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Describes an application of two-dimensional, nuclear magnetic resonance techniques for the study of proteins. Amino acid residue; Molecular weight; Glycyl residue spin systems; Spin-spin coupling constants; Protein conformation.

We have developed a semi-empirical method to determine the subunit molecular weight of a protein. The method is a minor modification of the Edman degradation and is based on a simple chemical procedure to modify specifically the N-terminal NH2 group. Initially, all NH2 groups, including the ε-NH2 groups of lysine residues and the N-terminal α-NH2 group, are reacted with phenylisothiocyanate and the protein derivative is subjected to one step of the Edman degradation. The newly exposed N terminus is then reacted with radioactively labelled phenylisothiocyanate. A value for the subunit molecular weight can be obtained from an analysis of the incorporated radioactivity and the amount of the protein in the sample. The molecular weight of five different proteins have been determined by this method. Our method is particularly useful for proteins containing lipid or sugar components and also for relatively small peptides. The procedure described in this paper for the specific modification of the N terminus has been found to be a powerful tool for protein sequencing. [ABSTRACT FROM AUTHOR]

Two 1,3; 1,4-β-glucan endohydrolases have been purified from extracts of germinating barley by ammonium sulphate precipitation, ion-exchange and gel filtration chromatography. Both enzymes are monomeric, basic proteins. Enzyme I has a molecular weight of 28000 and an isoelectric point of 8.5, while enzyme II has a molecular weight of 33000 and an isoelectric point greater than 10. Enzyme II is a glycoprotein containing 3.6% carbohydrate, of which three residues are probable N-acetylglucosamine, but enzyme I contains only traces of associated carbohydrate. The amino acid compositions of the two 1,3; 1,4-β-glucan endohydrolases are similar and the cross-reactivity of antibodies raised against the purified enzymes suggests that they share common antigenic determinants. [ABSTRACT FROM AUTHOR]

The biosynthesis and secretion of α1-antitrypsin was studied in rat hepatocyte primary cultures. After labeling with [35S]methionine an α1-antitrypsin with an apparent molecular weight of 49000 estimated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis was immunoprecipitated from the cell homogenate. This intracellular form of α1 -antitrypsin could be deglycosylated by endoglycosidase H treatment indicating that its oligosaccharide chains were of the high-mannose type. Pulse-chase experiments showed that about 30 min after its synthesis the transformation of the 49000-Mr α1 -antitrypsin to a protein with an apparent molecular weight of 54000 began. Only this 54000-Mr protein was secreted by the hepatocytes. The 54000-Mr α1 -antitrypsin was not sensitive to endoglycosidase H, but sensitive to neuraminidase, and it incorporated [³H]galactose and [³H]fucose indicating that its oligosaccharide chains were of the complex type. In the presence of tunicamycin, which blocks the formation of N-asparagine-linked oligosaccharide chains, an unglycosylated α1-antitrypsin with an apparent molecular weight of 41000 was found in the cells as well as in the medium. However, tunicamycin decreased the secretion of α1-antitrypsin by 60–70%, whereas the secretion of albumin remained unaffected. In the presence of colchicine the secretion of both α1-antitrypsin and albumin was impaired. The results demonstrate the importance of glycosylation for the secretion of α1-antitrypsin. [ABSTRACT FROM AUTHOR]

The ethylmaleimide-sensitive phosphate-transport system of heart mitochondria was isolated and the activity reconstituted in liposomes. The identification of the phosphate-carrier protein was complicated by several factors. 1. The phosphate-carrier fraction, isolated through different procedures in different laboratories, contains 4–5 protein components of very similar apparent molecular weight, as shown by gradient dodecylsulfate gel electrophoresis. 2. The amino acid composition of protein 1, 2 and 3(apparent Mr = 34500, 34000 and 33000, respectively) is quite similar although not identical. 3. Including several protease inhibitors during isolation of the phosphate-carrier fraction does not influence the protein pattern, 4. Labeled N-ethylmaleimide binds only protein 1 and 3, labeled N,N′-dicyclohexylcarbodiimide bonds only to protein 1 and 2. 5. N,.N′-Dicyclohexylcarbodiimide had no effect on the reconstituted phosphate-exchange activity after incubation with intact mitochondria or proteoliposomes. 6. In the presence of protecting mersalyl concentrations N-ethylmaleimide inhibits the phosphate transport in intact mitochondria. Under these conditions no label ethylmaleimide was bound to the phosphate-carrier fraction. 7. Comparison of the reconstituted transport activity with the protein pattern of fractions, isolated with or without cardiolipin, shows correlation of transport with the amount of protein 2 but not with that of protein 1, which binds ethylmaleimide. A model of the mitochondrial phosphate carrier is presented which suggests a proteolytic degradation of the phosphate-carrier protein during its isolation. The model explains the contradictory results of this investigation. [ABSTRACT FROM AUTHOR]

Clathrin purified to a high degree of homogeneity showed the presence of accompanying polypeptides of lower molecular weights and assembled into baskets or cages when the pH of its solution was adjusted from pH 7.5 to 6.5. Brief chymotrypsin treatment of this clathrin preparation at pH 6.5 cleaved clathrin-associated proteins rendering clathrin unable to reform baskets. Hydrolysis of clathrin-associated proteins did not affect clathrin's capacity to polymerize into open lattices or to bind actin and α-actinin from solution. When open lattices formed, the turbidity of the solution rose to levels that were higher than those produced when cages or baskets were formed by native clathrin. In both native or enzyme-treated clathrin, turbidity increase was inhibited by salts. The addition of certain cytoskeletal-disrupting agents, such as chlorpromazine and imipramine, increased clathrin's turbidity but did not result in formation of baskets. Colchicine and cytochalasin B did not affect turbidity or the assembly of cages. The addition of increasing amounts of vinblastine resulted in the precipitation of larger amounts of clathrin which, after solubilization, retained its ability to polymerize into baskets. It seems that clathrin exists as a complex with other protein molecules, clathrin-associated proteins, that modulate the extent of polymerization and assembly of clathrin into characteristic structures. [ABSTRACT FROM AUTHOR]

The major polypeptide chain of bovine β-crystallin, βBp, was fragmented by means of cyanogen bromide treatment and by enzymatic digestions. Manual and automated Edman degradation of the resulting peptides provided the complete amino acid sequence of the βBp chain. The N-terminal alanine residue was shown to be N-α-acetylated by mass spectrometry. The chain has a length of 204 residues and a calculated molecular weight of 23 210. There is a considerable degree of homology between the N-terminal and C-terminal halves of the chain, presumably reflecting a tandem duplication of a shorter ancestral gene. The sequence of βBp is sufficiently related to that of γ-crystallin II to place these proteins in the same superfamily. No sequence relationship was found with the α-crystallin chains. [ABSTRACT FROM AUTHOR]

Thioredoxin f has been isolated and purified to homogeneity from spinach chioroplasts. The protein is a dimer which dissociates in two apparently identical halves when ionic strength is raised. The dimer has a molecular weight of 16000. In its oxidized state, chloroplastic thioredoxin I has no sulihydry group directly available, even after denaturation of the protein. When reduced by dithiothreitol, four - SB groups become available on the dimer: Taken together, these results show that there exists one disulfide bridge per monomer. Reduction by dithiothreitol results in the breaking of two disulfide bridges and the appearance of four sulfliydryl groups. Monomeric thioredoxin f does not result in a significant activation of fructose 1 ,6-bisphosphatase. Full activation is obtained in presence of the dimeric protein. This is understandabJe since activation of fructose bisphosphatase necessitates reduction of two disulfide bridges of the enzyme. Full activation of fructose bisphosphatase is obtained in the presence of either an excess of dimeric thio- redoxin I, or in the presence of dithiothreitol. Moreover a large excess of oxidized thioredoxin deactivates fructose bisphosphatase. These results suggest that there exists an equilibrium where The and The stand for reduced and oxidized thioredoxin, oFruP2ase and rFruP2ase for oxidized and reduced fructose bisphosphatase. Moreover, this equilibrium must be strongly shifted towards the left, NADPH in presence of NADP-reductase may reduce thioredoxin, and conversely reduced thioredoxin may reduce NADP if the reductase is present. These results together with the existence of the above equilibrium provides a tentative scheme of fructose bisphosphatase inactivation in vivo in the dark. When the light has been turned off, electrons are transferred from thioredoxin to NADP via NADP-reductase. Thioredoxin becomes oxidized and then deactivates fructose bisphosphatase. [ABSTRACT FROM AUTHOR]

Characterizes the polysomal poly(A) protein particles in rat liver. Procedure of protein preparation by sucrose gradient centrifugation; Particle sedimentation; Tissue distribution; Molecular weight of identified polypeptides.

Protein L11 was isolated from the 50-S subunit of Escherichia coli ribosomes, using two salt extractions and two chromatographic separations on CM-cellulose. The unusual behavior of the protein when run on sodium dodecyl sulfate electrophoresis showed multiple bands. The complete primary structure of protein L11 is presented in detail. Its sequence was derived from peptides obtained by digesting the protein with trypsin, chymotrypsin, thermolysin, Staphylococcus aureus protease and, after modification, with trypsin. Chemical cleavage was performed with cyanogen bromide. Sequencing of the various peptides was achieved by manual micro-dansyl-Edman degradations and automatic methods. The N-terminal residue of the protein is blocked and was not degradable in the liquid-phase sequenator by the Edman method. It was identified by a combination of enzymatic cleavage and mass spectrometry. Protein L11 contains three methylated amino acid residues, a Nα-trimethylalanine, and two residues of Nϵ-trimethyllysine. Their behaviour and influence in the sequence elucidation are described. The protein contains 141 amino acid residues and has a molecular weight of 14874. Secondary structure predictions of the protein are given, and its sequence is compared with those of other E. coli ribosomal proteins. [ABSTRACT FROM AUTHOR]

Three groups of proteins can be clearly discriminated in the total protein of L cell polysomes by selective labelling in the presence of low doses of actinomycin D and two-dimensional polyacrylamide/ dodecylsulfate gel electrophoresis followed by autoradiography: (a) structural ribosomal proteins which are not labelled in the presence of actinomycin D and form stained non-radioactive spots in gels; (b) exchangeable ribosomal proteins which are labelled in the presence of actinomycin D and form stained radioactive spots; (c) non-ribosomal proteins which are detectable only by autoradiography of gels. The large and small subunits of L cell ribosomes contain respectively 45 and 34 ribosomal proteins with molecular weights ≤ 50000; seven of the large subunit proteins and nine of the small subunit proteins are exchangeable. Most of the non-ribosomal proteins migrate in the region of the second-dimension gel slab corresponding to the molecular weight range 50000-120000. Problems related to the separation of the ribosomal proteins of mammalian cells and the possible significance of the presence of non-ribosomal proteins in polysomes are discussed. [ABSTRACT FROM AUTHOR]

Characterization of a butanol-solubilized protein isolated from chloroplast membranes is reported. The proteolipid, which specifically and covalently binds dicyclohexylcarbodiimide, has an apparent molecular weight of 8000 in dodecylsulfate electrophoresis. The minimum molecular weight calculated from amino acid analysis data is 7700. N-Formyl-methionine was determined to be the N-terminal amino acid. Glycine, alanine and leucine were present in elevated amounts, resulting in a polarity of 29 %. Cysteine and histidine were lacking. In high-voltage electrophoresis the peptide appeared as a single homogenous spot which migrated, at pH 6.5, with the relative mobility of glycine. At concentrations where dicyclohexylcarbodiimide inhibited ATPase activity maximally (20 nmol per mg membrane protein), 0.17 nmol dicyclohexylcarbodiimide was covalently bound per nmol isolated proteolipid, indicating that one out of six molecules of proteolipid was labeled. [ABSTRACT FROM AUTHOR]

Eucaryotic L7/L12-type proteins are present in ethanol/salt extracts (P1 protein) of ribosomes from Artemia salina and rat liver. These proteins are partially phosphorylated and occur in two forms of closely related structure: a major form eL12 having methionine at the N-terminal position and a minor form of eL12 (eL12′) which seems slightly elongated and contains a blocked N terminus. Purification of the four different forms of this protein, eL12, eL12-P, eL12′ and eL12′-P, was performed by ion-exchange chromatography on carboxymethyl-cellulose and DEAE-cellulose. Using a radioimmuno assay, 1.8 copies of eL12 and 0.9 of ell2′ were found on the 80-S A. salina ribosome. In ribosomes of both rat liver and A. salina, eL12 is present in a larger quantity than eL12′. 40-S and 60-S ribosomal subunits extracted with ethanol/salt were essentially free of eL12 proteins. A large pool of eL12 was found in the cytosol after removal of the ribosomes by centrifugation or molecular sieving. The proteins of rat liver and A. salina are similar with regard to their isoelectric points and molecular weights. Sedimentation equilibrium studies indicated that the isolated protein eL12 occurs as a dimer. [ABSTRACT FROM AUTHOR]

A method for reducing the degree of heterogeneity in the electrophoretic enzyme activity pattern of hypoxanthine phosphoribosyltransferase preparations by incubation with a (magnesium) phosphoribosyl diphosphate substrate is described. Hypoxanthine phosphoribosyltransferase was isolated from human erythrocytes and Chinese hamster livers. A subunit molecular weight of 26000–27000 as reported by other authors was obtained for both enzymes by gel electrophoresis in the presence of dodecylsulfate. Gradient gel electrophoresis revealed that the native enzymes mainly have a molecular weight of 105000–110000 and are thus apparently tetrameric, when held in the active state by the presence of phosphoribosyl diphosphate. The dimeric enzyme with a molecular weight of 52000–55000, was also found under other conditions. The trimer occurred only in the absence of phosphoribosyl diphosphate, for instance by glycerol gradient centrifugation. The enzyme from human erythrocytes was partly degraded during purification in the absence of a protease inhibitor. The purified enzyme has a very low protease contamination level. Proteolysis is an additional cause of heterogeneity and might therefore explain earlier conflicting results. Since the heterogeneous nature of hypoxanthine phosphoribosyltransferase is caused only by the secondary processes of dissociation/association and, in the case of the human erythrocyte enzyme, degradation, we suggest that the use of the term ‘isozyme’ to describe the different forms should be avoided. [ABSTRACT FROM AUTHOR]

The properties of proteolytically non-modified phosphofructokinase from baker's yeast were investigated by means of hydrodynamic, electrophoretic, and chemical methods. The sedimentation coefficient shows a linear dependence on the protein concentration down to 0.01 mg/ml. The sedimentation coefficient has been determined to be s200.w = 20.81 S. At concentrations of the enzyme at less than 0.01 mg/ml a significant decrease of this value was found. The partial specific volume of the enzyme as determined by three different methods at 20 °C is v2 = 0.742 ml/g. From equilibrium sedimentation a molecular weight of 835000 + 32000 was calculated for the native enzyme. Amino acid analysis of the enzyme was performed and the number of half-cystine residues determined as 11⁄100000g is in good agreement with the number of titratable -SH groups after denaturation of the enzyme. The subunit molecular weight was determined by applying equilibrium sedimentation in the presence of sodium dodecylsulphate. Taking into account that 0.37 g of dodecylsulphate is bound per g of the enzyme protein as found by equilibrium dialysis, a subunit molecular weight of 104000 could be calculated. This value is about 15°/, smaller than that obtained by gel electrophoresis in the presence of sodium dodecylsulphate. Evidently, yeast phosphofructokinase seems to be octameric. Limited proteolysis in the presence of proteinase B from yeast was investigated by following the alterations of the sedimentation coefficient, the molecular weight, and the subunit pattern of the enzyme. During partial proteolytic degradation the molecular weight of the native enzyme decreases by about 230000. This diminution is in accordance with the difference in the molecular weight per subunit if an octameric composition is considered. [ABSTRACT FROM AUTHOR]

The cell envelope of Salmonella typhimurium SW 1061 contains five proteins which covalently bind benzyl-[14C]penicillin, numbered 1-5 in order of decreasing molecular weight. Proteins 1, 4 and 5 were extracted from membranes of the organism by treatment with Genapol X-100 in the presence of 1 M LiCl. This extract catalysed a DD-carboxypeptidase reaction, the synthesis of a cross-linked dimer from L-Ala-D-Glu-(ms-[1,7-14C]A2pm-D-Ala-D-Ala) in a natural model transpeptidation reaction, and the concomitant hydrolysis of this dimer, i.e. an endopeptidase reaction. Proteins 1, 4 and 5 have been separated by ion-exchange chromatography of the LiCl/Genapol X-100 extract on DEAE-Sepharose CL-6B, and protein 4 was found to possess the majority of the recovered DD-carboxypeptidase activity, and to be an efficient transpeptidase. However, this procedure yielded unstable preparations of protein 5 (the major penicillin-binding protein in the cell), which had very low enzymic activity but retained the ability to bind benzyl-[14C]penicillin. Stable, pure preparations of protein 5 were obtained by covalent affinity chromatography on ampicillin-affinose of material solubilized from membranes using Genapol X-100 in the absence of LiCl. Such preparations possessed DD-carboxypeptidase activity, and also catalysed the natural model transpeptidation reaction but with a lower efficiency than protein 4. Proteins 4 and 5 released benzylpenicillin with a concomitant recovery of catalytic activity, the half-times for this recovery being approximately 90 min and 5 min respectively at 37 °C. Protein 4 was found to have the highest affinity of the five membrane-bound penicillin-binding proteins for benzyl-[14C]penicillin. The complete separation of protein 1 from proteins 4 and 5 has not been achieved; however, our preliminary findings suggest that this protein also catalyses both the DD-carboxypeptidase and natural transpeptidase... [ABSTRACT FROM AUTHOR]

Using 14C-labeled Salmonella bacterial cells as the substrate, the enzymic and molecular properties of the base-plate parts of phage P22 were studied. The base-plate part consisted of a single protein species which cleaved extensively the O-antigen of Salmonella typhimurium, Salmonelly schottmuellerie and with somewhat slower rate that of Salmonella typhi, releasing oligosaccharide products with rhamnose at the reducing end. Much less cleavage was observed with a strain of S. typhimurium lysogenie for P22, and no significant reaction with Salmonella anatum. Salmonella newington and Salmonella minneapolis. The base-plate part enzyme was a very heat-stable protein and only 10–20% loss was observed after treatment at 85 °C for 5 min. The pH optimum of the enzyme was around 7.5. and the glycosidase activity was not influenced by the ionic strength (25–250 mM) of the medium or the presence of Mg2+. The molecular weight of the base-plate part was 320000 by sedimentation equilibrium. Dodecylsulphate-acrylamide gel electrophoresis revealed a single band of molecular weight 77000, indicating that a single base-plate part corresponds to a tetramer of identical subunits. Circular dichroism spectra of P22 base-plate parts showed a major contribution of β structure. The protein was rich in acidic amino acids, glycine and serine. [ABSTRACT FROM AUTHOR]

A procedure is described that from one batch of cells allows the isolation of all major proteins of the outer cell envelope membrane of Escherichia coli B/r. The method involves differential extraction of cell envelopes with ionic and non-ionic detergents with and without Mg2+ present, an the proteins are finally separated by molecular sieve chromatography in the presence of sodium dodecylsulfate. From 200 g cell paste in ten days (including the five days chromatography) ≈ 120 mg protein I (molecular weight ≈ 38000), ≈ 110 mg protein II* (molecular weight ≈ 33000), ≈ 50 mg protein III (molecular weight ≈ 17000), and ≈ 30 mg protein IV (molecular weight ≈ 7000) are obtained in pure state, and these yields are near the expected ones assuming quantitative recoveries. Protein II* is a heat-modifiable protein (perhaps due to complete unfolding and/or binding of sodium dodecylsulfate only at higher temperatures), and the isolated protein is completely in its unmodified form. Protein IV, Braun's lipoprotein, in the cell envelope exists in two forms, one covalently bound to the murein layer and the other not. The isolated protein IV represents the free form of the protein that so far had not been isolated; its protein part does not differ substantially from that of the bound form. [ABSTRACT FROM AUTHOR]

In the pH interval 10.5-11.8, 70% of the nonhistone proteins normally present in rat liver chromatin were dissociated. The rest remained complexed with DNA even at pH 13. Dodecylsulfate-polyacrylamide gel electrophoresis revealed that the majority of the high-molecular-weight nonhistone proteins together with a few characteristic fractions with molecular weights of 40 000 60 000 remained in the alkali-resistant group. L-[14C]Leucine pulse-labelling experiments showed that the specific radioactivity of the alkali-labile nonhistone proteins was 2-3 times after than that of the alkali-resistant nonhistone proteins, which, in turn, had the same specific radioactivity as that of the histones. The same held true for chromatin from regenerating rat liver. In the course of a 21-day chase of the specific radioactivity of the alkali-labile nonhistone proteins gradually decreased and finally became 3 times lower than that of the alkali-resistant nonhistone proteins. On the contrary, the ratio of the specific radioactivities of the alkali-resistant nonhistone proteins and of the histones to the specific radioactivity of DNA remained constant during the chase. A conclusion can be drawn that a fraction of liver nonhistone proteins exists which is alkali-resistant and is conserved in chromatin like histones. [ABSTRACT FROM AUTHOR]