Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora. VII. Isolation and sequences of three large cyanogen bromide peptides.

The isolation and sequences of three peptides of large size from a cyanogen bromide digest of the NAD-specific glutamate dehydrogenase of Neurospora crassa are reported. These three peptides comprise 86, 117, and 134 residues, respectively, and represent approximately 30% of the estimated 1030 residues in the peptide chain.     

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora crassa. Isolation and sequences of several cyanogen bromide peptides from the NH2-terminal portion of the peptide chain

Previous studies of the amino acid sequence of the NAD-specific glutamate dehydrogenase of Neurospora crassa (EC 1.4.1.2) resulted in the assignments of peptides to four fragments, the longest being the COOH-terminal 669 residues of the protein. A further study of peptides derived by cyanogen bromide cleavage by different separation methods has yielded additional peptides that have provided new information concerning the sequence and has given overlaps of previously known sequences. This has permitted establishment of 313 residues in one sequence (fragment II). This is in addition to a sequence of 43 residues (fragment I) at the NH2-terminal end and a sequence of 669 residues (fragment III) previously established at the COOH-terminal end of the molecule. The present status of our knowledge of the overall sequence is given in the accompanying papers, together with some views regarding the conformation of the protein (Haberland, M.E., Chen, C.-W., and Smith, E.L. (1980) J. Biol. Chem. 255, 7993-8000, and Austen, B.M., Haberland, M.E., and Smith, E.L. (1980) J. Biol. Chem. 255, 8001-8004).     

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora. VI. Isolation and sequences of eighteen fragments from the cyanogen bromide digest.

The 1030-residue polypeptide chain of the NAD-specific glutamate dehydrogenase of Neurospora crassa was fragmented by treatment with cyanogen bromide. The isolation and sequences of 18 fragments ranging in size from 4 to 51 residues are described. Some of these peptides proved to be cleavage products resulting from hydrolysis at acid-sensitive aspartyl-prolyl bonds. Some overlaps could be deduced on the basis of known sequences of peptides obtained by tryptic hydrolysis.     

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora. V. Tryptic peptides.

The isolation and sequences of an additional 80 peptides from a tryptic digest of the NAD-specific glutamate dehydrogenase of Neurospora crassa are reported. These include an additional peptide containing a lysine residue labeled at the epsilon-amino group with pyridoxal 5'-phosphate. The sequence of this peptide shows some homology with the reactive lysine residue of other glutamate dehydrogenases.     

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora. VIII. Isolation and sequences of peptides from a tryptic hydrolysate of the maleylated protein.

The NAD-specific glutamate dehydrogenase of Neurospora crassa was S-carboxymethylated with [14C]iodoacetate, maleylated, and hydrolyzed with trypsin. The isolation and sequences of the resulting peptides are described. These peptides gave information on the structure of the protein that was previously unknown and gave many overlaps of previously isolated segments of the protein.     

Nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase of Neurospora.

Neurospora NADP-specific glutamate dehydrogenase that was treated with iodoacetate, iodoacetamide, or N-ethylmaleimide to block the thiol groups was cleaved with cyanogen bromide. Of the expected 10 peptides, based on a methionine content of 9 residues, 8 were obtained in pure form and 2 were handled as a mixture. The fragments ranged in size from 9 to 109 residues. In addition, there were isolated 6 peptides, produced by anomalous cleavage at the carboxyl groups of tryptophan residues, and two by hydrolysis of an aspartyl-proline bond. Preliminary separation of these peptides was accomplished by gel filtration followed by either ion-exchange chromatography of the larger peptides or by paper chromatography and paper electrophoresis of the smaller fragments. Ordering of the CNBr fragments in sequence was based upon sequences of tryptic and chymotryptic peptides obtained in another laboratory. The complete sequence of the protein is presented. The amino acid sequences of the bovine and chicken liver glutamate dehydrogenases previously determined show considerable homology with the NADP-specific enzyme of Neurospora in the NH2-terminal half of the molecule; this includes the region of the specifically reactive lysine residue and the portion of the sequence that has been implicated in coenzyme binding. Particularly striking is the fact that most of the residues conserved among the three homologous proteins would be expected to be important for conformational, rather than catalytic, effects. This implies that the conformation of the Neurospora enzyme must be similar in parts of its structure to the vertebrate enzymes but undoubtedly differs in some regards.     

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora. IV. The COOH-terminal 669 residues of the peptide chain; comparison with other glutamate dehydrogenases.

A sequence is presented for the COOH-terminal 669 residues of the NAD-specific glutamate dehydrogenase of Neurospora crassa. Comparison of this sequence with those of the vertebrate glutamate dehydrogenases of chicken and bovine liver and with the NADP-specific enzyme of Neurospora shows some similarities in sequences around residues previously identified as important for the function of these enzymes. These are: (a) the reactive lysine residue of low pK in the NADP and the vertebrate enzymes; (b) the tyrosine residue of the NADP enzyme that is readily nitrated by tetranitromethane with inactivation, a residue protected by NADP or by NMN; and (c) the arginine residue of the NADP-enzyme that is reactive with 1,2-cyclohexanedione with inactivation. Despite these similarities, comparison of the sequence of the NAD-enzyme with those of the other glutamate dehydrogenases of known sequences revealed relatively little overall homology as determined by computer analysis.     

Mutants of Aspergillus nidulans lacking nicotinamide adenine dinucleotide-specific glutamate dehydrogenase.

Ten mutants of Aspergillus nidulans lacking nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (NAD-GDH) have been isolated, and their mutations (gdhB1 through gdhB10) have been shown to lie in the gdhB gene. In addition, a temperature-sensitive gdhB mutant (gdhB11) has been isolated. A revertant (designated R-5) of the mutant gdhB1 bears an additional lesion in the gdhB gene and has altered NAD-GDH activity with altered Km values for ammonia or ammonium ions and for alpha-ketoglutarate. These results suggest that gdhB specifies a structural component for NAD-GDH. The growth characteristics of gdhB mutants indicate the routes by which amino acids are utilized as nitrogen and carbon energy sources. The properties are described of the double mutants bearing the mutations gdhB1 and gdhA1 or tamA119, which have low NADP-GDH activity.     

Nicotinamide–adenine dinucleotide-specific isocitrate dehydrogenase from pea mitochondria: Purification and Properties

1. A method of stabilizing the enzyme by using glycerol is described. 2. A purification procedure is presented giving a higher purification than previously described. 3. Data showing substrate activation and activation by citrate are presented. 4. Kinetic constants for NAD(+), NADH and certain bivalent metal ions are given. 5. Pronounced inhibitory buffer effects are described. 6. A brief comparison between the NAD-specific isocitrate dehydrogenase from peas and that from other sources is made.