PubMed Health⌕ Search

Biomedical subjects

J Bubis

Publications and source records attributed to J Bubis.

25 records · Page 2Linked to original sources
PubMed1987

Limited proteolysis alters the photoaffinity labeling of adenosine 3',5'-monophosphate dependent protein kinase II with 8-azidoadenosine 3',5'-monophosphate.

Photoaffinity labeling of the regulatory subunits of cAMP-dependent protein kinase with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) has proved to be a very specific method for identifying amino acid residues that are in close proximity to the cAMP-binding sites. Each regulatory subunit contains two tandem cAMP-binding sites. The type II regulatory subunit (RII) from porcine heart was modified at a single site, Tyr-381 [Kerlavage, A., & Taylor, S.S. (1980) J. Biol. Chem. 255, 8483-8488]. When a proteolytic fragment of this RII subunit was photolabeled with 8-N3cAMP, two sites were covalently modified. One site corresponded to Tyr-381 and, thus, was analogous to the native RII. The other site of modification was identified as Tyr-196, which is not labeled in the native protein. Photoaffinity labeling was carried out in the presence of various analogues of cAMP that show a preference for one of the two tandem cAMP-binding sites. These studies established that the covalent modification of Tyr-381 was derived from 8-N3cAMP that was bound to the second cAMP-binding site (domain B) and that covalent modification to Tyr-196 was due to 8-N3cAMP that was bound to the first cAMP-binding site (domain A). These sites of covalent modification have been correlated with a model of each cAMP-binding site on the basis of the crystal structure of the catabolite gene activator protein (CAP), which is the major cAMP-binding protein in Escherichia coli.

Adenosine Triphosphate↗
PubMed1987

Correlation of photolabeling with occupancy of cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I.

Each regulatory subunit of the cAMP-dependent protein kinase contains two in-tandem cAMP binding sites. Photolabeling of holoenzyme I with 8-azidoadenosine 3',5'-monophosphate (8-N3-cAMP) leads to the covalent modification of two residues, Trp-260 and Tyr-371. In order to correlate photolabeling of these two residues with occupancy of each specific cAMP binding site, photolabeling was carried out in the presence of various analogues of cAMP that bind preferentially to one site. Photolabeling of holoenzyme I after dissociation of 60% of 8-N3-[3H]cAMP with an excess of N6-monobutyryl-cAMP nearly abolished the incorporation of 8-N3-cAMP into Trp-260, whereas the modification of Tyr-371 was reduced by 49%. When 8-N3-[32P]cAMP was bound under equilibrium conditions in the presence of various cAMP analogues, N6-monobutyryl-cAMP also selectively abolished incorporation of radioactivity into Trp-260, whereas 8-(methylamino)-cAMP preferentially reduced the covalent modification of Tyr-371. Photolabeling with trace amounts of 8-N3-[32P]cAMP in the presence of saturating amounts of N6-monobutyryl-cAMP led to the covalent modification of only Tyr-371. In addition, photolabeling of Tyr-371 was enhanced synergistically in the presence of N6-monobutyryl-cAMP. MgATP reduced the covalent modification of both Trp-260 and Tyr-371 but showed no selectivity for either site. These studies support a model that correlates photolabeling of Trp-260 with occupancy of cAMP binding site A and photolabeling of Tyr-371 with occupancy of cAMP binding site B.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1987

Predicted structures of cAMP binding domains of type I and II regulatory subunits of cAMP-dependent protein kinase.

The mammalian cAMP-dependent protein kinases have regulatory (R) subunits that show substantial homology in amino acid sequence with the catabolite gene activator protein (CAP), a cAMP-dependent gene regulatory protein from Escherichia coli. Each R subunit has two in-tandem cAMP binding domains, and the structure of each of these domains has been modeled by analogy with the crystal structure of CAP. Both the type I and II regulatory subunits have been considered, so that four cAMP binding domains have been modeled. The binding of cAMP in general is analogous in all the structures and has been correlated with previous results based on photolabeling and binding of cAMP analogues. The model predicts that the first cAMP binding domain correlates with the previously defined fast dissociation site, which preferentially binds N6-substituted analogues of cAMP. The second domain corresponds to the slow dissociation site, which has a preference for C8-substituted analogues. The model also is consistent with cAMP binding in the syn conformation in both sites. Finally, this model has targeted specific regions that are likely to be involved in interdomain contacts. This includes contacts between the two cAMP binding domains as well as contacts with the amino-terminal region of the R subunit and with the catalytic subunit.

Amino Acid Sequence↗
PubMed1985

Covalent modification of both cAMP binding sites in cAMP-dependent protein kinase I by 8-azidoadenosine 3',5'-monophosphate.

Reconstituted porcine cAMP-dependent protein kinase type I was labeled with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) to study cyclic nucleotide binding and to identify amino acid residues that are either in or in close proximity to the cAMP binding sites. The photoaffinity analogue 8-N3cAMP behaved as cAMP itself with respect to cyclic nucleotide binding. For both cAMP and 8-N3cAMP, 2 mol of nucleotide was bound per mole of type I regulatory subunit monomer (RI), the apparent Kd's observed were approximately 10-17 nM on the basis of either Millipore filtration assays, equilibrium dialysis, or ammonium sulfate precipitation, Scatchard plots showed positive cooperativity, and (4) the Hill coefficients were approximately 1.5-1.6. After photolysis and addition of an excess of cAMP, approximately 1 mol of 8-N3cAMP/mol of RI monomer was covalently incorporated. Tryptic digestion of the labeled protein revealed that two unique tryptic peptides were modified. Proline-271 and tyrosine-371 were identified as the two residues that were covalently modified by 8-N3cAMP in RI. These results contrast with the type II regulatory subunit (RII) where 8-N3cAMP modified covalently a single tyrosine residue [Kerlavage, A. R., & Taylor, S. S. (1980) J. Biol. Chem. 255, 8483-8488]. RI contains two adjacent regions of sequence homology in the COOH-terminal fragment that binds two molecules of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1983

Comparative studies of pig platelet membrane proteins.

The proteins and glycoproteins of pig platelet membranes have been studied using gel electrophoretic techniques. A nomenclature is suggested from the apparent molecular weights estimated by one-dimensional electrophoresis. Isoelectric focusing showed that the majority of the proteins are in the 4.0-7.0 pH range. Subunits have been inferred from oligoproteins by two-dimensional, reduced-nonreduced, electrophoresis techniques. High resolution two dimensional electrophoresis combining isoelectric focusing and sodium dodecyl sulphate allows the observations of 60 polypeptide bands. An identification of some of those bands based on a correlation from reported human blood platelet membrane proteins is presented for comparison.

Animals↗
PubMed1983

Use of antibodies to intermediate filaments in the diagnosis of metastatic amelanotic malignant melanoma.

Immunofluorescent staining of tissue from a lung tumor detected 12 years after excision of a primary malignant melanoma of the skin was negative for prekeratin and positive for vimentin, indicating that the tumor was not epithelial in origin and excluding carcinoma from the differential diagnosis. Complementary conventional staining with hematoxylin-eosin confirmed the melanocytic origin of the tumor, indicating that it was probably an amelanotic metastasis of the original malignant melanoma. The findings in this case demonstrate the potential usefulness of immunohistochemical microscopic characterization of specific intermediate filament proteins in the diagnosis of otherwise ambiguous cases of amelanotic melanoma.

Antibodies↗