A mutant form of ornithine transcarbamylase found in a strain of Neurospora carrying a pyrimidine-proline suppressor gene

doi:10.1016/0003-9861(62)90063-2

Archives of Biochemistry and Biophysics

Volume 97, Issue 1, April 1962, Pages 185-191

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Abstract

A mutation, s, of Neurospora, leads to a 98% reduction in ornithine transcarbamylase (OTC) activity without imposing an arginine requirement. It has been found that the enzyme present in the s strain is different from the normal enzyme in its affinities for ornithine and carbamyl phosphate. The affinity of the mutant enzyme for carbamyl phosphate is much greater than in the case of the wild type enzyme, and this characteristic could compensate to a large extent for the low specific activity in s strains. The data are used to indicate that the s locus is a gene which controls the structure of OTC in Neurospora. The possibility of there being two forms of this enzyme in wild type strains is discussed.

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Cited by (28)

Isolation and characterization of strains defective in vacuolar ornithine permease in Neurospora crassa
1998, Fungal Genetics and Biology
Vacuolar uptake of ornithine and lysine was characterized inNeurospora crassausing a cupric ion permeabilization system. Michaelis constants were measured as 1.4 mM for lysine and 11.0 mM for ornithine, and maximal velocities were determined. Vacuolar lysine uptake was shown to be inhibited competitively byl-arginine and histidine while ornithine uptake was inhibited by a variety of amino acids. Strains defective in the vacuolar ornithine permease were isolated using a filtration enrichment method. Two isolates—RSC-39 and RSC-63—had a reduced ability to accumulate ornithine. Vacuolar uptake of amino acids was measured using cupric ion-permeabilized mycelia; both strains had reduced ornithine uptake while lysine uptake and arginine uptake were normal. For both isolates, both the Michaelis constant and the maximal velocity for ornithine uptake were reduced compared to those of wild type. These results suggest that both strains are defective in the gene which encodes the vacuolar ornithine permease.
Characterization of vacuolar arginine uptake and amino acid efflux in Neurospora crassa using cupric ion to permeabilize the plasma membrane
1997, Fungal Genetics and Biology
Treatment ofNeurospora crassamycelia with cupric ion has been shown to permeabilize the plasma and mitochondrial membranes. Permeabilized mycelia were shown to take up arginine into the vacuoles. Uptake was ATP-independent and appeared to be driven by an existing K⁺-gradient. The kinetic characteristics of the observed uptake were similar to those observed using vacuolar membrane vesicles: theK_mfor arginine uptake was found to be 4.2–4.5 mM. Permeabilized mycelia were used to study the regulation of arginine uptake into vacuoles. The results suggest that uptake is relatively indifferent to the contents of the vacuoles and is not affected by growth of mycelia in amino acid-supplemented medium. Efflux of arginine, lysine, and ornithine from vacuoles was also measured using mycelia permeabilized with cupric ion. Arginine release was shown to be specifically enhanced by cytosolic ornithine and/or increases in the vacuolar pool of arginine or ornithine. Lysine efflux was shown be indifferent to the presence of other amino acids. These observations emphasize the importance of vacuolar compartmentation in controlling arginine and ornithine metabolism and suggest that vacuolar compartmentation may play an important role in nitrogen homeostasis of filamentous fungi.
Ornithine transcarbamylase from Neurospora crassa: Purification and properties
1985, Archives of Biochemistry and Biophysics
Ornithine transcarbamylase catalyzes the synthesis of citrulline from carbamyl phosphate and ornithine. This enzyme is involved in the biosynthesis of arginine in many organisms and participates in the urea cycle of mammals. The biosynthetic ornithine transcarbamylase has been purified from the filamentous fungus, Neurospora crassa. It was found to be a homotrimer with an apparent subunit molecular weight of 37,000 and a native molecular weight of about 110,000. Its catalytic activity has a pH optimum of 9.5 and K_m's of about 5 and 2.5 mm for the substrates, ornithine and carbamyl phosphate, respectively, at pH 9.5. The K_m's and pH optimum are much higher than those of previously characterized enzymes from bacteria, other fungi, and mammals. These unusual kinetic properties may be of significance with regard to the regulation of ornithine transcarbamylase in this organism, especially in the avoidance of a futile ornithine cycle. Polyclonal antibodies were raised against the purified enzyme. These antibodies and antibody raised against purified rat liver ornithine transcarbamylase were used to examine the structural similarities of the enzyme from a number of organisms. Cross-reactivity was observed only for mitochondrial ornithine transcarbamylases of related organisms.
Cloning and characterization of the ornithine carbamoyltransferase gene from Aspergillus nidulans
1983, Gene
An Aspergillus nidulans DNA fragment composed of two adjacent SalI subfragments (1.8 and 0.85 kb) that carries an argB complementing the yeast arg3 mutation has been isolated from two different gene libraries. Hybridization results and immunological tests indicate that the cloned fragment contains the A. nidulans structural gene coding for ornithine carbamoyltransferase (OTCase). Using the cloned gene as a probe, the specific mRNA was identified. The level of this RNA observed in A. nidulans strains grown under various conditions correlated with the level of the OTCase activity, suggesting transcriptional control of OTCase synthesis. Expression of the cloned gene in Saccharomyces cerevisiae does not depend on its orientation in the vector. In Escherichia coli, the cloned gene does not function; however arg- transformants revert to phototrophy with high frequency possibly due to DNA rearrangements within the recombinant plasmid.
Methods for mycelial breakage and isolation of mitochondria and vacuoles of neurospora
1983, Analytical Biochemistry
A new and inexpensive glass-bead blender allows rapid, easy, and controlled cell breakage of Neurospora, with good organellar survival. The yield of mitochondria and vacuoles is comparable to or better than methods involving sand grinding or snail-gut digestion of cell walls. A method for removing cell wall fragments from a crude homogenate is described. Isolation of mitochondria and vacuoles from the crude homogenate with little cross-contamination is accomplished by density-gradient centrifugation in a fixed-angle rotor (Sorvall).
Enzyme assays using permeabilized cells of Neurospora
1979, Analytical Biochemistry
A procedure is described for the preparation of homogeneous, steady-state cultures of germinated conidia of Neurospora crassa. Permeabilization of such cells has been accomplished by a combination of toluene-ethanol and freeze-thaw treatments. These permeabilized cells have been used for the determination of enzyme activities. The method has been shown to be rapid and rellable and is applicable to nuclear, mitochondrial, and cytosolic enzymes.

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^☆: Supported in part by an Institutional Research Grant to the University of Michigan from the American Cancer Society and by a grant (G18012) from the National Science Foundation.

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A mutant form of ornithine transcarbamylase found in a strain of Neurospora carrying a pyrimidine-proline suppressor gene☆

Abstract

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

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