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J B Howard

Publications and source records attributed to J B Howard.

At least 55 records · Page 3Linked to original sources
PubMed1985

Reaction of methylamine with human alpha 2-macroglobulin. Mechanism of inactivation.

The human protease inhibitor alpha 2-macroglobulin (alpha 2 M) is inactivated by reaction with methylamine. The site of reaction is a protein functional group having the properties of a thiol ester. To ascertain the relationship between thiol ester cleavage and protein inactivation, the rates of methylamine incorporation and thiol release were measured. As expected for a concerted reaction of a nucleophile with a thiol ester, the rates were identical. Furthermore, both rates were first order with respect to methylamine and second order overall. The methylamine inactivation of alpha 2M was determined by measuring the loss of total protease-binding capacity. This rate was slower than the thiol ester cleavage and had a substantial initial lag. However, the inactivation followed the same time course as a conformational change in alpha 2M that was measured by fluorescent dye binding, ultraviolet difference spectroscopy, and limited proteolysis. Thus, the methylamine inactivation of alpha 2M is a sequential two-step process where thiol ester cleavage is followed by a protein conformational change. It is the latter that results in the loss of total protease-binding capacity. A second assay was used to monitor the effect of methylamine on alpha 2M. The assay measures the fraction of alpha 2M-bound protease (less than 50%) that is resistant to inactivation by 100 microM soybean trypsin inhibitor. In contrast to the total protease-binding capacity, this subclass disappeared with a rate coincident with methylamine cleavage of the thiol ester. alpha 2M-bound protease that is resistant to a high soybean trypsin inhibitor concentration may reflect the fraction of the protease randomly cross-linked to alpha 2M. Both the thiol ester cleavage and the protein conformational change rates were dependent on methylamine concentration. However, the thiol ester cleavage depended on methylamine acting as a nucleophile, while the conformational change was accelerated by the ionic strength of methylamine. Other salts and buffers that do not cleave the thiol ester increased the rate of the conformational change. A detailed kinetic analysis and model of the methylamine reaction with alpha 2M is presented. The methylamine reaction was exploited to study the mechanism of protease binding by alpha 2M. At low ionic strength, the protein conformational change was considerably slower than thiol ester cleavage by methylamine. Thus, at some time points, a substantial fraction of the alpha 2M had all four thiol esters cleaved, yet had not undergone the conformational change. This fraction (approximately 50%) retained full protease-binding capacity.(ABSTRACT TRUNCATED AT 400 WORDS)

Humans↗
PubMed1984

Reactions with the oxidized iron protein of Azotobacter vinelandii nitrogenase: formation of a 2Fe center.

The Fe-S center of oxidized Fe protein from Azotobacter vinelandii nitrogenase is decomposed by alpha,alpha'-dipyridyl in a biphasic process. In the presence of MgATP, 2 Fe are immediately removed by chelation while the additional irons are removed only after several hours. A slower biphasic Fe release also was observed in the presence of chelator alone. MgADP prevented the Fe release by chelator. An intermediate in the reaction was isolated containing 2 Fe. The visible spectrum of the intermediate was similar to that of 2Fe-2S ferredoxins (epsilon max at 325, 416, and 460 nm of 16.1, 11.3, and 9.0 mM-1 cm-1). The 2Fe form was electron paramagnetic resonance (EPR) silent until partially reduced with sodium dithionite. The EPR spectral properties were similar to 2Fe-2S ferredoxins; namely, the Fe center had resonances at g = 2.00, 1.94, and 1.92 which were detectable, essentially unbroadened at 70 K. The results suggest that in the oxidized (2+) state Fe protein can undergo a 4Fe to 2Fe conversion.

2,2'-Dipyridyl↗
PubMed1983

Thiol reactivity of the nitrogenase Fe-protein from Azotobacter vinelandii.

A procedure has been developed to examine some of the functional roles of the 14 cysteinyl residues in the nitrogenase Fe-protein (Av2) from Azotobacter vinelandii. The reduced form of Av2 was alkylated with iodo[2-14C]acetic acid under a variety of experimental conditions, e.g. reaction in the presence of nucleotides, alpha,alpha'-dipyridyl and nucleotides, or denaturants. The labeled cysteinyl residues were identified and quantified using an analytical DEAE-Sepharose ion exchange chromatography peptide mapping technique based upon the known amino acid sequence (Hausinger, R. P., and Howard, J. B. (1982) J. Biol. Chem. 257, 2483-2490). From the results of the labeling experiments, the following features of the Av2 structure have been proposed. 1) Av2 contains no disulfides, hyperreactive thiols, or surface thiols as defined by reaction with iodoacetic acid. 2) Cysteines 97 and 132 are the probable ligands for the Av2 Fe:S center which is bound symmetrically between subunits. 3) MgATP partially protects cysteine 85 from carboxymethylation by iodoacetic acid and may be part of the nucleotide-binding site. 4) Of the five nonligand thiols only cysteines 5 and 184 are completely alkylated when Av2 is denatured in hexamethylphosphoramide, whereas all five nonligand thiols appear to rapidly exchange at the Fe:S center if the protein is denatured in the absence of alkylating reagents. 5) Both Av2 and apo-Av2 appear to undergo a reversible conformational change upon binding MgATP.

Azotobacter↗
PubMed1983

Crystallization of the Azotobacter vinelandii nitrogenase iron protein.

The iron protein from Azotobacter vinelandii nitrogenase has been crystallized in the reduced form. The needle-shaped crystals are in space group P2(1)2(1)2 (a = 94.6 A, b = 179.9 A, c = 74.1 A) and diffract to at least 3.5-A resolution. Five or six Fe-protein monomers are present in the asymmetric unit.

Azotobacter↗
PubMed1983

Structure of Azotobacter vinelandii 7Fe ferredoxin. Amino acid sequence and electron density maps of residues.

The complete amino acid sequence of the 7Fe ferredoxin from Azotobacter vinelandii (Av Fd) was determined by repetitive Edman degradation of the whole protein and of peptides derived from CNBr cleavage or chymotrypsin digestion. The sequence was confirmed by the 2A electron density maps for the residues calculated with difference Fourier coefficients. The density maps for all residues are included in the paper. Av Fd has several important differences with the clostridial-type ferredoxins: (i) Av Fd is 106 residues (versus 55-60 for other bacterial ferredoxins); (ii) Av Fd has 9 cysteines, one of which (residue 24) is not homologous with the bacterial ferredoxins; (iii) Av Fd has 2 extra residues between 2 cysteines (residues 11 and 16) homologous to cysteines in the bacterial ferredoxins; and (iv) Av Fd has the unique sequence -Cys-Val-Glu-Val-Cys- (residues 16-20) which are two of the ligands of the 3Fe:3S center. These sequence features are compared to the sequences of various ferredoxin groups. Structure predictions for other suspected 7Fe ferredoxins are discussed.

Amino Acid Sequence↗
PubMed1983

The methylamine reactive site and protease inhibition in alpha 2-macroglobulin.

The inactivation of alpha 2M by the nucleophiles, NH4+, hydrazine, and methylamine, follows pseudo-first-order (second order, overall) rates. The rate of incorporation of nucleophiles however, is biphasic with the faster rate consistent with inactivation. Protease cleavage of alpha 2M is prevented by complete inactivation of alpha 2M by methylamine. Loss of protease cleavage is slower than methylamine incorporation but parallels inactivation. Our results are consistent with a complex model of sequential conformation changes leading to binding and inactivation of proteases by alpha 2M. Our results suggest that cross-linking of the protease to alpha 2M by aminolysis of the thiolester is not required for inactivation of the protease.

Humans↗
PubMed1982

Amino acid sequence of a 3Fe:3S ferredoxin from the "archaebacterium" Methanosarcina barkeri (DSM 800).

The complete amino acid sequence for a 3Fe:3S ferredoxin from the "archaebacterium" Methanosarcina barkeri (DSM 800) was determined by repetitive Edman degradation on the whole protein and peptides derived from trypsin, thermolysin, and Staphylococcus aureus protease digestion. The protein has 59 residues of which 8 are cysteines. The latter have the same spacing and distribution as found for the clostridial-type 2 x 4Fe:4S ferredoxins. Also, the sequence had evidence of internal homology which is indicative of gene duplication prior to the divergence of the archaebacteria and the eubacteria. This is the first sequence to be reported for a methanogen ferredoxin and only the fourth for a 3Fe:3S ferredoxin from any source.

Amino Acid Sequence↗
PubMed1981

Isolation and sequences of the cysteinyl tryptic peptides from the MoFe-protein of Azotobacter vinelandii nitrogenase.

The cysteinyl residues in the alpha and beta subunits of the MoFe-protein from Azotobacter vinelandii nitrogenase were radiolabeled by carboxymethylation with iodo[14C]acetic acid. The tryptic peptides from the isolated subunits were separated by ion-exchange chromatography on DEAE-Sephadex and SP-Sephadex. The radiolabeled (cysteinyl) peptides were sequenced by Edman degradation. The isolation procedure and sequences of the peptides provide a method for the identification of the potential cysteinyl ligands for the protein Fe:S and Mo:Fe:S centers. Although the cysteinyl peptides account for approximately 20% of the total sequence, little sequence homology was observed between the peptides from the two subunits. In contrast to the ferredoxins, the cysteinyl residues are not grouped within the protein. Only one peptide from the beta subunit has sequence homology with other Fe:S proteins. This peptide has 15 of 22 residues identical or conservative with residues 11 to 32 of the bacterial ferredoxins. The conservation of this region suggests that it may contain an important secondary structure unique to 4Fe:4S proteins, namely, the sequence (formula, see text) where (formula, see text) represents a variable residue. One peptide from the alpha subunit has a sequence identical with the conserved residues 7 to 11 of the bacterial ferredoxins. This alpha-peptide has the correct sequence, -Glu-Pro-Val-Ser-Cys-Val-Ser-Asp-Ser-, for the glutamyl, cysteinyl, and aspartyl residues to ligand a single 4Fe:4S cluster.

Amino Acid Sequence↗
PubMed1981

Reactive site in human alpha 2-macroglobulin: circumstantial evidence for a thiolester.

The reaction of methylamine with alpha 2-macroglobulin (alpha 2M) results in the covalent modification of one glutamic residue per subunit as gamma-glutamylmethylamide [Swenson, R. & Howard, J. B. (1979) Proc. Natl. Acad. Sci. USA 76, 4313--4316]. Furthermore, alpha 2M can undergo specific peptide autolysis involving the same reactive glutamic residue [Howard, J. B., Vermeulen, M. & Swenson, R. (1980) J. Biol Chem. 255, 3820--3823]. During both reactions, a cysteinyl thiol is exposed and can be alkylated by iodoacetic acid. After alpha 2M was modified with [14C]methylamine and iodo[2-3H]acetic acid, a tryptic peptide was isolated that contained both labels in the same ratio as in the original protein. From the chymotryptic digest of the tryptic peptide, a single radiolabeled peptide was isolated. The amino acid sequence of the chymotryptic peptide was the same as that previously reported to include gamma-glutamylmethylamide. This is circumstantial evidence for a thiolester between the cysteine and a glutamic acid located three residues away in the primary sequence. A reaction mechanism involving a pyroglutamyl intermediate derived from the thiolester is suggested to explain the autolysis. Kinetic analysis of the autolysis reaction is consistent with this intermediate and mechanism.

Cysteine↗
PubMed1980

ADP-ribosylation of elongation factor 2 by diphtheria toxin. Isolation and properties of the novel ribosyl-amino acid and its hydrolysis products.

We have isolated the novel amino acid, amino acid X, in elongation factor 2 (EF-2) which is ADP-ribosylated by diphtheria toxin (Robinson, E. A., Hendriksen, O., and Maxwell, E. S. (1974) J. Biol. Chem. 249, 5088-5093). The pure ribosyl-amino acid was obtained in milligram quantities following sequential enzymatic degradation of yeast ADP-ribosyl-EF-2 (Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1978) J. Biol. Chem. 253, 8687-8690) with an overall yield of 30 to 60%. In the course of acid hydrolysis, the ribosyl-amino acid was converted first to an intermediate compound which did not contain ribose. Further acid hydrolysis converted the intermediate to amino acid X and ammonia. Alkaline hydrolysis of the ribosyl-amino acid produced a different intermediate which contained ribose. We have isolated the two hydrolysis intermediates and on the basis of the properties of all four compounds we conclude that the ribosyl-amino acid contains an amido function which is not involved in a glycosidic linkage and a net positive charge of 2 at acid pH. We believe that the amino acid amide occurs in EF-2 and for this compound we propose the trivial name diphthamide. Acid hydrolysis of diphthamide yields amino acid X for which we propose the trivial name diphthine.

Adenosine Diphosphate Ribose↗
PubMed1980

ADP-ribosylation of elongation factor 2 by diphtheria toxin. NMR spectra and proposed structures of ribosyl-diphthamide and its hydrolysis products.

NMR spectral analysis of the novel amino acid, diphthamide, in elongation factor 2 which is ADP-ribosylated by diphtheria toxin suggests that it is 2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine. Ribosyl-diphthamide was prepared by enzymatic hydrolysis of ADP-ribosyl-elongation factor 2 and three compounds were produced by its chemical hydrolysis (Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) J. Biol. Chem. 255, 10717-10720). Proton NMR spectroscopy in 2H2O of diphthine demonstrated the elements of histidine minus the carbon 2 proton plus 14 additional nonexchangeable protons. These protons were attributed to an extensive modification at carbon 2 of the imidazole ring. Proton NMR spectroscopy in 2H2O of ribosyl-diphthamide showed only those protons seen in diphthine plus those expected of a ribofuranosylmoiety. Chemical shift dependence on pH was demonstrated for the histidine-derived protons as well as several protons of the modifying side chain. Natural abundance carbon 13 NMR spectroscopy of ribosyldiphthamide also showed the elements of histidine and ribose plus 7 additional carbon atoms attributed to the modification of the imidazole ring. Based on the NMR spectral properties of the anomeric proton of ribosyldiphthamide we propose that the ribose is linked to one of the nitrogens of the histidine imidazole ring.

Adenosine Diphosphate Ribose↗
PubMed1980

Amino acid sequence of the tryptic peptide containing the alkylamine-reactive site from human alpha 2-macroglobulin. Identification of gamma-glutamylmethylamide.

Human alpha 2-macroglobulin (alpha 2M) is inhibited by covalent reaction with alkylamines. The site of methylamine incorporation has been proposed to be an activated glutamyl residue (Swenson, R. P., and Howard, J. B. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4313-4316). A large, 56-amino acid residue glycopeptide derived from tryptic cleavage of [14C]methylamine-labeled alpha 2M was isolated. Based upon recovery of the specific radioactivity in the peptide, there appears to be only a single site of incorporation per Mr = 185,000 subunit. The complete amino acid sequence was deduced from Edman degradation and carboxypeptidase Y digestion of the tryptic peptide and of several small peptides derived from it. The structure of the radiolabeled amino acid was determined to be gamma-glutamylmethylamide by mass spectral analysis of the phenylthiohydantoin and N-benzoyl-O-methylester derivatives. The putative structure was confirmed by a comparison of the mass spectral and chromatographic properties of the authentic compound and the protein-derived amino acid residue. The 10 amino acid residues following the methylamine-reactive glutamyl residue were identical with the first 10 amino acid residues of the pyroglutaminase-deblocked, Mr = 65,000 fragment generated by heat denaturation of alpha 2M (Howard, J. B., Vermeulen, M., and Swenson, R. P. (1980) J. Biol. Chem. 255, 3820-3823).

Amino Acid Sequence↗
PubMed1980

Methylamine reaction and denaturation-dependent fragmentation of complement component 3. Comparison with alpha2-macroglobulin.

Complement protein C-3 can covalently incoporate [14C]methylamine with a stoichiometry of 0.85 +/- 0.11 mol/mol of protein. The reactive site is located in the larger, Mr = 135,000 peptide subunit of C-3. The methylamine is incorporated as a derivative of glutamic acid, viz. as gamma-glutamylmethylamide, which was identified by high performance liquid chromatography and low resolution mass spectroscopy. C-3 was shown to undergo a specific, denaturation-dependent protein fragmentation in sodium dodecyl sulfate at 90 degrees C. The cleavage results in the partial conversion of the Mr = 135,000 subunit to fragments of Mr = 84,000 and 53,000. The cleavage is completely prevented by reaction of C-3 with methylamine prior to the 90 degrees C incubation. The site of methylamine incorporation (the glutamyl residue) and the peptide fragmentation reaction have been reported for alpha2-macroglobulin (Howard, J.B., Vermeulen, M., and Swenson, R. (1980) J. Biol. Chem. 255, 3820-3823). A comparison of the results for the two proteins suggests that they have a common reactive site.

Binding Sites↗