Biomedical subjects

R L Weiss

Publications and source records attributed to R L Weiss.

At least 73 records · Page 4Linked to original sources

PubMed1986

N-acetyl-L-glutamate synthase of Neurospora crassa. Characteristics, localization, regulation, and genetic control.

N-Acetylglutamate synthase, an early enzyme of the arginine pathway, provides acetylglutamate for ornithine synthesis in the so-called "acetylglutamate cycle." Because acetylglutamate is regenerated as ornithine is formed, the enzyme has only a catalytic or anaplerotic role in the pathway, maintaining "bound" acetyl groups during growth. We have detected this enzyme in crude extracts of Neurospora crassa and have localized it to the mitochondria along with other ornithine biosynthetic enzymes. The enzyme is bound to the mitochondrial membrane. The enzyme has a pH optimum of 9.0 and Km values for glutamate and CoASAc of 6.3 and 1.6 mM, respectively. It is feedback-inhibited by L-arginine (I0.5 = 0.16 mM), and its specific activity is augmented 2-3-fold by arginine starvation of the mycelium. Mutants of the newly recognized arg-14 locus lack activity for the enzyme. Because these mutants are complete auxotrophs, we conclude that N-acetylglutamate synthase is an indispensible enzyme of arginine biosynthesis in N. crassa. This work completes the assignment of enzymes of the arginine pathway of N. crassa to corresponding genetic loci. The membrane localization of the enzyme suggests a novel mechanism by which feedback inhibition might occur across a semipermeable membrane.

Acetyltransferases↗

PubMed1986

Simultaneous purification of three mitochondrial enzymes. Acetylglutamate kinase, acetylglutamyl-phosphate reductase and carbamoyl-phosphate synthetase from Neurospora crassa.

The early enzymes of arginine biosynthesis in Neurospora crassa are localized in the mitochondrion and catalyze the conversion of glutamate to citrulline. The final conversion of citrulline to arginine occurs via two enzymatic steps in the cytoplasm. We have devised a method for the isolation and purification of three of the mitochondrial arginine biosynthetic enzymes from a single extract. Acetylglutamate kinase and acetylglutamyl-phosphate reductase (both products of the complex arg-6 locus) were purified to homogeneity and near homogeneity, respectively. The large catalytic subunit of carbamoyl-phosphate synthetase was also purified to homogeneity. The three enzymes were resolved into separate fractions by chromatography on three dye-ligand affinity resins, which are specific for nucleotide binding enzymes and have a high protein binding capacity. High performance liquid chromatography was employed in the final stages of purification and was extremely effective in fractionating both acetylglutamate kinase and acetylglutamyl-phosphate reductase from proteins with very similar properties, which were not removed by other techniques. The purified proteins were used to raise specific antisera against these proteins. Acetylglutamate kinase and acetylglutamyl-phosphate reductase were shown to be immunologically unrelated. This finding suggests that the arg-6 locus encompasses two nonoverlapping cistrons. The antisera raised against carbamoyl-phosphate synthetase has been shown to cross-react with related enzymes in Saccharomyces cerevisiae, Escherichia coli, and rat liver (Ness, S. A., and Weiss, R. L. (1985) J. Biol. Chem. 260, 14355-14362). Acetylglutamate kinase is a regulatory enzyme and has been shown to be feedback-inhibited by arginine. We have determined the submitochondrial localization of acetylglutamate kinase and the second arg-6 product, acetylglutamyl-phosphate reductase. Both enzymes were shown to be soluble matrix enzymes. We discuss the relevance of this finding with respect to possible mechanisms for end product inhibition of a mitochondrial enzyme by a cytoplasmic effector.

Aldehyde Oxidoreductases↗

PubMed1986

Effect of chloramphenicol and ethidium bromide on the level of ornithine carbamoyltransferase in Neurospora crassa.

The specific activity of the nuclear-gene-encoded, mitochondrial arginine biosynthetic enzyme ornithine carbamoyltransferase (EC 2.1.3.3) in Neurospora crassa was elevated in mycelia treated with chloramphenicol or ethidium bromide. The increase in specific activity was caused by an increase in the number of mature enzyme molecules rather than by the activation of a preexisting enzyme. Chloramphenicol and ethidium bromide appeared to act indirectly via arginine-mediated derepression. However, derepression did not appear to result from a drug-mediated decrease in the arginine pool.

Arginine↗

PubMed1986

Scheuermann's dorsal kyphosis and spinal cord compression: case report.

Although Scheuermann's disease (juvenile dorsal kyphosis) is a common problem of late childhood and adolescence, its potential for neurological complications is not widely appreciated. In rare instances, spinal cord compression appears to be produced by the kyphotic protrusion alone, and we present an example of this unusual problem. Although the results of surgical treatment in this situation cannot be substantiated, anterior spondylotomy and decompression followed by posterior fixation appear to offer the best mechanical relief. Spinal cord compression can also be produced by extradural cysts, with which Scheuermann's disease is frequently associated. Scheuermann's disease also is reported to occur in combination with thoracic disc protrusion, but the coincidence here may be random. Pertinent literature is reviewed.

Adolescent↗

PubMed1985

Carbamoyl-phosphate synthetases from Neurospora crassa. Immunological relatedness of the enzymes from Neurospora, bacteria, yeast, and mammals.

Neurospora crassa contains two carbamoyl-phosphate synthetases: a mitochondrial enzyme (CPS-A) which supplies carbamoyl phosphate for arginine biosynthesis, and a nuclear enzyme whose product is used for the synthesis of pyrimidines. We have prepared antiserum against a highly purified preparation of the large subunit of CPS-A and have used the antiserum to demonstrate that the large subunit is, like most mitochondrially localized proteins, initially synthesized as a higher molecular weight precursor. The CPS-A antiserum cross-reacts with the nuclear enzyme, allowing us to identify the product of the complex N. crassa pyr-3 genetic locus as a protein with a subunit molecular weight of 180,000. Finally, we have found that the CPS-A antiserum also cross-reacts with carbamoyl-phosphate synthetases from bacteria, yeast, and mammals. The immunological relatedness of carbamoyl-phosphate synthetases from such diverse species suggests that the protein sequences required for carbamoyl phosphate production have been highly conserved during the course of evolution.

Animals↗

PubMed1985

Acetylglutamate kinase-acetylglutamyl-phosphate reductase complex of Neurospora crassa. Evidence for two polypeptides.

Mutations at the arg-6 locus in Neurospora crassa are divided into two complementation groups (A and B) and a third noncomplementing group. There are many suppressible nonsense mutations among mutants in complementation group B and one in the noncomplementing group; no nonsense mutations exist among mutants in complementation group A (Davis, R. H., and Weiss, R. L. (1983) Mol. Gen. Genet. 192, 46-50). We show here that the mutants are defective in either or both of two enzymes of arginine biosynthesis, acetylglutamate kinase and/or acetylglutamyl-phosphate reductase. Mutants in complementation group A lack acetylglutamate kinase, those in complementation group B lack acetylglutamyl-phosphate reductase, and those in the noncomplementing group lack both activities. Mutants in group B also have reduced levels of acetylglutamate kinase. The enzymes from purified mitochondria are readily separable by gel filtration and by Blue A dye affinity chromatography. Acetylglutamate kinase appears to be an octamer with a molecular weight of 400,000, whereas acetylglutamyl-phosphate reductase appears to be a dimer with a molecular weight of 93,000. This suggests that the two activities reside on distinct polypeptides. These results are best accommodated by the following model: the arg-6 locus encodes a single mRNA which is translated into a single polypeptide; the latter is then cleaved post-translationally to yield two physically separable enzymes.

Aldehyde Oxidoreductases↗

PubMed1985

Ornithine transcarbamylase from Neurospora crassa: purification and properties.

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 Km's of about 5 and 2.5 mM for the substrates, ornithine and carbamyl phosphate, respectively, at pH 9.5. The Km'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.

Amino Acids↗

PubMed1985

Fungal contamination of balanced salt solution.

A fungal organism, identified as Ulocladium, was isolated from an unopened 15 ml bottle of balanced salt solution. The authors note the importance of visually inspecting solutions used in ophthalmic surgery.

Drug Contamination↗

PubMed1984

Mobilization of vacuolar arginine in Neurospora crassa. Mechanism and role of glutamine.

Nitrogen starvation has been shown to increase the cytosolic arginine concentration and to accelerate protein turnover in mycelia of Neurospora crassa. The cytosolic arginine is derived from a metabolically inactive vacuolar pool. Redistribution of arginine between cytosolic and vacuolar compartments is the result of mobilization of this metabolite in response to nitrogen starvation. Mobilization of arginine (and purines) also occurred in response to glutamine limitation, but arginine accumulated upon proline starvation. These observations indicate that mobilization is a consequence of glutamine limitation rather than a general response to amino acid starvation (or limitation). Analysis of the amino acid pools in mycelia subjected to starvation or limitation suggests that glutamine (or a metabolite derived from glutamine) provides a signal which determines the metabolic fate of vacuolar arginine. The results are consistent with the hypothesis that vacuolar compartmentation provides a readily available store of nitrogen-rich compounds to be utilized during differentiation or under conditions of nutritional stress.

Arginine↗

PubMed1984

Improved coating and fixation methods for scanning electron microscope autoradiography.

A simple apparatus for emulsion coating is described. The apparatus is inexpensive and easily assembled in a standard glass shop. Emulsion coating for scanning electron microscope autoradiography with this apparatus consistently yields uniform layers. When used in conjunction with newly described fixation methods, this new approach produces reliable autoradiographs of undamaged specimens.

Arginine↗

PubMed1984

Distribution of concanavalin a binding carbohydrates during mating in Chlamydomonas.

Cell surface carbohydrates, detected by fluorescein isothiocyanate/concanavalin A (FITC-ConA), were identified at four locations on gametes of Chlamydomonas reinhardtii. (1) The cell wall: uniform labelling with FITC-ConA was observed; a substantial number of sites were localized in the sodium dodecyl sulphate-insoluble inner wall, which contains the flagellar collars. (2) The periplasm: a crescent-shaped area was visualized with FITC-ConA and localized by ferritin-ConA. We were able to recover autolytic activity on a ConA affinity column from the mating medium of wild-type cells after the release of these periplasmic sites. The cell-wall-less mutant CW15 displays no periplasmic sites and demonstrates a corresponding inability to release autolytic activity after mating for 60 min. A model for wall lysis is presented, which considers the involvement of these sites in the lytic process. (3) The mating structure: during mating a small fluorescent plaque-like site was observed on cells at a location corresponding to the carbohydrate-like zone of the mating type minus mating structure and may indicate the involvement of ConA binding material in gametic cell fusion. (4) Secreted products: following cell fusion zygotes begin to secrete ConA positive material at about 1 1/2 h. After 24 h a ConA positive zygote wall and pellicle appear.

Binding Sites↗

PubMed1984

Ultrastructure of the flagellar roots in Chlamydomonas gametes.

The cytoskeleton of Chlamydomonas reinhardtii gametes has been studied by electron microscopy. The microtubular system, consisting of four flagellar roots inserted into the basal apparatus, is shown to include two daughter basal bodies and two striated fibres, newly described in this report. One new fibre associates with the 3-over-1 root and is similar to its counterpart, the striated fibre of the 2-member root. These similar root fibres connect each daughter basal body to the V-shaped microtubular root pair. The other new striated fibre joins the daughter basal body to both flagellar roots and is similar to the proximal striated fibre. In mt+ gametes, the conventional root microtubules make direct contact with the doublet zone of the non-activated mating structure. During activation, doublet zone microfilaments associate with the daughter basal body and the finely striated fibre of the 3-over-1 root. These observations suggest that the cytoskeleton acts as a scaffolding for membrane extension by the mt+ mating structure microfilaments.

Chlamydomonas↗

PubMed1984

Localization of concanavalin A binding carbohydrate in Chlamydomonas flagella.

Chlamydomonas flagella are shown to possess two zones of concanavalin A (ConA) binding carbohydrate. The first zone, distinguished by a requirement for a prolonged labelling period for visualization of fluorescein isothiocyanate (FITC)-ConA fluorescence, is localized in the flagellar coat. The second zone is characterized by a rapid FITC- and [125I]ConA labelling subsequent to disruption of the flagellar membrane, but is unaffected by reagents that act only on the flagellar surface coat. Electron microscopy and ferritin-ConA labelling indicate that this subsurface zone is localized between the flagellar membrane and axoneme in the space that we term the flagelloplasm. These results are used to suggest possible functions for ConA binding glycosyl residues in flagella.

Agglutinins↗

PubMed1983

Measurements of cytoplasmic and vacuolar pH in Neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy.

The nitrogen-15 chemical shift of the N1 (tau)-nitrogen of 15N-labeled histidine and the half-height line widths of proton-coupled resonances of the delta- and omega,omega'-nitrogens of 15N-labeled arginine and of the alpha-nitrogens of 15N-labeled alanine and proline were measured in intact mycelia of Neurospora crassa to obtain to estimates of intracellular pH. For intracellular 15N-labeled histidine, the N1 (tau)-nitrogen chemical shift was 200.2 ppm. In vitro measurements showed that the chemical shift was slightly affected by the presence of phosphate, with which the basic amino acids may be associated in vivo. These considerations indicate a pH of 5.7-6.0 for the environment of intracellular histidine. The half-height line widths of the delta- and omega,omega'-nitrogens of [15N]arginine were 15 and 26 Hz, respectively. In vitro studies showed that these line widths also are influenced by the presence of phosphate, and, after suitable allowance for this, the line widths indicate pH 6.1-6.5 for intracellular arginine. The half-height line widths for intracellular alanine and proline were 17 and 12 Hz, respectively, which are consistent with an intracellular pH of 7.1-7.2. Pools of histidine and arginine are found principally in the vacuole of Neurospora, most likely in association with polyphosphates. Proline and alanine are cytoplasmic. The results reported here are consistent with these localizations and indicate that the vacuolar pH is 6.1 +/- 0.4 while the cytoplasmic pH is 7.15 +/- 0.10. Comparisons of these estimates with those obtained by other techniques and their implications for vacuolar function are discussed.

Arginine↗

PubMed1983

Cloning and characterization of the ornithine carbamoyltransferase gene from Aspergillus nidulans.

An Aspergillus nidulans DNA fragment composed of two adjacent SalI subfragments (1.8 and 0.85 kb) that carries an argB gene 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 prototrophy with high frequency possibly due to DNA rearrangements within the recombinant plasmid.

Aspergillus nidulans↗

PubMed1982

Effect of the nitrogen source on glutamine and alanine biosynthesis in Neurospora crassa. An in vivo 15N nuclear magnetic resonance study.

The influences of different nitrogen sources on the relative rates of biosynthesis of glutamine and alanine have been studied by 15N nuclear magnetic resonance spectroscopy of intact Neurospora crassa mycelia suspensions. The rate of glutamine synthesis was fastest after growth in media deficient in free ammonium ion, whereas it was slowest following growth in media containing both glutamic acid and glutamine. The reverse trend was observed for the biosynthesis of alanine. A competition between the two biosynthetic pathways for the same substrate, glutamic acid, was found to limit the rate of alanine synthesis when glutamine synthesis was rapid. The observed in vivo rates of these reactions are compared to the reported specific activities of the enzymes catalyzing the reactions, and implications of these results for nitrogen regulation of these pathways under various physiological conditions are discussed.

Alanine↗

PubMed1982

Nitrogen-15 spin-lattice relaxation times of amino acids in Neurospora crassa as a probe of intracellular environment.

The nitrogen-15 spin-lattice relaxation time, T1, and the nuclear Overhauser enhancement (NOE) have been measured for intracellular glutamine, alanine, and arginine in intact Neurospora crassa mycelia to probe their various intracellular environments. The relaxations of 15N gamma of glutamine, 15N alpha of alanine, and 15N omega, omega ' of arginine in N. crassa were found, on the basis of their NOE values, to be predominantly the result of 15N-H dipolar relaxation. These relaxations are therefore related to the microviscosities of the various environments and associations of the respective molecules with other cellular components that act to increase the effective molecular sizes. For 15N gamma of glutamine in the cytoplasm, the intracellular T1 (4.1 s) was only slightly shorter than that in the culture medium (4.9 s). This indicates that the microviscosity of the cytoplasm surrounding the glutamine molecules is not much greater than 1.3 cP. By contrast, for 15N omega, omega ' of arginine, which is sequestered in vacuoles containing polyphosphates, the intracellular T1 (1.1 s) was only one-fourth of that in the medium (4.6 s). In model systems, the T1 of 15N omega, omega ' in a 1 M aqueous solution of arginine containing 0.2 M pentaphosphate was 0.95 s, whereas in an isoviscous (2.8 cP) solution without pentaphosphate, the T1 was 1.8 s. These results suggest either that the vacuolar viscosity is substantially above 2.8 cP or that the omega, omega '-nitrogens of vacuolar arginine are associated with a polyanion, possibly polyphosphate. The implications of these results for the properties of the vacuolar interior are discussed in relation to the mechanism of amino acid compartmentation.

Alanine↗

PubMed1982

Energetics of vacuolar compartmentation of arginine in Neurospora crassa.

The energy requirements for the uptake and retention of arginine by vacuoles of Neurospora crassa have been studied. Exponentially growing mycelial cultures were treated with inhibitors of respiration or glycolysis or an uncoupler of respiration. Catabolism of arginine was monitored as urea production in urease-less strains. The rationale was that the rate and extent of such catabolism was indicative of the cytosolic arginine concentration. No catabolism was observed in cultures treated with an inhibitor or an uncoupler of respiration, but cultures treated with inhibitors of glycolysis rapidly degraded arginine. These differences could not be accounted for by alterations in the level or activity of arginase. Mycelia growing in arginine-supplemented medium and treated with an inhibitor or uncoupler of respiration degraded an amount of arginine equivalent to the cytosolic fraction of the arginine pool. The inhibitors and the uncoupler of respiration reduced the ATP pool and the energy charge. The inhibitors of glycolysis reduced the ATP pool but did not affect the energy charge. The results suggest that metabolic energy is required for the transport of arginine into the vacuoles but not for its retention. The latter is affected by inhibitors of glycolysis. The form of energy and the nature of the vacuolar transport mechanism(s) are discussed.

Amino Acids↗