Biomedical subjects

M S Madden

Publications and source records attributed to M S Madden.

7 recordsLinked to original sources

PubMed1995

Reverse isoelectric focusing procedure resolves charge variants of basic proteins.

Several powerful methods exist which effectively resolve proteins based on their differences in overall change. Among these, two-dimensional gel electrophoresis, a method combining isoelectric focusing with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, can resolve over 1000 proteins with isoelectric points in the range of pH 4-7. However, resolution of basic proteins is poor, even when the pH gradient is extended to a higher pH. Nonequilibrium pH gradient electrophoresis was developed as an alternative procedure to resolve basic proteins. However, samples must be dissolved in urea prior to analysis and this method requires 2000-8000 V-h for optimal resolution. In this report we demonstrate that a simple, rapid procedure, based on nonequilibrium pH gradient electrophoresis, can resolve the single charge variants of two fungal enzymes with isoelectric points greater than 8. This procedure utilizes ready-made materials and requires less than 500 V-h to complete (total run time is less than 2 h).

Aspergillus↗

PubMed1995

Fusarium graminearum A 3/5 as a novel host for heterologous protein production.

We describe a novel fungal expression system which utilizes the Quorn myco-protein fungus Fusarium graminearum A 3/5. A transformation system was developed for F. graminearum and was used to introduce the coding and regulatory regions of a trypsin gene from Fusarium oxysporum. The protein was efficiently expressed, processed and secreted by the recombinant host strain. In addition, the promoter and terminator of the F. oxysporum trypsin gene have been successfully utilized to drive the expression of a cellulase gene from Scytalidium thermophilum and a lipase gene from Thermomyces lanuginosus in F. graminearum.

Base Sequence↗

PubMed1994

Biosynthesis of the iron-molybdenum cofactor of nitrogenase.

The iron-molybdenum cofactor (FeMo-co) of nitrogenase is a unique molybdenum-containing prosthetic group that has been proposed to form an integral part of the active site of dinitrogenase. In Klebsiella pneumoniae, at least six nif (nitrogen fixation) gene products are required for the biosynthesis of FeMo-co, including NIFB, NIFNE, NIFH, NIFQ, and NIFV. An in vitro system for the synthesis of FeMo-co, which requires MgATP, molybdate, homocitrate, and at least the products of nifN, E, B, and H, has provided an enzymatic assay for the purification of many of the gene products required for FeMo-co biosynthesis. Although the structure of the cofactor has been solved recently, much about the biosynthetic pathway remains unknown. This article reviews what is known about the various components required for FeMo-co biosynthesis.

Genes, Bacterial↗

PubMed1992

Plausible structure of the iron-molybdenum cofactor of nitrogenase.

A plausible structure of the iron-molybdenum cofactor of nitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1] is presented based on altered substrate reduction properties of dinitrogenase containing homocitrate analogs within the cofactor. Alterations on each carbon of the four-carbon homocitrate backbone were correlated with altered substrate reduction properties of dinitrogenase containing these analogs. Altered substrate reduction properties are the basis for a model in which homocitrate is oriented about two cubane metal clusters.

Formates↗

PubMed1991

Effects of homocitrate, homocitrate lactone, and fluorohomocitrate on nitrogenase in NifV- mutants of Azotobacter vinelandii.

Azotobacter vinelandii DJ71, which contains a mutation in the nifV gene, was derepressed for nitrogenase in the presence of homocitrate. When dinitrogenase was isolated from this culture, it was found to be identical to the wild-type dinitrogenase. However, when the same NifV- strain was derepressed in the presence of erythrofluorohomocitrate, a homocitrate analog which produces a nitrogenase with wild-type properties in vitro, the isolated dinitrogenase was characteristic of the NifV- enzyme. These data show that homocitrate, but not fluorohomocitrate, is utilized by NifV- mutant cells. Fluorohomocitrate does not inhibit the uptake of homocitrate because the wild-type phenotype resulted when both compounds were added to the medium during nitrogenase derepression. Homocitrate lactone failed to cure the NifV- phenotype.

Azotobacter↗

PubMed1990

Diastereomer-dependent substrate reduction properties of a dinitrogenase containing 1-fluorohomocitrate in the iron-molybdenum cofactor.

In vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase using homocitrate and its analogs allows the formation of modified forms of FeMo-co that show altered substrate specificities (N2, acetylene, cyanide, or proton reduction) of nitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1]. The (1R,2S)-threo- and (1S,2S)-erythro-fluorinated diastereomers of homocitrate have been incorporated in vitro into dinitrogenase in place of homocitrate. Dinitrogenase activated with FeMo-co synthesized using threo-fluorohomocitrate reduces protons, cyanide, and acetylene but cannot reduce N2. In addition, proton reduction is inhibited by carbon monoxide (CO), a characteristic of dinitrogenase from NifV- mutants. Dinitrogenase activated with FeMo-co synthesized using erythro-fluorohomocitrate reduces protons, cyanide, acetylene, and N2. In this case proton reduction is not inhibited by CO, a characteristic of the wild-type enzyme. Cyanide reduction properties of dinitrogenase activated with FeMo-co containing either fluorohomocitrate diastereomer are similar, and CO strongly inhibits cyanide reduction. Dinitrogenases activated with FeMo-co containing homocitrate analogs with a hydroxyl group on the C-1 position are much less susceptible to CO inhibition of cyanide reduction. However, proton and cyanide reduction by dinitrogenase containing FeMo-co activated with (1R,2S) threo-isocitrate is only one-third that of dinitrogenase activated with the racemic mixture of -isocitrate and shows strong CO inhibition of substrate reduction. These results suggest that CO inhibition of proton and cyanide reduction occurs when the hydroxyl group on the C-1 position of analogs is "trans" to the C-2 carboxyl group (i.e., in the threo conformation). When racemic mixtures of these analogs are used in the system, it seems that the erythro form is preferentially incorporated into dinitrogenase. Finally, carbonyl sulfide inhibition of substrate reduction by dinitrogenase is dependent on the homocitrate analog incorporated into FeMo-co.

Carbon Monoxide↗

PubMed1989

Substrate reduction properties of dinitrogenase activated in vitro are dependent upon the presence of homocitrate or its analogues during iron-molybdenum cofactor synthesis.

(R)-2-Hydroxy-1,2,4-butanetricarboxylic acid [(R)-homocitrate] has been has been recently reported to be an integral constituent of the otherwise thought to be inorganic iron-molybdenum cofactor of dinitrogenase [Hoover, T.R., Imperial, J., Ludden, P.W., & Shah, V.K. (1989) Biochemistry 28,2768-2771]. Different organic acids can substitute for homocitrate in an in vitro system for iron-molybdenum cofactor synthesis and incorporation into dinitrogenase [Hoover, T.R., Imperial, J., Ludden, P.W., & Shah, V. K. (1988) Biochemistry 27, 3647-3652]. Dinitrogenase activated with homocitrate-FeMo-co was able to reduce dinitrogen, acetylene, and protons efficiently. Homoisocitrate and isocitrate dinitrogenases did not reduce dinitrogen or acetylene, but showed very high proton reduction activities. Citrate and citramalate dinitrogenases had very low dinitrogen reduction activities and intermediate acetylene and proton reduction activities. CO inhibited proton reduction in both these cases but not in the case of dinitrogenases activated with other homocitrate analogues. By use of these and other commercially available homocitrate analogues in the in vitro system, the structural features of the homocitrate molecule absolutely required for the synthesis of a catalytically competent iron-molybdenum cofactor were determined to be the hydroxyl group, the 1- and 2-carboxyl groups, and the R configuration of the chiral center. The stringency of the structural requirements was dependent on the nitrogenase substrate used for the assay, with dinitrogen having the most stringent requirements followed by acetylene and protons.

Bacteria↗