Subunit structure of a class A aspartate transcarbamoylase from Pseudomonas fluorescens.

The class A aspartate transcarbamoylase (ATCase, EC 2.1.3.2) from Pseudomonas fluorescens was purified to homogeneity with retention of full catalytic and regulatory functions. Careful determinations under conditions that minimized proteolysis showed that the molecule is a 1:1 stoichiometric complex of two polypeptide chains of 34 and 45 kDa. Pyridoxal phosphate is a competitive inhibitor of the enzyme (Ki = 1 microM). Reduction of the pyridoxal phosphate enzyme adduct with sodium boro[3H]hydride showed that the active site is located on the 34-kDa polypeptide. Affinity labeling with 5'-[p-(fluorosulfonyl)benzoyl]adenosine, an ATP analog, suggested that the regulatory site is also located on the 34-kDa species. While the function of the 45-kDa subunit is unknown, neither carbamoyl phosphate synthetase nor dihydroorotase activities are associated with the ATCase. The molecular mass of the enzyme was determined by gel filtration, sedimentation velocity, and electron microscopy to be 464 kDa. Thus the enzyme is composed of six copies of the 34-kDa polypeptide and six copies of the 45-kDa polypeptide. The molecule has a Stokes' ratio of 70.9 A and a frictional ratio of 1.37, suggesting a compact globular shape. We propose that the P. fluorescens ATCase is composed of two trimers of 34-kDa catalytic chains and is likely to be a D3 dodecamer with an arrangement of subunits analogous to that of the class B ATCase molecules.

Mammalian aspartate transcarbamylase (ATCase): sequence of the ATCase domain and interdomain linker in the CAD multifunctional polypeptide and properties of the isolated domain.

Mammalian aspartate transcarbamylase (ATCase; carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) is part of a 240-kDa multifunctional polypeptide called CAD, which also has carbamoyl-phosphate synthetase and dihydroorotase activities. We have sequenced selected restriction fragments of a Syrian hamster CAD cDNA that are clearly homologous to three prokaryotic ATCases. These studies, combined with previous sequence data, showed that the ATCase domain of CAD is encoded by 924 base pairs and has a mass of 34,323 Da and a pI of 9.8. While the bacterial pyrimidine biosynthetic enzymes are separate proteins, in mammals the ATCase domain is fused to the carboxyl end of the CAD chimera via a 133-amino acid (14-kDa) linker with an unusual amino acid composition, a pI of 10.2, and pronounced hydrophilic character. The fully active domain isolated from proteolytic digests was characterized by partial amino acid sequencing and amino acid analysis. Trypsin cleavage produced the ATCase domain with a 20-residue amino-terminal extension. Hydrodynamic studies showed that the isolated domain is a 110-kDa trimer with a Stokes radius of 41 A. The mammalian ATCase domain and the prokaryotic enzymes have virtually identical active-site residues and are likely to have the same tertiary fold.

Intraviral homology and subgenomic RNAs of pepper ringspot virus.

The Tobraviruses constitute a group of rod-shaped, bipartite, plus-stranded RNA viruses. We report on homologies between the two viral genomic RNA molecules of pepper ringspot virus (PRV) and on the subgenomic components generated from them during infection. It has previously been shown that the 3'-terminal 459 nucleotides of PRV RNA-1 and RNA-2 are identical. Here it is shown that there is a second homology between RNA-1 and a region of RNA-2, located at least 240 nucleotides downstream from its 5' terminus. In agreement with strains in another Tobravirus group, we observe three subgenomic components in extracts of plants infected with PRV. Two of these, RNAs-1a (1.6 kb) and -1b (0.8 kb), are derived from RNA-1 (the larger genomic RNA). They are probably mRNAs for 29- and 16-kDa nonstructural virus proteins. The smaller genomic RNA, RNA-2, generates the subgenomic component RNA-2a (1.3-1.45 kb) which is probably an efficient capsid protein mRNA. Previously reported subgenomic components of 2.8 and 1.1 kb are shown to be electrophoretic artifacts. RNA-2a, but not 1a or 1b, is present in virion RNA preparations, indicating that it is the only subgenomic species to be encapsidated.

The nucleotide sequence of tobacco rattle virus RNA-2 (CAM strain).

The nucleotide sequence of the smaller genomic strand (RNA-2) of the bipartite tobacco rattle virus (CAM strain) has been determined. RNA-2 is capped at the 5' terminus and contains 1799 nucleotide residues. There is a single 223 codon long open reading frame extending from nucleotide 574 to 1242 which designates a protein of Mr 23,654. The derived amino acid composition, in percent, matches that previously determined for the virus capsid protein. The long open reading frame is flanked by 5' and 3' untranslated regions of 573 and 554 nucleotides, respectively. The 5' leader sequence contains two different sets of direct repeats, one of 119 nucleotides and the other of 76. It also contains 13 apparently unused AUG codons, four of which lie in the same frame as the capsid protein cistron. The 3' terminal sequence of RNA-2 is identical to that of the larger genomic strand (RNA-1) for 459 nucleotides.