Kinds Of Polypeptide Chains Research Articles

Quaternary structural analysis of nucleoside diphosphate kinases using capillary electrophoresis

Capillary electrophoresis was used to monitor the quaternary structure and structural changes by denaturants of nucleoside diphosphate kinase (NDP kinase). NDP kinase from human erythrocyte consists of two kinds of polypeptide chains: A (Nm23-H1) and B (Nm23-H2), which are the products of nm23-H1 and -H2 genes, respectively. Structural characteristics of native NDP kinase, recombinant Nm23-H1 and -H2 were examined using capillary electrophoresis employing high pH running buffer in an uncoated fused-silica capillary as a separation column. The results were compared with those of the conventional sodium dodecyl sulfate and non-denaturing polyacrylamide gel electrophoresis. It shows that capillary electrophoresis is a possible tool to investigate protein structure, including the quaternary structure. This protocol was applied to the investigation of the denaturation process of each enzyme by denaturants (6 M urea or heating to 70°C) and to the monitoring of the interaction between denatured Nm23-H1 and -H2. The structural information of NDP kinase obtained by analysis using capillary electrophoresis is valuable for understanding its suggested biological function as a tumor metastasis suppressor and c- myc transcription factor, etc.

Isolation and characterization of circulating complex between human pregnancy-associated plasma protein-A and proform of eosinophil major basic protein

The plasma protein previously known as pregnancy associated plasma protein-A (PAPP-A) and believed to contain only one kind of polypeptide chain has recently been shown to be a complex containing two different chains in equimolar amounts. One of the chains is now defined as the PAPP-A submit, and the other has been identified as the proform of eosinophil major basic protein (proMBP) (Oxvig et al. (1993) J. Biol. Chem. 268, 12243–12246). A procedure for large scale preparation of the circulating complex (PAPP-A/proMBP) from pooled pregnancy serum is described. The amino acid and carbohydrate compositions of the isolated reduced and carboxymethylated PAPP-A (199 kDa) and proMBP subunits (38 kDa), and of the intact PAPP-A/proMBP have been determined. The PAPP-A and proMBP subunits contain 13.4% (w/w) and 38.6% (w/w) carbohydrate, respectively, and the intact complex contains 17.4% (w/w) carbohydrate. The PAPP-A subunit contains N-bound carbohydrate groups. In contrast, the proMBP subunit contains both N- and O-bound groups as well as glycosaminoglycan, previously found among plasma proteins only in inter-α-trypsin inhibitor and pre-α-trypsin inhibitor. It is shown that PAPP-A/proMBP can competitively inhibit human leucocyte elastase ( K I = (5–10) · 10 −9 M) at an ionic strength of 0.075, but the inhibition is negligible at ionic strengths greater than 0.15. Human cathepsin G is also competitively inhibited ( K I approx. 1 · 10 −6 M). The inhibition of both enzymes is most likely due to interactions with the glycosaminoglycan moiety of PAPP-A/proMBP. It is concluded that PAPP-A/proMBP is neither a potent nor a specific inhibitor of human leucocyte elastase.

Nucleoside diphosphate kinase from human erythrocytes. Structural characterization of the two polypeptide chains responsible for heterogeneity of the hexameric enzyme

Human erythrocyte nucleoside-diphosphate kinase (NDP kinase) is a hexameric enzyme consisting of two kinds of polypeptide chains, A and B. By random association (A6, A5B...AB5, B6) these polypeptides form isoenzymes differing in their isoelectric point. Chains A and B of NDP kinase were purified by ion-exchange chromatography under denaturing conditions. Upon mixing and renaturation, the isozymic pattern of NDP kinase obtained by conventional methods was restored. Antibodies raised against purified chains showed significant cross-reactivity, both in immunoblot experiments and activity inhibition studies. Sequence determination showed that both chains consisted of 152 amino acid residues corresponding to Mr or 17,143 (chain A) and 17,294 (chain B), respectively. There was high homology between the two sequences (88% identity). The phosphorylation site on the enzyme is located at His-118. Chain A was identical with human Nm23 protein, which has been reported as a potential suppressor protein in tumor metastasis and chain B was identical with Nm23-H2 protein.

Proglobulin Processing Enzyme in Vacuoles Isolated from Developing Pumpkin Cotyledons

The enzymic conversion of proglobulin to globulin catalyzed by the extracts of vacuoles isolated from developing pumpkin (Cucurbita sp. cv Kurokawa Amakuri Nankin) cotyledons was investigated. The endoplasmic reticulum fraction isolated from the developing cotyledons pulselabeled with [(35)S]methionine was shown to contain mainly the radiolabeled proglobulin, which was used as a substrate for assaying the proteolytic processing in vitro. The vacuolar extracts catalyzed the proteolytic processing of the proglobulin molecule to produce globulin containing two kinds of polypeptide chains, gamma and delta. The pH optimum for the vacuole-mediated conversion was at pH 5.0. The proteolytic processing of proglobulin by the vacuolar extracts was inhibited in the presence of various thiol reagents, e.g. p-chloromercuribenzoate, N-ethylmaleimide, iodoacetic acid, Hg(2+), and Cu(2+), but not phenylmethylsulfonyl fluoride, EDTA, o-phenanthroline, leupeptin, antipain, pepstatin, chymostatin, or pumpkin trypsin inhibitor, and was activated in the presence of dithiothreitol and cysteine, indicating that the processing enzyme is a thiol protease. The suborganellar fractionation of the vacuoles showed that the processing activity was localized in the matrix fraction, but not in the membrane or crystalloid fractions. During the seed development, the enzyme was shown to increase, exhibiting the maximal activity at the late developmental stage. The matrix fraction of the protein bodies isolated from the dry castor bean (Ricinus communis) exhibited the processing activity toward the pumpkin proglobulin molecules in the same manner as that by the matrix fraction of pumpkin vacuoles.

Preliminary results for the primary structure of bacterioferritin of Escherichia coli.

Bacterioferritins are type-b cytochromes which resemble ferritin. Amino acid analysis combined with chemical modification and partial sequence analysis characterize bacterioferritin of Escherichia coli in terms of its primary structure. It is a protein composed of one kind of polypeptide chain that commences with methionine and terminates with glutamic acid. The length of the polypeptide chain is, tentatively, 146 residues. Besides the N-terminal methionine residue there are three more methionine residues, which yield four CNBr peptides, which have been aligned. The identity of the following positions in the sequence has been ascertained: residues 1-25, 30-37, 83-88, 127-132 and 143-146. No homology with ferritin was found.

Flow of information in the light-triggered cyclic nucleotide cascade of vision.

Photolyzed rhodopsin catalyzes the exchange of GTP for FDP bound to a protein in retinal rod outer segments. We previously proposed that the GTP complex of this protein regulates the cyclic GMP phosphodiesterase and that it may be the first amplified intermediate in visual excitation [Fung, B. K.-K. & Stryer, L. (1980) Proc. Natl. Acad. Sci. USA 77, 2500-2504]. We report here the identification and characterization of transducin, a regulatory protein consisting of three kinds of polypeptide chains: T alpha (39 kilodaltons), T beta (36 kilodaltons), and T gamma (approximately 10 kilodaltons). Reconstituted membranes containing transducin and rhodopsin but no phosphodiesterase exhibit GTPase activity and amplified binding of guanosine 5'[beta, gamma-imido]triphosphate (p[NH]ppG), a nonhydrolyzable analog of GTP, on illumination. A single photolyzed rhodopsin molecule led to the uptake of p[NH]ppG by 71 molecules of transducin. High-pressure liquid chromatography showed that the binding site for GTP is on the alpha subunit of transducin. The isolation of the complex of ;[NH]ppG with T alpha enabled us to determine whether this species is the activator of the phosphodiesterase. We found that phosphodiesterase on unilluminated disc membranes can indeed be fully activated by addition of T alpha containing bound p[NH]ppG. These findings strongly suggest that transducin is the first amplified information-carrying intermediate in the cyclic nucleotide cascade of vision.

Genes for the alpha and beta subunits of the phenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli.

The phenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli is a tetramer that contains two different kinds of polypeptide chains. To locate the genes for the two polypeptides, we analyzed temperature-sensitive mutants with defective phenylalanyl-transfer ribonucleic acid synthetases to see which subunit was altered. The method was in vitro complementation; mutant cell extracts were mixed with purified separated alpha or beta subunits of the wild-type enzyme to generate an active hybrid enzyme. With three mutants, enzyme activity appeared when alpha was added, but not when beta was added: these are, therefore, assumed to carry lesions in the gene for the alpha subunit. Two other mutants gave the opposite response and are presumably beta mutants. Enzyme activity is also generated when alpha and beta mutant extracts are mixed, but not when two alpha or two beta mutant extracts are mixed. The inactive mutant enzymes appear to be dissociated, as judged by their sedimentation in sucrose density gradients, but the dissociation may be only partial. The active enzyme generated by complementation occurred in two forms, one that resembled the native wild-type enzyme and one that sedimented more slowly. Both alpha and beta mutants are capable of generating the native form, although alpha mutants require prior urea denaturation of the defective enzyme. With the mutants thus characterized, the genes for the alpha and beta subunits (designated pheS and heT, respectively) were mapped. The gene order, as determined by transduction is aroD-pps-pheT-pheS. The pheS and pheT genes are close together and may be immediately adjacent.

Isolation, properties, and structure of human IgA myeloma globulins.

For the past few years our work has been based on the principle that the characteristic covalent structure of 99% of all human immunoglobulins could be established by determining the amino acid sequence of five kinds of polypeptide chains: κ and λ light chains, and γ, μ, and a heavy chains. We reported the first complete sequence of κ (1) and λ (2,3) light chains. Others sequenced the γ chain (4,5) and we recently reported the complete sequence of the μ chain (6). The a chain is the last of the big five, and many laboratories are now engaged in its study.

Amino acid compositions of all the tryptic peptides from the and polypeptide chains of adult hemoglobin of the slow loris (Nycticebus coucang). Biochemical studies on hemoglobins and myoglobins. X.

Hemoglobin was prepared from blood of adult animals of slow loris, and furthermore a and β polypeptide chains of the globin were separated by column chromatography. These two kinds of polypeptide chains were respectively digested with trypsin. The tryptic peptides thus obtained were isolated and purified by column chromatography and paper chromatography, and then each amino acid composition was analyzed.

Nuclear DHT-receptor in Tfm-Y kidney cell.

OUR previous studies on the X-linked testicular feminization (Tfm) mutation1 of the mouse2–4 showed that the so-called cytosol and nuclear 5αx-dihydrotestosterone (DHT) receptor protein5–7 might be a regulatory protein specified by the Tfm locus. The dual role of being a translational repressor in the cytoplasma and a mediator of hypertrophy in the nucleus was envisaged8. We found, however, another class of androgen-receptor in the polysome fraction of kidney proximal tubule cells which seems better qualified to be a translational regulator. Since a single gene locus specifies only one kind of polypeptide chain, we re-examined whether the cytosol and nuclear DHT-receptor protein underwent a true mutational change in Tfm/Y individuals.

Ontogeny of haemoglobin in the chicken

ABSTRACT The controversy as to whether haemoglobin embryology in the chicken involves qualitative or merely quantitative changes is settled by the observation of consistent electrophoretic differences between the haemoglobins of early embryos and the haemoglobins of foetuses, chicks and adult chickens. Studies on polypeptide chain composition indicate that the multiple haemoglobins of 3-to 5-day incubation embryos are A2C2, B2C2 and D2F2. The major and minor haemoglobins of foetuses and adult chickens are A2F2 and A2E2. Thus, although embryos and adults have no haemoglobins in common, they appear to have two kinds of polypeptide chains in common, A and F. Embryos have three unique polypeptide chain types and adults have one, on the basis of differences in electrophoretic mobility. Some degree of polypeptide chain interchange occurs with at least one of the chicken haemoglobins in that one of the adult haemoglobins will hybridize with human adult haemoglobin, α 2β 2. Although two of the three 3-to 5-day chick embryonic haemoglobin components have one of their polypeptide chain types in common with the major adult haemoglobin, the oxygen equilibria and Bohr effect of adult and early embryonic haemoglobin are very different. Chick embryo haemoglobin has an extremely high oxygen affinity and a Bohr effect only two-thirds that of the adult. The haemoglobin ‘switchover’ at the end of day 5 of incubation is not paralleled by changes in erythrocyte esterases, lactate dehydrogenase or malate dehydrogenase isoenzymes.

Comparisons of Bence-Jones proteins and L polypeptide chains of myeloma globulins after hydrolysis with trypsin.

L polypeptide chains of myeloma globulin and Bence-Jones protein isolated from the same patient were found to be identical after comparison of their tryptic hydrolysates by two-dimensional high voltage electrophoresis. The patterns of peptides from proteins belonging to antigenic group I differed markedly from those of proteins in antigenic group II. A partially purified H chain fraction was compared with L chains from the same myeloma protein. The tryptic hydrolysates yielded dissimilar patterns of peptides. These data indicate that gamma-myeloma proteins contain two kinds of polypeptide chains, Hgamma chains and either L(I) or L(II) chains. The L chains appear to be identical with those comprising the Bence-Jones protein from the same patient.

The structure of influenza viruses: II. C-terminal amino acid analyses

Reaction of influenza B virus (LEE strain) with carboxypeptidase resulted in the rapid and simultaneous release of leucine and tyrosine in the approximate molar ratio of 1.5:1, followed by smaller amounts of other, unidentified amino acids. This result was obtained only when the virus was denatured with formamide or cuprammonium sulphite; practically no amino acids were released from native virus under similar conditions of digestion. Leucine and tyrosine were released only from the fraction of cuprammonium sulphite-treated LEE virus previously shown to be lacking N-terminal aspartic acid. The protein which possessed an N-terminal aspartic acid residue was much less susceptible to the action of carboxypeptidase, and its C-terminal residue was not identified. Fractions obtained by electrophoresis of dodecyl sulphate-disrupted LEE virus were reacted with carboxypeptidase. Equimolar amounts of leucine and tyrosine were split off from the fastest fraction, but from the slowest, the ratio of leucine to tyrosine released was 7.5:1. These results suggest that particles of LEE virus contain at least three different kinds of polypeptide chains. Reaction with carboxypeptidase of the MEL, BEL, and PR8 strains of influenza A virus (denatured by cuprammonium sulphite), resulted in the slow and simultaneous release of about five amino acids from each strain. None of these could be unequivocally identified as C-terminal.

A Quantitative Study of the Hydrolysis of Human Dinitrophenyl(DNP)globin: The Number and Kind of Polypeptide Chains in Normal Adult Human Hemoglobin

A quantitative investigation of the partial hydrolysis of DNP-globin in refluxing 6 N hydrochloric acid has led to an explanation of our earlier conclusion that normal adult human hemoglobin contains a non-integral number, 3.6, of N-terminal valyl residues per molecule. It is now concluded that 4 E-terminal residues are present. Moreover, it has been found that the molecule contains two kinds of polypeptide chains, with respect to the K-termini. Under the above hydrolytic conditions the N-terminal valyl residues are released as DNP-Val-leu almost quantitatively from two chains (A chains) within 15 min. On continued hydrolysis the other two chains (B chains) release DNP-valine. No N-terminal peptides originating from the B chains have been definitely identified.