Subunit structure of anthranilate synthetase from Neurospora crassa.

Freshly purified preparations of anthranilate synthetase complex from Neurospora crassa appeared to be homogeneous on polyacrylamide disc gels and were composed of two distinct subunits, 94,000 and 70,000 daltons, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Carboxymethylation of the complex or treatment with guanidine hydrochloride and urea before sodium dodecyl sulfate treatment did not alter the subunit pattern. When the purified complex was iodinated with 125I- or methylated with [14C]dimethylsulfate, no labeled components other than the two subunits stained with Coomassie blue were detected after electrophoresis in the presence of sodium dodecyl sulfate. Although some purified preparations were stable, most were unstable upon storage. Analysis of the unstable preparations on nondenaturing and sodium dodecyl sulfate polyacrylamide disc gels revealed that the complex in these preparations was progressively fragmented to smaller components and subunits upon repeated freeze-thaw treatment or prolonged incubation at or above 4 degrees. Distinct fragments were generated ranging in size down to 25,000 daltons, and some fragments retained some of the activities associated with the anthranilate synthetase complex. On the basis of these and earlier studies, we conclude that anthranilate synthetase from Neurospora crassa is composed of two distinct subunits in an alpha2beta2 structure; one subunit is a trifunctional peptide which contains the catalytic sites for the phosphoribosylanthranilate isomerase and indoleglycerol phosphate synthetase reactions, and associates with the second subunit to form glutamine-dependent anthranilate synthetase. The smaller subunits and components previously reported for this complex are apparently due to protease activity present in purified preparations.     

Purification and characterization of alanine carrier isolated from H-proteins of Bacillus subtilis.

Alanine transport carrier was isolated and purified from H-proteins of Bacillus subtilis. The purified carrier preparation was homogeneous in migration on polyacrylamide gels containing urea or sodium dodecyl sulfate. Electrophoresis on polyacrylamide gels containing dodecyl sulfate showed a single band of molecular weight of about 7500. 1 mol alanine was bound/mol carrier protein with a dissociation constant of 0.2 micron. The binding was inhibited by p-chloromercuribenzoate and the inhibition was reversed by dithiothreitol.     

Purification and partial characterization of the Mr 30,000 integral membrane protein associated with the erythrocyte Rh(D) antigen

Erythrocytes bearing the Rh(D) antigen have an Mr 30,000 integral membrane protein which can be surface-labeled with 125I and can be quantitatively immunoprecipitated from Triton X-100-solubilized spectrin-depleted membrane vesicles. The 125I-labeled Rh(D)-associated protein was purified to radiochemical homogeneity from membrane skeletons solubilized in sodium dodecyl sulfate and urea by hydroxylapatite chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis. The Rh(D)-associated protein was purified nearly 200-fold from 2 units of erythrocytes from DD individuals by employing similar methods on a large scale using the purified 125I-labeled Rh(D)-associated protein as a tracer. The product appeared to be greater than 95% pure and migrated as a diffuse band of Mr approximately 30,000-32,000 on silver-stained sodium dodecyl sulfate electrophoresis gels poured from 12% acrylamide. It is estimated that the Rh(D)-associated protein makes up approximately 0.5% of the original membrane protein. When concentrated, partially purified Rh(D)-associated protein forms dimers and larger oligomers which are stable in sodium dodecyl sulfate and urea. The Rh(D)-associated protein was protected from degradation when intact erythrocytes or inside out membrane vesicles were enzymatically digested. These studies indicate that the Mr 30,000 protein associated with the Rh(D) antigen is linked to the membrane skeleton, resides within the lipid bilayer with minimal extra- or intracellular protrusions, exists normally as an oligomer, and can be purified in denatured form.     

Purification and Characterization of Axial Filaments from Treponema phagedenis Biotype reiterii (the Reiter Treponeme)

Axial filaments have been purified from Treponema phagedenis biotype reiterii (the Reiter treponeme) and partially characterized chemically. The preparations consist largely of protein but also contain small amounts of hexose (3%). Filaments dissociate to subunits in acid, alkali, urea, guanidine, and various detergents. Amino acid analyses show an overall resemblance to other spirochetal axial filaments and to bacterial flagella. Dissociated filaments migrate as a single band upon acrylamide gel electrophoresis at pH 4.3 (in 4 M urea and 10 (3) M ethylenediaminetetraacetate) and at pH 12, but in sodium dodecyl sulfate gels, three bands are obtained under a wide variety of conditions. Two of these bands migrate very close together, with molecular weights of 33,000 +/- 500. The other band has a molecular weight of 36,500 +/- 500. Analysis of axial filaments by the dansyl chloride method yields both methionine and glutamic acid as amino terminal end groups. Sedimentation equilibrium measurements on dissociated axial filaments in 7 M guanidine hydrochloride yield plots of log C against varkappa(2) which vary with the speed and initial protein concentration used. Molecular weight values calculated from these plots are consistent with a model in which axial filament subunits are heterogeneous with respect to molecular weight in the approximate range of 32,000 to 36,000.     

The localization of tightly bound cardiolipin in cytochrome oxidase

One to two molecules of tightly bound cardiolipin are associated with resolved fractions of cytochrome oxidase containing subunits I to III or I to IV. Large scale isolation of subunits I to IV indicates the presence of approximately 0.5 molecule of cardiolipin per molecule of subunit I. Lipoprotein staining of sodium dodecyl sulfate/urea/acrylamide gels of cytochrome oxidase support the findings that subunit I is a lipoprotein. The resistance of this tightly bound cardiolipin to organic solvent extraction suggests a specific association of some tenacity with the protein.     

Exchange of colicin receptor capacity between strains of Escherichia coli sensitive or resistant to colicin K-K235

Phage and colicin-resistant mutants were derived from Escherichia coli K-12P678. Two classes of phage T6 and colicin K-resistant mutants (genotype tsx) were isolated. Tsx-2 mutants, which demonstrated mucoid growth and increased sensitivities to many antibiotics, became sensitive to colicin K when pretreated with ethylenediaminetetraacetate (EDTA), whereas Tsx-1 mutants did not. Reassociation of EDTA-released material partially restored resistance to colicin K for Tsx-2 mutants. When EDTA-released material from strain P678 was associated with either class of K-resistant mutant, an increase in colicin K sensitivity resulted. Observations suggest that colicin K can act on its target site once it penetrates the cell surface. In addition, results suggest that functional colicin K receptors can be transferred from sensitive to resistant strains, thus conferring colicin sensitivity.Non-standard Abbreviations SDS sodium dodecyl sulfate     

Fat cell protein phosphorylation. Identification of phosphoprotein-2 as ATP-citrate lyase.

We have purified to apparent homogeneity a phosphoprotein from rat adipose tissue which is rapidly phosphorylated in vitro by ATP. The native phosphoprotein has an approximate sedimentation coefficient of 14.8 S. On sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the protein dissociated into identical subunits of Mr = 128,000. A phosphoprotein with similar properties was also isolated from liver. Purified phosphoproteins from fat cells and liver had ATP-citrate lyase activity and co-migrated on sodium dodecyl sulfate gels with fat cell phosphoprotein-2, the phosphorylation of which is increased by incubating fat cells with insulin. The phosphoamino acid residue of the cells with insulin. The phosphoamino acid residue of the phosphoprotein was identified as tau-phosphohistidine. These evidences suggest that fat cell phosphoprotein-2 is ATP-citrate lyase.     

Studies on the protein composition of human serum very low density lipoproteins: demonstration of the beta 2-glycoprotein-I.

Human serum VLDL isolated by polyanion precipitation and ultracentrifugation have been delipidated with ethanal/diethyl ether. By electrophoresis in 10% polyacrylamide gels containing 8M urea, we found a protein which comigrated with apolipoprotein E. This protein was purified by column chromatography and turned out to be identical with beta 2-glycoprotein-I, the serum factor which is necessary for the precipitation of triglyceride-rich lipoproteins with sodium decyl sulfate or sodium dodecyl sulfate. Upon analytical isoelectric focusing, beta 2-glycoprotein-I gave four major bands in the pH region 5.7--6.6. All four bands gave an immunochemical reaction of identity with a monospecific antiserum. From its unique amino acid composition we conclude that beta 2-glycoprotein-I is distinct from all apolipoproteins described previously in the literature.     

Dissociation of thyroglobulin by tetraphenylborate ion

Purified bovine and ovine thyroglobulins (19 S) are partially dissociated into 12-S subunits after treatment with sodium tetraphenyl borate. The extent of dissociation obtained by sodium tetraphenyl borate or sodium dodecyl sulfate treatment is the same. The electrophoretic mobilities on acrylamide gels of sodium tetraphenyl borate-resistant molecules and of native thyroglobulin are identical. Sodium dodecyl sulfate-resistant molecules move more slowly than the native protein.     

Presence of two subunit types in ribulose 1,5-bisphosphate carboxylase from Thiobacillus intermedius.

Ribulose bisphosphate carboxylase (EC 4.1.1.39) has been purified to homogeneity from glutamate-CO2-thiosulfate-grown Thiobacillus intermedius by pelleting the protein from the 93,000 X g supernatant fluid followed by ammonium sulfate fractionation and sedimentation into a discontinuous sucrose density gradient. The molecular weight of the native protein approximated that of the higher plant enzyme (550,000) based on its relative electrophoretic mobility in polyacrylamide disc gels compared with that of standards of known molecular weight, including crystalline tobacco ribulose bisphosphate carboxylase. Sodium dodecyl sulfate electrophoresis in 12% polyacrylamide disc gels and Sephadex G-100 chromatography in the presence of sodium dodecyl sulfate indicated that the purified Thiobacillus protein, like the tobacco enzyme, consisted of two types of nonidentical subunits. The molecular weights of the large and small subunits were estimated to be about 55,000 and 13,000, respectively, by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carboxylase activity of the protein purified from spinach leaves and T. intermedius responded similarly to the effectors reduced nicotinamide adenine dinucleotide phosphate and 6-phosphogluconate. Contrary to a previous report (K. Purohit, B. A. McFadden, and A. L. Cohen, J. Bacteriol. 127:505-515, 1976), these results indicate that ribulose bisphosphate carboxylase purified from Thiobacillus intermedius closely resembles the higher plant enzyme with respect to quaternary structure, molecular weight, and regulatory properties.     

Partial purification and characterization of the multiple molecular forms of staphylococcal clotting activity (coagulase).

The clotting activity of Staphylococcus aureus strain 104 was purified 46,000-fold, but absolute purity was not achieved. Carbohydrate content of the purified material was not more than 5%. Elution of clotting activity from denaturing and nondenaturing polyacrylamide gels revealed the presence of four distinct molecular forms. Molecular weights of the forms were approximately 31,500, 34,800, 44,800, and 56,800 as determined by gel filtration in 8 M urea, by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis, and by calculation with determined values for the Stokes radius and sedimentation coefficient. Molecular weights determined on sodium dodecyl sulfate-urea gels were found to decrease as the gel concentration increased, suggesting that the amount of sodium dodecyl sulfate bound was less than normal. Estimated frictional ratios for the forms showed that they differ in shape from one another and that they are all highly asymmetrical. Each of the forms had an isoelectric point between pH 5.44 and 5.47 when focused in 6% polyacrylamide gels for 9 h; however, prolonged focusing altered the isoelectric point of the forms to within the range of pH 4.35 to 4.65. The multiple clotting forms were not artifacts of the purification procedure and did not appear to be products of the proteolytic degradation of a larger protein.     

The photochemical reaction center of Chloroflexus aurantiacus is composed of two structurally similar polypeptides

A method has been devised which allowed the isolation of highly purified reaction center from the thermophilic green bacterium, Chloroflexus aurantiacus. The procedure consisted of three chromatography steps. The final step was fast protein liquid chromatography on Mono Q in the presence of nonanoyl-N-methylglucamide (Mega-9). The purified reaction center complex was photochemically active and had an A280/A813 of 1.4 or less. Under non-denaturing conditions, a pigmented protein band having a Mr of 52,000-55,000 was observed in sodium dodecyl sulfate gels. When the isolated complex was heat-dissociated in the presence of sodium dodecyl sulfate, just two polypeptides having very similar Mr (24,000 and 24,500) were observed. Two protein bands were also observed in two-dimensional isoelectric focusing/sodium-dodecyl-sulfate polyacrylamide gel electrophoresis; the PI values of the two polypeptides were 6.5 and 6.7. Partial peptide mapping of the two isolated subunits, using both enzymatic and chemical cleavage techniques, yielded almost identical patterns which indicated a high degree of sequence homology between the two polypeptides. The N-terminal amino acid sequences of the two polypeptides were identical and did not exhibit any homology to reaction center subunits of purple sulfur bacteria. The Chloroflexus reaction center is believed to be composed of one molecule of each polypeptide, the photoactive bacteriochlorophyll a dimer and, as accessory pigments, an additional bacteriochlorophyll a and three bacteriopheophytins. Hence, it appears to be the smallest photochemically active reaction center isolated to date.     

Denaturation behavior of phaseolin in urea, guanidine hydrochloride, and sodium dodecyl sulfate solutions

The denaturation behavior of phaseolin in urea, guanidine hydrochloride, and sodium dodecyl sulfate solutions was examined by monitoring changes in the intrinsic fluorescence of tryptophan and tyrosyl residues. Changes in various fluorescence parameters, such as quantum yield, emission maximum, spectral half-width, fluorescence depolarization, and fluorescence quenching by acrylamide, have indicated that while phaseolin is relatively stable up to 8 M urea, it is completely destabilized in 6 M guanidine hydrochloride and 6 mM sodium dodecyl sulfate. Furthermore, while the denaturation of phaseolin in urea solutions followed a two-step process, that in guanidine hydrochloride and sodium dodecyl sulfate followed a single-step process. While the accessibility of tryptophan residues to the nonionic acrylamide quencher is almost 100% in 6 M guanidine hydrochloride and 6 mM sodium dodecyl sulfate, only about 72% was accessible in 8 M urea compared to 52% in native phaseolin. The results presented here suggest that the protomeric structure of phaseolin is quite stable to changes in the environment. This structural stability may be partly responsible for its resistance to proteolysis by various proteinases.     

Solubilization, isolation, and immunochemical characterization of the major outer membrane protein from Rhodopseudomonas sphaeroides

Solubilization of the major outer membrane protein of Rhodopseudomonas sphaeroides, and subsequent isolation, has been achieved by both non-detergent- and detergent-based methods. The protein was differentially solubilized from other outer membrane proteins in 5 M guanidine thiocyanate which was exchanged by dialysis for 7 M urea. The urea-soluble protein was purified to homogeneity by a combination of DEAE-Sephadex chromatography and preparative electrophoretic techniques. Similar to the peptidoglycan-associated proteins of other Gram-negative bacteria, the protein was also purified by differential temperature extraction of the outer membrane in the presence of sodium dodecyl sulfate (SDS) followed by preparative SDS-polyacrylamide gel electrophoresis. Immunochemical analysis of the proteins isolated by the two techniques established the immunochemical identity and homogeneity of each preparation. Immunoblots of SDS-polyacrylamide gels revealed that antibody directed against the major outer membrane protein reacted with the three high molecular weight aggregates present in the outer membrane which we have previously shown to be composed of the major outer membrane protein and three nonidentical small molecular weight proteins.     

Multiple molecular forms of endothelial cell factor VIII related antigen

Heterogeneous factor VIII related antigen isolated from endothelial cell postculture medium was characterized. On crossed immunoelectrophoresis, slow moving less anodal populations of factor VIII related antigen molecules were more prominent in endothelial cell postculture medium than in plasma. The protein synthesized by endothelial cells appeared as two discrete protein bands of different molecular weight in sodium dodecyl sulfate polyacrylamide gels. In contrast the factor VIII related antigen isolated from plasma moved as a single protein band in an identical gel system. The factor VIII related antigen from endothelial cell postculture medium was immunoisolated using monospecific rabbit antibody to normal human plasma factor VIII related antigen, electrophoresed on sodium dodecyl sulfate polyacrylamide gels, radiolabeled with ¹²⁵I, trypsinized and subjected to peptide mapping using two-dimensional high voltage electrophoresis and thin-layer chromatography. The two forms of endothelial cell factor VIII antigen had virtually identical peptide maps. These studies suggest that the molecular heterogeneity of the factor VIII related antigen sytem reflects polymeric associations of identical subunits. Circulation in vivo may alter the ratio of polymer subsets.     

Escherichia coli Mutants Permissive for T4 Bacteriophage with Deletion in e Gene (Phage Lysozyme)

Escherichia coli mutants have been isolated that are permissive for the infection by T4 phage with deletion in the cistron for the phage lysozyme, the e gene. Some, but not all, of these mutants are simultaneously permissive for the infection by T4 phage defective in the t gene, the product of which has also been implicated in the release of progeny phages. Most of these mutants shared the following properties: temperature sensitivity in growth and cell division, increased sensitivity towards a number of unrelated antibiotics and colicins, and increased sensitivity towards anionic detergents (sodium dodecyl sulfate and sodium deoxycholate). The possible biochemical basis for these phenotypes is discussed.     

Protein Components of the Purified Sodium Channel from Rat Skeletal Muscle Sarcolemma

Abstract: Sensitive detection systems have been used to study the protein components of the sodium channel purified from rat skeletal muscle sarcolemma. This functional, purified sodium channel contains at least three subunits on 7–20% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: a large glycoprotein which migrates anomalously in the high-molecular-weight range, a 45,000 molecular weight polypeptide, and a third protein often seen as a doublet at 38,000. The large glycoprotein runs as a diffuse band and stains very poorly with Coomassie blue, but is adequately visualized with silver staining or iodination followed by autoradiography. This glycoprotein exhibits anomalous electrophoretic behavior in SDS-polyacrylamide gels. The apparent molecular weight of the center of the band varies from ～230,000 on 13% acrylamide gels to ～130,000 on 5% gels; on 7–20% gradient gels a value of 160,000 is found. Plots of relative migration versus gel concentration suggest an unusually high apparent free solution mobility. Lectin binding to purified channel peptides separated by gel electrophoresis indicates that the large glycoprotein is the only subunit that binds either concanavalin A or wheat germ agglutinin, and this component has high binding capacity for both lectins. The smaller channel components run consistently at 45,000 and 38,000 molecular weight in a variety of gel systems and do not appear to be glycosylated.     

Inhibitor of uracil-DNA glycosylase induced by bacteriophage PBS2. Purification and preliminary characterization

A PBS2 phage-coded inhibitor of uracil-DNA glycosylase activity from Bacillus subtilis has been purified extensively and characterized preliminary. The inhibitor has a relative S value of 1.44 +/- 0.08 measured by sedimentation in 15 to 40% glycerol density gradients. It is unusually stable to heating and to the presence of sodium dodecyl sulfate and/or 8 M urea. The inhibitor has no known cofactor requirement and is active in the presence of 10 mM EDTA. Inhibitor activity is sensitive to digestion with proteinase K, but is insensitive to DNase or RNase digestion. The purified inhibitor behaves anomalously during electrophoresis in poly-acrylamide gels containing sodium dodecyl sulfate; however, experiments designed to show that the inhibitor is a glycoprotein were negative. The inhibitor clearly contains a protein required for activity, however, the possibility that some other molecular component is part of the active inhibitor cannot be excluded.     

Unusual stability of the Methanospirillum hungatei sheath.

The proteinaceous sheath of Methanospirillum hungatei was isolated by lysing cells in 50 mM dithiothreitol, separating the sheath from other cellular material by discontinuous sucrose density centrifugation, and removing the "cell spacers" with dilute NaOH. The isolated sheath material consisted of hollow tubes which had a highly ordered surface array. The stability of the sheath to treatment with denaturants and to enzymatic digestion was examined by a turbidimetric assay in conjunction with electron microscopy and optical or electron diffraction. The sheath was resistant to a range of proteases and also was not digested by peptidoglycan-degrading enzymes, a lipase, a cellulase, a glucosidase, or Rhozyme (a mixture of galactosidases, acetylglucosaminidase, acetylgalactosaminidase, fucosidase, and mannosidases). In addition to being unaffected by common salts, thiol-reducing agents, and EDTA, the layer was resistant to powerful denaturants such as 6 M urea, 6 M guanidinium hydrochloride, 10 M LiSCN, cyanogen bromide, sodium periodate, and 1% sodium dodecyl sulfate. Strong bases, boiling 3 N HCl, and performic acid did attack the sheath; in these cases, the array was systematically disassembled in a progressive manner, which was followed by electron microscopy. The layer was slightly modified by N-bromosuccinimide in urea, but the array remained intact. The stability of the sheath was remarkable, not only as compared to other bacterial surface arrays, but also as compared to proteins generally, and possibly indicated the presence of covalent cross-links between protein subunits.     

Partial resolution of the enzymes catalyzing photophosphorylation. XV. Approaches to the active site of coupling factor I.

1. Prolonged treatment of coupling factor I (CF1) from spinach chloroplasts with trypsin free of chymotrypsin yielded an active ATPase. The isolated preparation showed only two polypeptide chains (mol wt 55,000 to 60,000) on acrylamide gels run in the presence of sodium dodecyl sulfate. The three smaller subunits of CF1 were not detectable. The preparation no longer served as a coupling factor for photophosphorylation in either EDTA- or silicotungstate-treated chloroplasts. 2. An antiserum prepared against coupling factor I from chloroplasts inhibited the ATPase activity of the trypsin-treated CF1. In contrast, antisera prepared against the two individual (denatured) subunits did not inhibit the ATPase activity when tested either alone or together, although each interacted with the trypsin-treated protein, forming precipitin lines in Ouchterlony plates. 3. The trypsin-treated enzyme was still cold-labile, showing that the three smaller subunits are not required for this property. However, the enzyme was no longer sensitive to the natural inhibitor protein which is one of its subunits (subunit epislon), but was still sensitive to inhibition by the flavonoid quercetin. 4. Two equivalents of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole were sufficient to inhibit about 80% of the ATPase activity of the coupling factor, irrespective of whether it contained two of five subunits. The inhibition was completely reversed by dithiothreitol. 5. Triated 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was prepared. Treatment of the coupling factor with this tritium-labeled inhibitor followed by electrophoresis on acrylamide gels revealed that most of the radioactivity was incorporated into the beta subunit of the enzyme (molecular weight 56,000).