The subunit structure of the arom multienzyme complex of Neurospora crassa. A possible pentafunctional polypeptide chain.

A new procedure for the purification of the arom multienzyme complex from Neurospora crassa is presented. Important factors are the inactivation of proteinases by phenylmethanesulphonyl fluoride and the use of cellulose phosphate as an affinity adsorbent. A homogeneous enzyme, with a specific shikimate dehydrogenase activity of 70 units/mg of protein, is obtained in 25% yield. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, combined with cross-linking studies using dimethyl suberimidate, suggest that the complex is composed of two subunits of molecular weight 165000. Glycerol-density-gradient centrifugation indicates a molecular weight for the intact complex of about 270000. Evidence for the effects of proteolysis, both during the preparation and on storage of the purified complex, is presented, and previous reports in the literature of the occurrence of multiple subunits are discussed in this light.     

The subunit structure of the arom multienzyme complex of Neurospora crassa. Evidence from peptide ''maps'' for the identity of the subunits.

Evidence was obtained, from polyacrylamide-gel electrophoresis in the presence of urea and from peptide 'mapping' of specifically labelled cysteine-and methionine-containing peptides, that the two subunits of the arom multienzyme complex of Neurospora crassa are chemically very similar and possibly identical.     

Purification of the arom multienzyme aggregate from Euglena gracilis.

The arom multienzyme complex that catalyzes steps two through six in the prechorismate polyaromatic amino acid biosynthetic pathway has been purified up to 2000-fold from Euglena gracilis. The native arom aggregate has a molecular weight of approx. 249 000 based on a sedimentation coefficient of 9.5 and Stokes radius of 60 angstrom. A comparison between the arom aggregates of Neurospora crassa and Euglena gracilis and the possible phylogenetic relationships between the organisms are discussed.     

The functional unit of the arom conjugate in Neurospora

The functional unit of arom polyenzyme conjugate of Neurospora crassa was determined by analysis of radiation inactivation of each of the five activities in the conjugate. The functional targets for all five enzymes were in close agreement with the value of 300,000 obtained by conventional hydrodynamic procedure for the native dimeric structure. These data indicate that at least 95% of the functional enzyme system in crude extracts exists in a dimeric form and that both polypeptide chains of the homodimer are required for full activity of each of the five enzymes.     

The protease problem in Neurospora. Variable stability of enzymes in aromatic amino acid metabolism.

A proteolytic activity isolated from Neurospora crassa is shown to be responsible for the variable stability observed in vitro for enzymes involved in aromatic amino acid metabolism. For example, the activity of kynurenine formamidase was insensitive to the action of this protease preparation over a 24-h period of incubation at 25 °C, whereas chorismate synthase, anthranilate synthase, kynureninase, and the five activities of the arom multienzyme system were inactivated during this time. Anthranilate synthase and two of the arom system activities (dehydroquinate synthase and shikimate kinase) were inactivated by the protease preparation within 2 h. Phenylmethanesulfonylfluoride and a specific proteolytic inhibitor from N. crassa prevented inactivation of these enzymes. Spontaneous loss of activity at 25 °C of purified samples of anthranilate synthase, dehydroquinate synthase and shikimate kinase was also prevented by the inhibitors. A method for purifying the inhibitor from N. crassa is described, and its use as a reagent in the analysis of proteolytic action is demonstrated.     

Purification and isolation of the arom aggregates of Schizosaccharomyces pombe

The five enzymes that catalyzing steps two through six in the prechorismate polyaromatic amino acid biosynthetic pathway are physically associated and have been purified up to 400-fold from Schizosaccharomyces pombe. The native arom aggregate has a molecular weight of approx. 140,000-145,000 based on gel filtration, glycerol-density-gradient centrifugation, and polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Similarities between the S. pombe arom aggregate and that of Neurospora crassa and Euglena gracilis are discussed.     

溶菌酶提取过程中出现的问题及改进措施

溶菌酶的提取是一个比较复杂的过程,每一步的操作对最终的实验结果都是至关重要的。先简单介绍了溶菌酶提取的实验条件及基本过程,而后阐述了实验过程中一些常见的细节问题,并针对这些问题提出了相应的改进措施,最后总结归纳出了实验操作中的其他注意事项,这对于溶菌酶的成功提取具有积极的作用。     

The 3-dehydroquinate synthase activity of the pentafunctional arom enzyme complex of Neurospora crassa is Zn2+-dependent.

We have demonstrated the co-purification in constant ratio of all five activities of the pentafunctional arom enzyme complex from Neurospora crassa. Progressive inactivation of the 3-dehydroquinate synthase component of the purified enzyme complex by chelating agents was blocked by the substrate, 3-deoxy-D-arabino-heptulosonate 7-phosphate, but not by the cofactor NAD+. Full activity was restored at Zn2+ concentrations as low as 0.05 nM. Atomic absorption data indicated that the intact enzyme complex contained 1 atom per subunit of tightly bound zinc. The arom 3-dehydroquinate synthase had a calculated turnover number of 19s-1, this being within the narrow range of values obtained for the other four activities of the intact multifunctional enzyme. The Km for 3-deoxy-D-arabino-heptulosonate 7-phosphate was 1.4 microM in a phosphate-free buffer; inorganic phosphate was a competitive inhibitor with respect to 3-deoxy-D-arabino-heptulosonate 7-phosphate.     

Beta-oxidation system of the filamentous fungus Neurospora crassa. Structural characterization of the trifunctional protein.

Treatment of the trifunctional protein from Neurospora crassa with various proteases produced almost identical patterns of proteolytic fragments. To study the structural features of the protein in more detail limited proteolysis with trypsin was carried out. Polyclonal antibodies were raised against three different tryptic fragments. With the help of immunological methods and amino-terminal sequence analysis we were able to monitor the sequential cleavage steps during proteolysis. Two major fragments (an amino-terminal one of 51 kDa and a carboxyl-terminal one of 46 kDa) were identified at the first cleavage step, dividing the 93-kDa subunit of the trifunctional protein almost in half. Additional proteolysis products, deriving from either half, were formed in subsequent proteolytic steps. Combining these results with those obtained from enzyme analysis of the proteolyzed protein, a domain structure of the trifunctional protein is proposed. According to our model each subunit of the tetrameric protein consists of at least two large domains, the amino-terminal one possessing 2-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase activity and the carboxyl-terminal one bearing 3-hydroxyacyl-CoA epimerase activity.     

Revertants and Secondary arom-2 Mutants Induced in Non-Complementing Mutants in the arom Gene Cluster of Neurospora Crassa

Extensive genetical and biochemical studies have been performed with revertants and secondary arom-2 mutants induced in two different primary non-complementing mutants which map within the arom gene cluster of Neurospora crassa. These studies indicate that mutant M54 but not M25 can revert by super-suppressor mutations in unlinked genes, thus confirming previous evidence that M54 contains a nonsense codon. At least three new super suppressors of M54 have been detected. All four super suppressors (including one previously detected) when combined with M54 result in high levels of all five of the arom enzymic activities in the form of arom multienzyme complexes very similar to (but not necessarily identical with) that in wild type (WT).-Evidence has also been obtained that the two non-complementing mutants can yield revertants which appear to result from true back mutations and produce arom aggregates essentially indistinguishable from that of WT. In addition, M25, but not M54, when plated on quinic acid yields revertants (secondary mutants) some of which are phenotypically indistinguishable from arom-2 primary mutants and others of which, although also mapping within the arom-2 gene, exhibit unusual properties. Genetic evidence indicates that the M25 secondary mutants are localized within the arom-2 gene, but that they arise from mutational events more complex than ones resulting in single base pair changes in the M25 codon.-The recovery of secondary arom-2 mutants as revertants of non-complementing arom mutants provides strong evidence, independent of earlier recombination data, that non-complementing arom mutants are located within the arom-2 structural gene of the arom gene cluster. In addition, the occurrence and characteristics of these secondary arom-2 mutants provide strong evidence, independent of the results with nonsense suppressors, that the arom gene cluster is transcribed, beginning with the arom-2 gene, as a single polycistronic messenger ribonucleic acid (mRNA) molecule which is subsequently translated into the arom multienzyme complex.     

Large scale isolation and storage of Neurospora plasma membranes.

Functionally inverted plasma membrane vesieles isolated from the eukaryotic microorganism Neurospora crassa generate and maintain a transmembrane electrical potential via ATP hydrolysis catalyzed by a plasma membrane ATPase (G. A. Scarborough, 1976, Proc. Nat. Acad. Sci. USA73, 1485–1488). In order to facilitate investigation of the molecular mechanism of the electrogenic ATPase, and other transport systems, we have developed a method for the large scale isolation and storage of Neurospora plasma membranes in a stable form. Large quantities of open plasma membrane sheets (ghosts) are isolated by a scaled-up modification of the original method (G. A. Scarborough, 1975, J. Biol. Chem.250, 1106–1111) and stored at ?26°C in 60% glycerol ($\text{v}\text{v}$). As needed, the ghosts are washed free of glycerol and then converted to closed vesicles by a modification of the original method. With this technique, plasma membrane vesicles with normal electrogenic pump activity can be prepared daily in approximately 2.5 h.     

Structural determinants of post-translational modification and catalytic specificity for the lipoyl domains of the pyruvate dehydrogenase multienzyme complex of Escherichia coli

The lipoyl domains of the dihydrolipoyl acyltransferase (E2p, E2o) components of the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes are specifically recognised by their cognate 2-oxo acid decarboxylase (E1p, E1o). A prominent surface loop links the first and second beta-strands in all lipoyl domains, close in space to the lipoyl-lysine beta-turn. This loop was subjected to various modifications by directed mutagenesis of a sub-gene encoding a lipoyl domain of Escherichia coli E2p. Deletion of the loop (four residues) rendered the domain incapable of reductive acetylation by E. coli E1p in the presence of pyruvate, but insertion of a new loop (six residues) corresponding to that in the E2o lipoyl domain partly restored this ability, albeit with a much lower rate. However, the modified domain remained unable to undergo reductive succinylation by E1o in the presence of 2-oxoglutarate. Additional exchange of the two residues on the C-terminal side of the loop (V14A, E15T) had no effect. Insertion of a different four-residue loop also restored a limited ability to undergo reductive acetylation, but still significantly less than that of the wild-type domain. Exchanging the residue on the N-terminal side of the lipoyl-lysine beta-turn in the E2p and E2o domains (G39T), both singly and in conjunction with the loop exchange, had no effect on the ability of the E2p domain to be reductively acetylated but did confer a slight increase in susceptibility to reductive succinylation. All mutant E2p domains, apart from that with the loop deletion (LD), were readily lipoylated in vitro by E. coli lipoate protein ligase A; the E2p LD mutant could be lipoylated only at a significantly lower rate. Likewise, this domain exhibited 1D and 2D NMR spectra characteristic of a partially folded protein, whereas the spectra of mutants with modified loops were similar to those of the wild-type domain. The surface loop is evidently important to the structural integrity of the domain and may help to stabilize the thioester bond linking the acyl group to the reduced lipoyl-lysine swinging arm as part of the catalytic mechanism. Recognition of the lipoyl domain by its partner E1 appears to be a complex process and not attributable to any single determinant on the domain.     

The dicyclohexylcarbodiimide-binding protein of the mitochondrial ATPase complex from Neurospora crassa and Saccharomyces cerevisiae. Identification and isolation.

Incubation of mitochondria from Neurospora crassa and Saccharomyces cerevisiae with the radioactive ATPase inhibitor [14C]dicyclohexylcarbodiimide results in the irreversible and rather specific labelling of a low-molecular-weight polypeptide. This dicyclohexylcarbodiimide-binding protein is identical with the smallest subunit (Mr 8000) of the mitochondrial ATPase complex, and it occurs as oligomer, probably as hexamer, in the enzyme protein. The dicyclohexylcarbodiimide-binding protein is extracted from whole mitochondria with neutral chloroform/methanol both in the free and in the inhibitor-modified form. In Neurospora and yeast, this extraction is highly selective and the protein is obtained in homogeneous form when the mitochondria have been prewashed with certain organic solvents. The bound dicyclohexylcarbodiimide label is enriched in the purified protein up to 50-fold compared to whole mitochondria. Based on the amino acid analysis, the dicyclohexylcarbodiimide-binding protein from Neurospora and yeast consists of at least 81 and 76 residues, respectively. The content of hydrophobic residues is extremely high. Histidine and tryptophan are absent. The N-terminal amino acid is tyrosine in Neurospora and formylmethionine in yeast.