Mitochondrial phosphoenolpyruvate carboxykinase from the chicken. Comparison of the cDNA and protein sequences with the cytosolic isozyme.

The amino acid sequence of the mitochondrial form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK-M) from the chicken was deduced from the 3571 nucleotide sequence of three overlapping cDNA clones. The derived protein sequence, which includes 607 amino acids of the mature enzyme and a leader sequence, was aligned with nine tryptic peptides of PEPCK-M and the primary sequence of the cytosolic form of PEPCK from chicken. Secondary structure predictions for the two PEPCK isozymes indicated similar packing elements of conserved, hydrophobic beta strands in the central core of the primary sequence. This core protein, which contained three GTP-binding consensus elements, was 80% identical in the two chicken isozymes, although the overall level of identity was only 63% for amino acids and 60% for nucleotides. The untranslated regions of the two cDNAs were dissimilar, although both mRNAs have potential for significant secondary structure. The PEPCK-M mRNA contained several G-C-rich regions which demonstrated free energies of formation in dyad symmetry programs up to -70 kcal/mol. The 1.6-kilobase (kb) 3'-untranslated region contained several repeat elements including one of 11 base pairs, which was present 30 times; but, a signal sequence for polyadenylation was not present. Each of the three PEPCK-M cDNA clones recognized two mRNAs of 4.2 and 3.4 kb in the livers and kidneys of starved or normally fed chickens. However, the level of these two related PEPCK-M mRNAs changed in response to cAMP treatment, with the larger mRNA predominant at 20 and 160 min and the 3.4-kb mRNA present at intermediate times. In contrast, the level of the 2.8-kb PEPCK-C mRNA increased dramatically upon addition of the cyclic nucleotide, particularly in the liver where it was not detected without cAMP induction. Thus, PEPCK-M and PEPCK-C, clearly represented the products of two distinct genes, which were distinguished by altered protein sequences and non-cross-hybridizing, differentially regulated mRNAs.

Nucleotide sequence of the mRNA encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken.

We have determined the sequence of the mRNA encoding cytosolic phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] from the chicken and have deduced the primary structure of the protein. The message for the enzyme is 2762 bases long and encodes a protein of 622 amino acids with a molecular mass of 69,522 daltons. The 5' untranslated region is 246 nucleotides long and contains two nonfunctional AUG initiator codons. The 3' untranslated sequence is 649 bases long and contains multiple polyadenylylation signals. There are regions of dyad symmetry and an A + U-rich region within the 3' translated and untranslated sequences of the message. Such regions are also present in the mRNA for the enzyme from the rat and may be of functional significance. Conserved regions of the enzyme, that may interact with substrates, were identified by comparing the amino acid sequence of phosphoenolpyruvate carboxykinase with that of other proteins that use guanine nucleotides and phosphoenolpyruvate as substrates.

The regulatory subunit of neural cAMP-dependent protein kinase II represents a unique gene product.

Although the major form of soluble cAMP-dependent protein kinase in bovine cerebral cortex can be classified as a type II kinase, the regulatory subunit (RII) can be distinguished from RII found in other tissues such as heart. Heart and brain RII were distinguished qualitatively by autophosphorylation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mobility of dephosphorylated heart RII shifted from an apparent Mr of 55,000 to 57,000 following autophosphorylation. In contrast, when RII purified from brain was autophosphorylated with [gamma-32P]ATP, two radiolabeled bands were visualized, a minor band (less than or equal to 20%) which migrated with an Mr of 57,000 similar to the heart protein and a band with Mr = 55,000 which did not shift its mobility in response to autophosphorylation. Brain RII was further distinguished from heart RII on the basis of cAMP binding. Millipore filtration and equilibrium dialysis indicated that 2 mol of cAMP bound/mol of RII in contrast to 4 mol/mol with heart RII. Immunological differences were also apparent. Radioimmunoassays using monoclonal antibodies to RII showed that the brain protein had less than 4% of the cross-reactivity of heart RII. Both immunoblotting and immunoprecipitation using monoclonal as well as serum antibodies established that the cross-reactivity in phosphorylated brain RII was associated exclusively with the 57,000 component that behaved like heart RII. The lack of cross-reactivity of neural RII with two different monoclonal antibodies targeted the hinge region of RII as an area where structural differences might be anticipated, and comparative sequence analysis of this region definitively established that the major form of RII in brain is a unique gene product from the RII expressed in heart.

Monoclonal antibodies as probes for functional domains in cAMP-dependent protein kinase II.

The antigenic regions of the type II regulatory subunit of cAMP-dependent kinase from bovine heart have been correlated with the previously established domain structure of the molecule. Immunoblotting with both serum and monoclonal antibodies of fragments generated by limited proteolysis or chemical cleavage of the R-subunit established that the major antigenic sites were confined to the amino-terminal portion of the polypeptide chain (residues 1-145). Radioimmunoassays using two different antisera suggested that one or more of the high affinity serum antibody recognition sites were further restricted to residues 91-145. This amino-terminal portion of the R-subunit includes the hinge region which is particularly sensitive to proteolysis, allowing the R-subunit to be cleaved readily into a COOH-terminal domain which retains the cAMP-binding sites and an NH2-terminal fragment which appears to be the major site for interaction of the R-subunits in the native dimer. Monoclonal antibodies that recognized determinants on both sides of this hinge region were characterized and their specific recognition sites localized. Accessibility of antigenic sites in the holoenzyme in contrast to free R2 was compared. Although cAMP did tend to slightly increase the affinity of the holoenzyme for one of the monoclonal antibodies, all of the antigenic sites clearly were exposed and accessible in the holoenzyme. Furthermore, despite the presumed close proximity of antigenic sites to interaction sites between the R- and C-subunits, in no case did binding of antibody to the holoenzyme promote dissociation of the complex. The fact that the monoclonal antibodies would precipitate holoenzyme as well as free R2 was used to ascertain the importance of specific amino acid residues in the interaction of the R- and C-subunits. cAMP-binding domains were isolated following limited proteolysis with chymotrypsin and thermolysin. These fragments differed by only three amino acid residues at the NH2-terminal end. U of these fragments in conjunction with immunoadsorption established that the chymotryptic fragment, which contained the Asp-Arg-Arg preceding the site of autophosphorylation, was capable of forming a stable complex with the C-subunit. In contrast, the thermolytic fragment which differed by only those three residues no longer complexed with the C-subunit, indicating that the arginine residues not only contribute to the specificity of the phosphorylation site but also are an essential component for energetically stabilizing the holoenzyme complex.

Monoclonal antibodies as probes of structure, function and isoenzyme forms of the type II regulatory subunit of cyclic AMP-dependent protein kinase.

We have attempted to review studies which have utilized monoclonal antibodies in the analysis of various aspects of type II cAMP-dependent protein kinase. It is readily apparent that the monoclonal antibodies directed against RII can be used in a number of ways to assess the structure and function of this protein. Monoclonal antibodies have been used to identify specific structural aspects of the protein, down to the level of amino acid sequence. These have included identification of relationships between several functional domains and the antigenic sites recognized by different antibodies. Monoclonal antibodies also have been used to specifically identify distinct isoenzyme forms of RII. The antibodies were shown to have virtually complete specificity for heart-type RII despite relatively few amino acid substitutions in neural-type RII. This discrimination was utilized to show that purified brain RII is composed of a small fraction of protein similar to the heart isozyme while the bulk of the protein is a distinct isozyme form. It is anticipated that future studies using antibodies specific for individual forms will be a valuable approach to analyzing the distribution and functions of different forms. Monoclonal antibodies have also been used as probes of RII in tissue extracts to examine phosphorylation of this subunit in intact tissue. Monoclonal antibodies should continue to provide powerful probes of the structure and function of this and other protein kinases.

Monoclonal antibodies as structural probes of surface residues in the regulatory subunit of cAMP-dependent protein kinase II from porcine heart.

Two murine monoclonal antibodies (H5 and B6) generated against bovine heart type II regulatory subunit of cAMP-dependent protein kinase were shown to cross-react equally well with the homologous subunit from porcine heart. The antibodies demonstrated specificity for only the type II regulatory subunit and showed negligible cross-reactivity with the type I regulatory subunit, the catalytic subunit, and cGMP-dependent protein kinase. Following limited proteolysis of type II regulatory subunit with chymotrypsin, the H5 monoclonal antibody was shown to cross-react with the Mr = 37,000 cAMP-binding domain corresponding to the COOH-terminal region of the polypeptide chain. To more specifically localize the antigenic sites, the porcine type II regulatory subunit was carboxymethylated and cleaved with cyanogen bromide. Both monoclonal antibodies cross-reacted with the NH2-terminal CNBr peptide, and this peptide demonstrated affinities similar to native bovine type II regulatory subunit in competitive displacement radioimmunoassays. Tryptic cleavage of this CNBr fragment destroyed all antigenicity for both monoclonal antibodies, whereas antigenicity was retained following chymotryptic digestion. A single major immunoreactive chymotryptic fragment that cross-reacted with H5 was isolated by gel filtration and reverse phase high performance liquid chromatography. this peptide retained the complete antigenic site and had the following sequence: Asn-Pro-Asp-Glu-Glu-Glu-Glu-Asp-Thr-Asp-Pro-Arg-Val-Ile-His-Pro-Lys-Thr-Asp-Gl n. This antigenic site was localized just beyond the major site of autophosphorylation, approximately a third of the distance from the NH2-terminal end of the polypeptide chain.

Isolation of picornavirus from feces and semen from an infertile bull.

Virus was isolated from semen and fecal samples from a bull with orchitis, testicular degeneration, aspermatogenesis, and loss of libido. Both isolates were classified as picornavirus, bovine enterovirus serotype I, on the basis of physical, chemical, and serologic characteristics. Veterinary practitioners that may suspect viral infection as a cause of bovine infertility should submit both semen and fecal samples for virus isolation and identification.