PA28, an activator of the 20 S proteasome, is composed of two nonidentical but homologous subunits.

PA28, one of a series of a positive allosteric regulators of the 20 S proteasome, stimulates the enzyme's peptidase activities in an ATP-independent manner by binding to the terminal rings of the 20 S complex. PA28 has a native molecular mass of 180,000 Da and contains at least six subunits of approximately 28,000 Da. In this study we show that PA28 prepared from bovine heart contains two different subunits separable by reverse phase high performance liquid chromatography and that these subunits occur in approximately equal abundance. The subunits display mass values of 27,290 +/- 3.7 and 28,606 +/- 2.8 Da by electrospray mass spectrometry, showing that they differ in covalent structure. Partial amino acid sequence analysis of the subunits indicates that the subunits are the products of two different but homologous genes. A pair of subunits has also been isolated from rabbit heart, and partial amino acid sequence analysis shows each to be homologous to the corresponding subunit in bovine tissues. This indicates that the genes encoding two different polypeptide components of PA28 have been conserved during evolution and suggests the possibility that the two subunits play functionally distinct roles. Isolation of complexes formed between purified PA28 and the 20 S proteasome using density gradient centrifugation reveals that both PA28 subunits bind to the proteasome, indicating that both are components of functional PA28 molecules. These results are consistent with two alternative models for the subunit structure of PA28. There may exist two different PA28 molecules that are homooligomers of the 27,290- and 28,606-Da subunits, respectively. Alternatively, PA28 oligomers may contain mixtures of the 27,290- and 28,606-Da subunits either of fixed or variable stoichiometry.

Molecular cloning and expression of palmitoyl-protein thioesterase.

We have previously reported the purification of a palmitoyl-protein thioesterase (PPT) from bovine brain that removes palmitate from Ha-Ras (Camp, L. A., and Hofmann, S. L. (1993) J. Biol. Chem. 268, 22566-22574). In the current paper, we have isolated bovine and rat cDNA clones encoding PPT. The deduced amino acid sequence of PPT predicts a protein of 306 amino acids that contains amino acid motifs characteristic of thioesterases: "Gly-X-Ser-X-Gly" positioned near the NH2 terminus and "Gly-Asp-His" positioned near the COOH terminus of the protein. The identity of the PPT cDNA was further confirmed by expression in simian COS cells and insect Sf9 cells. Comparison of the DNA and protein sequence data suggests that a hydrophobic NH2-terminal sequence of 27 amino acid residues is removed from the primary translation product. Furthermore, the recombinant protein and the native protein purified from bovine brain contain complex asparagine-linked oligosaccharides and a large proportion of the expressed PPT is secreted from COS and Sf9 cells. Thus, while the palmitoyl-protein thioesterase will deacylate intracellular palmitoylated proteins such as Ha-Ras and the alpha subunits of heterotrimeric G proteins, the physiologic substrates are likely to be externally oriented or secreted proteins.

PA700, an ATP-dependent activator of the 20 S proteasome, is an ATPase containing multiple members of a nucleotide-binding protein family.

PA700 is a 700,000-dalton multisubunit protein that activates multiple proteolytic activities of the 20 S proteasome by a mechanism dependent upon ATP hydrolysis (Ma, C.-P., Vu, J.H., Proske, R.J., Slaughter, C.A., and DeMartino, G.N. (1994) J. Biol. Chem. 269, 3539-3547). In order to determine the identities of and structural relationships among the subunits of PA700, individual PA700 subunits were isolated by a combination of reverse phase high performance liquid chromatography (HPLC) and SDS-polyacrylamide gel electrophoresis. Seven of the 16 subunits of PA700 so isolated were subjected to solid phase protease digestion followed by reverse phase HPLC. Selected peptides from each protein were sequenced by automated Edman degradation. Comparison of the resulting amino acid sequences with those in current data bases indicated that three of the subunits represented novel proteins, whereas four subunits were homologous to previously describe proteins. Three subunits of the latter group were, in turn, homologous to one another and are members of a large family of proteins containing a consensus sequence for ATP binding. Purified PA700 demonstrated ATPase activity. Treatment of PA700 with alkylating agents, such as N-ethylmaleimide, inhibited with similar kinetics both proteasome activation and ATPase activity, suggesting that these two activities are functionally linked. Thus, PA700 is composed of multiple members of a protein family that may function in the ATP-dependent regulation of proteasome activity.

Distribution of a monoclonal antibody-recognized protective protein immunogen on the outer membranes of Pasteurella multocida rabbit isolates.

The distribution of a monoclonal antibody (MAb)-recognized protective protein immunogen on the outer membrane of 153 Pasteurella multocida rabbit isolates was determined by dot blot (DB) analysis. MAb 1608 reacted with 36 (24%) of the 153 clinical isolates. The DB-positive clinical isolates expressed capsular antigens A, D, and nontypable and somatic antigens 2, 3, 10, 12, 15, and nontypable. Western blot (immunoblot) analysis with adsorbed and eluted MAb 1608 confirmed that the antigenic determinant identified was located on the cell surface. With MAb 1608 as a probe for antibody-accessible radioimmunoassay, 31 of 36 DB-positive P. multocida rabbit isolates were shown to have surface-exposed and antibody-accessible antigenic determinants, while 44 of 44 DB-negative isolates were negative by antibody-accessible radioimmunoassay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed DB-negative P. multocida isolates both with (6 of 13, 46%) and without (7 of 13, 54%) the 37.5-kilodalton protein. This study establishes that the protective antigenic determinant of the 37.5-kilodalton outer membrane protein is present in 24% of rabbit clinical isolates tested and is detectable in P. multocida strains distributed among the major somatic types (3, 10, 12, and 15) and the capsular types (A and D) commonly isolated from rabbits in North America.

Identification of immunogenic outer membrane proteins of Pasteurella multocida 3:A in rabbits.

Four groups of protective rabbit immune sera were used to identify Pasteurella multocida outer membrane immunogens by a radioimmunoprecipitation procedure and Western blot (immunoblot) analysis. These are rabbit hyperimmune sera against KSCN extract of P. multocida (group 1) and rabbit immune sera against the KSCN extract of P. multocida (group 2), the outer membrane of P. multocida (group 3), and live P. multocida cells (group 4). Rabbits mounted an antibody response to 18 proteins found in the outer membrane of P. multocida, and the major antibody activities were directed to the 27,000-molecular-weight outer membrane protein (27K protein), as well as the 37.5K, 49.5K, 58.7K, and 64.4K outer membrane proteins. These outer membrane immunogens appear to be exposed on the cell surface and accessible to antibodies, since adsorption of these immune sera with intact P. multocida cells resulted in a significant reduction of antibody activities directed against these proteins, especially the 37.5K protein. Antibodies eluted from immune serum-P. multocida cell complexes were reactive to the 37.5K immunogen, confirming that this protein is exposed on cell surface and accessible to antibodies. Western blot analyses with group 1, 3, and 4 immune sera confirmed that the 27K, 37.5K, 49.5K, 58.7K, and 64.4K proteins are the major outer membrane immunogens of P. multocida in rabbits. Lung lavages of immunized rabbits also contained similar antibody activities directed against several outer membrane proteins, with major activities against the 37.5K and 64.4K proteins.