A subfraction of fragment D isolated from a plasmin hydrolysate of human fibrinogen.

Fragment D from a 4-hour plasminolysate of human fibrinogen was chromatographed on DEAE-cellulose and a nearly homogeneous subfraction obtained. It migrated as a single band in dodecylsulfate gel electrophoresis. Reduction yielded three peptide chains with approximate molecular weights of 45000, 295000 and 13000 as estimated from the electrophoretic migration rate in dodecylsulfate acrylamide gels. From these data the molecular weight of the Fragment D subfraction was calculated to be ca. 87500. The S-carboxymethylated peptide chains were separated by chromatography on DEAE-cellulose. They were correlated electrophoretically and their amino acid composition was determined. The peptide chains of molecular weight 45000 and 29500 showed a chromatographic microheterogeneity. The subfractions of these two chains, however, were not distinguished by their electrophoretic mobility in dodecylsulfate acry lamide gels and showed only insignificant differences in their amino acid composition.     

Isolation and partial characterization of a tubulin-like protein from human and swine synaptosomal membranes.

Synaptic membranes from human and swine brains were solubilized with 8 M urea and the proteins were reduced and alkylated. A protein was isolated from both sources and had identical amino acid compositions and molecular weights as determined by electrophoresis on polyacrylamide-sodium dodecylsulfate gels and by ion-exchange chromatography and gel filtration on Bioglas 1000. The apparent molecular weight of the protein was 53 000 on the acrylamide-sodium dodecylsulfate gels. Neither neutral sugars nor sialic acid was a significant component of the protein. When the proteins were digested with trypsin and the resultant peptides subjected to chromatography (n-butanol/acetic acid/water) and electrophoresis (pH 3.7) the peptide maps were identical. The protein comprises 1-2 percent of the total synaptosomal protein. With regard to amino acid composition, molecular weight, peptide map characteristics, behavior on DEAE-cellulose columns, electrophoretic mobility and sugar content, the synaptic protein is quite similar to the monomer of swine tubulin.     

Preparation of polypeptide subunits of cytochrome oxidase from Neurospora crassa.

Cytochrome oxidase was purified from Neurospora crassa by ammonium sulfate fractionation in the presence of bile salts. The enzyme preparations contained 10-13 nmol of heme a per mg of protein; no other hemoproteins could be detected. Dodecylsulfate gel electrophoresis resolved the enzyme complex into seven major bands, representing seven polypeptide subunits. A procedure is described that allows the isolation of these enzyme subunits on a large scale starting from a single batch of oxidase preparation. It involves dissociation of the enzyme complex by dodecylsulfate and subsequent separation of the obtained polypeptides by chromatography in the presence of various dodecylsulfate concentrations. Purification of subunits 3, 4, 5, 6 and 7 was achieved by column chromatography using molecular sieves (Sephadex G-100, Bio Gel P-60) and hydroxylapatite. For the purification of subunits 1 and 2 an electrophoretic separation on a preparative polyacrylamide gel was required. The advantages and disadvantages of the separation procedure of the enzyme polypeptides are discussed. As a special point of interest, the conservation of antigenic determinants of the polypeptide chains during the dodecylsulfate treatment is considered.     

Sarcoplasmic reticulum. X. The protein composition of sarcoplasmic reticulum membranes

The proteins of Sarcoplasmic reticulum membranes were resolved by polyacrylamide gel electrophoresis into several fractions ranging in mol wt from 300,000 to about 30,000. The ATPase enzyme involved in Ca²⁺ transport is associated with a major protein fraction and its molecular weight based on its electrophoretic mobility on polyacrylamide gels in the presence of sodium dodecylsulfate is about 106,000. Reducing agents (β-mercaptoethanol or dithiothreitol) cause the dissociation of membrane proteins into subunits of 20,000–60,000 mol wt, which can be separated by electrophoresis or Sephadex G-150 chromatography.     

Multiple methylation of methyl-accepting chemotaxis proteins during adaptation of E. coli to chemical stimuli

In bacterial chemotaxis, adaptation is correlated with methylation or demethylation of methyl-accepting chemotaxis proteins (MCPs). Each protein migrates as a characteristic set of multiple bands in sodium dodecylsulfate polyacrylamide gel electrophoresis. The changes in MCP methylation that accompany adaptation are not the same for all bands of a set. Adaptation to a type II repellent stimulus results in an overall decrease in MCP II methylation, but also in an increase in the amount of radioactive methyl groups in the upper band of the set. We demonstrate that this increase is not due to new methylation, but rather to reduced electrophoretic mobility of previously methylated molecules that have lost some but not all of their methyl groups. We suggest that the pattern of multiple bands is a direct reflection of multiple sites for methylation on MCP molecules, and that the distribution of radiolabel among the bands is determined by the total extent of methylation. The patterns of methylated peptides produced by limited proteolysis of different MCP bands imply that methylation of the multiple sites on a molecule may occur in a specific order.     

Increased activity of testosterone hydroxylases in liver microsomes of diabetic rats treated with insulin

Liver microsomes from alloxan diabetic rats displayed decreased activity to hydroxylate testosterone only at the 2-alpha and 6-beta positions. Diabetic insulin-treated rats showed higher hydroxylase activities than diabetic and control rats in the formation of all testosterone metabolites analyzed. The sodium dodecylsulfate electrophoretic profile of liver microsomal proteins from each group of rats exhibited distinct increases as well as decreases in the cytochrome P-450 region. Stimulation of testosterone metabolism by insulin may be associated with a higher synthesis of certain cytochrome P-450 isozymes.     

Characterization of the terminal degradation products of canine fibrinogen by plasmin.

Fibrinogen, isolated from canine plasma by the successive procedures of (1) freezing and thawing, (2) fractional precipitation with 25% saturated (HN4)2SO4 and (3) Sepharose 6B gel-filtration, had a molecular weight of 282 000 by the rapid sedimentation equilibrium method. However, a molecular weight for canine fibrinogen of 332 000, which is closer to that reported for human and bovine fibrinogens (340 000 plus or minus 20 000), was obtained from the sum of the molecular weights of the Aalpha, Bbeta and gamma chains, determined from dodecylsulfate gel electrophoretic patterns of reduced fibrinogen. Canine fibrinogen, subjected to proteolysis by urokinase-activated plasminogen for 24 h, contained degradation fragments D and E which were isolated by starch block electrophoresis and Sephadex G-200 gel-filtration. The purified D and E fragments with sedimentation coefficients of 5.0 S and 2.5 S had weight average molecular weights of 89 000 and 42 000, respectively by the rapid sedimentation equilibrium method. The ratio of D to E was 2:1 per parent fibrinogen molecule. Antigenic analysis according to anti-fibrinogen antiserum showed that both D and E fragments were antigenically deficient to native fibrinogen and revealed a reaction of non-identity with each other. Upon immunoelectrophoresis at pH 8.2, D and E had different electrophoretic mobilities. Preliminary studies indicate that based on thrombin time alone, D has anticoagulant activity while E appears to be a coagulation potentiator. Canine fibrinogen apparently consist of two core fragments with dissimilar chemical characteristics in common with the fundamental structures of human and bovine fibrinogens.     

Heat shock proteins from cell cultures of higher plants and their somatic hybrids

The species specificity of heat shock proteins of callus cultures of Nicotiana chinensis, Nicotiana glauca, Nicotiana tabacum, Atropa belladonna, Lycopersicon peruvianum, as well as some somatic hybrids of A. belladonna + N. chinensis, was investigated by means of polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Despite the general similarity of electrophoretic mobility of heat shock proteins from different plants, a number of species-specific differences between the proteins of distantly related genera were found. Heat shock proteins may thus serve as genetic markers in somatic hybridization.Abbreviations hs heat shock - hsp heat shock proteins - SDS sodium dodecylsulfate - PAGel'phoresis polyacrylamide gel electrophoresis - TCA trichloroacetic aeid - NA-1(3;5;7;11;15) Atropa belladonna + Nicotiana chinensis, clone 1(3;5;7;11;15)     

Evidence for the existence of actomyosin ATPase in the rat pancreas. Isolation and biochemical characterization

In a crude extract of rat pancreas, myosin was associated with a protein having the same electrophoretic mobility as actin. This myosin was purified after dissociation of the actomyosin complex with KI-ATP. On sodium dodecylsulfate/acrylamide gel electrophoresis, the isolated pancreatic myosin showed a major component of approximately 200 kDa, and two smaller components with apparent molecular weight of 22 and 15 kDa, respectively. This purified myosin exhibited high ATPase activity in the presence of K+ + EDTA or Ca2+ and very little activity in the presence of Mg2+. (K+ + EDTA)-ATPase activity showed one pH optimum at 8.0, while Ca2+-ATPase activity showed two pH optima at 6.0 and 9.0, respectively. (K+ + EDTA)-stimulated enzyme activity was specific for ATP whereas Ca2+-stimulated activity showed low specificity for nucleoside triphosphates.     

Thyroid microsomal membrane proteins. Effects of solubilization on molecular size.

The molecular size of microsomal membrane proteins from frozen porcine thyroids before and after solubilization by proteolytic and non-proteolytic techniques has been investigated by means of polyacrylamide-gel electrophoresis in the presence of 1% sodium dodecylsulfate. When thyroid microsomal membrane proteins are solubilized by non-proteolytic methods such as high pH, n-butanol, or deoxycholate, no major change in the electrophoretic pattern compared to untreated microsomes has been observed, thereby suggesting that these non-proteolytic methods are capable of extracting membrane proteins from thyroid microsomes without altering their molecular size. However, treatment of microsomes with protein-solubilizing levels of trypsin (1-5 mug trypsin per mg thyroid protein) results in degradation of all major proteins with a molecular weight greater than 30 000. The high-molecular-weight proteins are particularly susceptible to attack by trypsin. Thus, these experiments indicate that the use of trypsin to solubilize thyroid microsomal membrane proteins, particularly thyroid peroxidase, will result in fragmented proteins and should be avoided if intact membrane proteins are desired.     

Detergent-resistant phospholipase A of Escherichia coli K-12. Purification and properties.

Detergent-resistant phospholipase A, which is tightly bound to the outer membranes of Escherichia coli K-12 cells, was purified approximately 2000-fold to near homogeneity by solubilization with sodium dodecylsulfate and butan-1-ol, acid precipitation, acetone fractionation and column chromatographies on Sephadex G-100 in the presence of sodium dodecylsulfate and on DEAE-cellulose in the presence of Triton X-100. The final preparation showed a single band in the sodium dodecylsulfate gel system. The enzyme hydrolyzes both the 1-acyl and 2-acyl chains of phosphatidylethanolamine or phosphatidylcholine. It also attacks 1-acyl and 2-acylglycerylphosphorylethanolamine. Thus, this enzyme shows not only phospholipase A1 and lysophospholipase L1 activities but also phospholipase A2 and lysophospholipase L2 activities. The enzyme lost its activity completely on incubation at 80 degrees C for 5 min at either pH 6.4 or pH 8.0. It was stable in 0.5% sodium dodecylsulfate at below 40 degrees C. The enzyme was inactivated on incubation for 5 min at 90 degrees C in 1% sodium dodecylsulfate/1% 2-mercaptoethanol/4 M urea. The native and inactivated enzymes showed different protein bands with RF values corresponding to Mr 21 000 and Mr 28 000 respectively, in a sodium dodecylsulfate gel system. Triton X-100 seemed to protect the enzyme from inactivation. The purified enzyme was fully active on phosphatidylethanolamine in the presence of 0.0002% or 0.05% Triton X-100. The enzyme requires Ca2+. From its properties this enzyme seems to be identical with the enzyme purified from crude extracts of Escherichia coli B by Scandella and Kornberg. However, it differs from the latter in its positional specificity and susceptibility to sodium dodecylsulfate. Possible explanation of the difference of positional specificity of the two preparations is also described.     

High resolution two-dimensional gel electrophoresis of human erythrocyte membrane proteins.

Three different two-dimensional (2-D) gel electrophoretic techniques have been modified to provide high resolution of human erythrocyte membrane proteins. The resulting gels were referenced to the established one-dimensional (1-D) sodium dodecylsulfate (SDS) gel electrophoretic profile, and the effects of endogenous proteolysis and cytosolic contamination were studied. It is concluded that in vitro proteolysis and cytosolic contamination do not contribute significantly to the patterns observed on the 2-D gels, under the conditions used for erythrocyte ghost preparation. The procedures require only small quantities of blood; as many as twenty 2-D gel profiles can be obtained from 5 ml of blood. The combination of nonequilibrium isoelectric focusing (IEF) in the first dimension, SDS electrophoresis in the second dimension, and very sensitive silver staining techniques resolves more than 250 individual protein spots. This appears to be the most useful single procedure for the analysis of red cell membrane proteins. Membrane protein profiles from patients with Duchenne muscular dystrophy, Wernicke-Korsakoff syndrome, and acanthocytosis with degeneration of the basal ganglia were compared with normal controls. The patterns for Duchenne muscular dystrophy and Wernicke-Korsakoff syndrome were not different from normal patterns. The pattern for the patient with acanthocytosis and degeneration of the basal ganglia consistently showed a high level for one protein in the 100,000 mol. wt. range.     

On the heterogeneity and organ specificity of chicken tropomyosins.

1. Tropomyosins from chicken cardiac, skeletal, and gizzard muscles were each resolved into two subunits by polyacrylamide gel electrophoresis in a system containing sodium dodecylsulfate (SDS), urea and sodium borate, and were designated C1 C2, S1 S2, and G1 G2, respectively, in descending order of mobility on electrophoresis. S1, S2, G1, and G2 were prepared as pure samples by electrophoresis. 2. The apparent molecular weights of C (C1 + C2), S1, S2, G1, and G2 were calculated to be 36,000, 36,000, 37,500, 36,000, and 40,000, respectively, based on SDS gel electrophoretic mobility according to the method of Weber and Osborn. C and S1 showed nearly the same mobility in all electrophoretic systems tried. S1 and G1, which comigrated in an SDS-sodium borate system, showed different mobilities upon addition of 5 M urea to the system. 3. Immunological evidence presented indicates that each subunit has a specific antigenic site(s) in addition to an identical one(s) in common with the others. 4. As each tropomyosin subunit formed two precipitin lines with the homologous antiserum, as many as ten kinds of subunits may exist in chicken muscles.     

Preparative electrophoresis of human apolipoprotein E: an improved method

Apolipoprotein E was isolated from human very low density lipoproteins by a two-step electrophoretic procedure derived from that of Méndez (1982. Anal. Biochem. 126: 403-408). It included separation in a sodium dodecylsulfate polyacrylamide slab gel, transfer into an agarose gel, and extraction by ultracentrifugation for 30 min. No protein labeling, dialysis, or concentration procedures were needed. The method was fast, showed an excellent protein recovery, and could be suitable as a general method of protein isolation by polyacrylamide gel electrophoresis.     

The cold-insoluble globulin of human plasma: studies of its essential structural features.

These investigations were directed at furnishing information on the essential structural features of the cold-insoluble globulin of human plasma. Amino acid and carbohydrate analyses showed that it is a glycoprotein (1.2% sialic acid, 1.8% hexose, 2.1% hexosamine) containing all of the amino acids usually found in proteins. Circular dichroic spectral analysis suggested that cold-insoluble globulin contained a very high proportion of beta-structure; no evidence for the presence of alpha-helix was found. Sedimentation velocity experiments at pH 7.0, in the presence or absence of dithiothreitol, plus related gel electrophoretic experiments at pH 8.4, indicated that the integrity of certain disulfide bridges was necessary for its solubility under "physiologic" conditions. In experiments in urea-containing solution, two sedimenting peaks were observed. The major one, amounting to more than 95% of the total, had an s20,w of 5.6 S, the minor peak had an s20,w of 7.3 S. Following disulfide bridge reduction a single symmetrical peak of 3.9 S was formed. Such behavior suggested that cold-insoluble globulin is a multichain molecule whose subunit chains are linked by disulfide bridging. Strong support for this conclusion was obtained from electrophoretic analyses in gels containing dodecylsulfate, in that cold-insoluble globulin manifested an increased rate of migration after reduction of disulfide bridges. The reduced cold-insoluble globulin band could be resolved into a closely spaced doublet, the components of which had molecular weights of 220 000 and 215 000, respectively. Since in sedimentation equilibrium experiments the molecular weight of the unreduced molecule was estimated to be 450 000, values in this range for the size of the subunit chain suggested that each cold-insoluble globulin molecule is composed of two covalently linked chains. The nature of the size heterogeneity of the reduced subunit chains is uncertain. However, the finding of a single type of NH2-terminal sequence ([Glu-Ala) in cold-insoluble globulin preparations, is consistent with the speculation that the smaller subunit may be a catabolic intermediate arising via release of peptide material containing the COOH-terminus of a parent chain.     

Characterization of porins from the outer membrane of Salmonella typhimurium. 2. Physical properties of the functional oligomeric aggregates.

We have purified to homogeneity, from mutant strains of Salmonella typhimurium, the small oligomers of porin that confer permeability channels to artificial vesicle membranes reconstituted from phospholipids and lipopolysaccharide. The molecular weights of the porin oligomers from the strains SH5551 and SH6017 appeared to be 130000 and 125000, respectively, and those of the monomers were 41000 and 37500, respectively, when determined by sedimentation equilibrium in the presence of dodecylsulfate. It was thus concluded that the functional porin oligomers consisted of three identical subunits. The Stokes' radius of the trimer . dodecylsulfate complex was around 5 nm. The trimer bound less dodecylsulfate than the monomer. The trimer . dodecylsulfate complex retained at room temperature the native conformation of porin, which is rich in beta-structure. When the trimers were dissociated further by various treatments, only the porin monomers were recovered in significant amounts, and the permeability-conferring activity was lost simultaneously. We propose, therefore, that the trimer is the minimal functional unit of porin that is capable of forming permeability channels in the outer membrane of Salmonella typhimurium.     

Stimulation of proteinase K action by denaturing agents: application to the isolation of nucleic acids and the degradation of 'masked' proteins.

Hydrolysis of serum albumin by proteinase K was strongly (greater than 7-fold) stimulated by urea and dodecylsulfate in a dose-dependent manner. With an oligopeptide as substrate, however, proteinase K was inactivated by dodecylsulfate. This indicates that the apparent activation of proteinase K by urea and dodecylsulfate is caused primarily by denaturation of the protein substrates. Although dodecylsulfate inhibited ribonuclease activity in the test-tube completely, it could not prevent RNA degradation during isolation of polysomal RNA, to which ribonuclease had been added, because of the reversible nature of the dodecylsulfate inhibition. Complete protection of RNA, however, was achieved by a combination of dodecylsulfate and proteinase K. The combined action of the detergent and proteinase K was also effective in degrading "masked" proteins in a poly(adenosine diphosphoribose) preparation which could not be attacked by the proteinase alone.     

Characteristics of membrane protein phosphorylation in Plasmodium berghei-infected mouse erythrocytes

Membrane protein phosphorylation in Plasmodium berghei-infected erythrocytes was studied by incubating intact cells with (32P)orthophosphate and incubating isolated membrane with (gamma-32P)ATP. Phosphorylated proteins were detected by autoradiography after sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis or isoelectric focusing followed by gel electrophoresis. New phosphorylated proteins were found in membrane from infected erythrocytes, including a protein with electrophoretic mobility identical to band 5, with Mr 43,000. The molar ratio of phosphate to protein ranged between 0.1 and 0.5. Isoelectric focusing-SDS polyacrylamide gel electrophoresis, peptide mapping, extractability properties, and reduction of susceptibility to DNase I inhibition suggested that this protein is phosphorylated actin. In contrast, spectrin phosphorylation in infected erythrocytes was mostly unchanged.     

Antibodies against chromosomal HMG proteins stain the cytoplasm of mammalian cells.

Antibodies specific to protein HMG-1 were purified by affinity chromatography on Sepharose columns to which HMG-1 was covalently bound. Immunofluorescence studies with these antibodies reveal that HMG-1 or components which immunologically cross-react with HMG-1 are present in the cytoplasm of Chinese hamster V-79, rat liver TR-12 and bovine trachea EBTr-NBL-4 cells. At selected antibody concentrations, the fluorescence present in the cytoplasm is more intense than that observed in the nucleus. The presence of HMG-1 protein in the cytoplasm of rat liver cells was verified by direct examination of the protein content of selected cytoplasmic fractions. A protein with electrophoretic mobility identical to HMG-1 was detected by electrophoresis on polyacrylamide gels containing either sodium dodecylsulfate or urea. Furthermore, the cytoplasmic extracts yielded a positive complement fixation with anti-HMG-1, while no reaction was obtained with control anti-H1 sera. We suggest that HMG protins, rather than functioning in the nucleus alone, are important structural elements of the entire cell.     

Resynthesis of insulin from its A and B chains in the presence of denaturants

Reoxidation of the reduced insulin A and B chains in 8 M urea leads to a recovery of native insulin of 2-7% whereas in 6 M guanidine or 0.5 mM dodecylsulfate very little, if any, resynthesis of insulin could be detected. Considering all the possibilities of different oligomeric forms containing one or both of the chains, the yield in 8 M urea is well over the yield as calculated from random joining of the chains and the yield in guanidine or dodecylsulfate is to be expected. It is concluded that some interaction and pairing of the chains occur even in the presence of 8 M urea.