new polypeptide was unreactive to concanavalin A, as opposed to the preexisting polypeptide, suggesting the absence of the glycan portion. Trifluoromethanesulfonic acid and N-glycanase were used to cleave the carbohydrate from the preexisting concanavalin A binding polypeptide. In each case a deglycosylated polypeptide of the same isoelectric point and molecular weight as the new polypeptide from tunicamycin-treated tissue resulted. Since the absence of carbohydrate from the new endomembrane polypeptide did not prevent its appearance on autoradiograms of Golgi and plasma membrane, intracellular transport and intercalation of newly synthesized glycoproteins into plant cell membranes may not require the presence of polysaccharide moieties.     

Assembly of the sarcoplasmic reticulum. Cell-free synthesis of te Ca2+ + Mg2+-adenosine triphosphatase and calsequestrin

Polyadenylated RNA prepared from neonatal rat muscle was translated in a rabbit reticulocyte cell-free system. Two sarcoplasmic reticulum proteins, the Ca2+ + Mg2+-dependent adenosine triphosphatase (ATPase) and calsequestrin, were isolated from the translation mixture by immunoprecipitation, followed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The [35S]methionine-labeled translation products were characterized by molecular weight, peptide mapping, and NH2-terminal sequence analysis. The ATPase synthesized in the cell-free system was found to have the same molecular weight (Mr = 100,000) and [35S]-methionine-labeled peptide map as the mature ATPase. The methionine residue present at the NH2 terminus of the mature ATPase was donated by initiator methionyl-tRNArMet and it became acetylated during translation. These results suggest that the ATPase was synthesized without an NH2-terminal signal sequence. Calsequestrin (Mr - 63,000) was synthesized as a higher molecular weight precursor (Mr = 66,000) that contained an additional [35S]methionine-labeled peptide when compared to mature calsequestrin. The NH2-terminal sequence of the precursor was different from the mature protein. The precursor was processed to a polypeptide with a molecular weight identical with mature calsequestrin when microsomal membranes prepared from canine pancreas were included during translation. These results show that calsequestrin is synthesized with an NH2-terminal signal sequence that is removed during translation. These data add to the evidence that the ATPase and calsequestrin follow distinctly different biosynthetic pathways, even though, ultimately, they are both located in the same membrane.