Biomedical subjects

Barry S Cooperman

Publications and source records attributed to Barry S Cooperman.

22 records · Page 2Linked to original sources

PubMed2002

Inhibition of prostate-specific antigen (PSA) by alpha(1)-antichymotrypsin: salt-dependent activation mediated by a conformational change.

Prostate-specific antigen (PSA) and its SDS-stable complex with the serine proteinase inhibitor (serpin) alpha(1)-antichymotrypsin (ACT), which is the dominant form of PSA in serum, are in widespread use as markers for the diagnosis of prostate cancer, and there is increasing evidence for the involvement of PSA proteinase activity itself in the development of prostate and other cancers. However, both the formation and degradation of the PSA-ACT complex, denoted PSA*ACT* to indicate substantial changes in the structure of both proteins on complex formation, have been incompletely studied. Here we determine rate and equilibrium constants for the steps involved in PSA*ACT* formation and demonstrate that (a) the effects of added NaCl, polyamines, and Zn(2+) on this process parallel their effects on PSA catalytic activity [Hsieh, M.-C., and Cooperman, B. S. (2000) Biochim. Biophys. Acta 1481, 75-87], (b) the effect of added NaCl in dramatically increasing the rate of ACT inhibition of PSA correlates with salt-induced changes in PSA conformation, and (c) the PSA*ACT* complex is subject to proteolysis by human neutrophil elastase. Possible clinical implications of these findings are considered.

Electrophoresis, Polyacrylamide Gel↗

PubMed2002

Affinity-driven selection of tripeptide inhibitors of ribonucleotide reductase.

Tripeptide libraries of the type Fmoc(W/F)XF were screened for binding to the large subunit of mouse ribonucleotide reductase (mRR), using a new, affinity chromatography method. A high-affinity tripeptide, FmocWFF, was found that inhibited mRR activity with a K(i) equal to that of AcFTLDADF, the heptapeptide corresponding to the C-terminus of the small subunit of mRR.

Amino Acids↗

PubMed2002

A comprehensive model for the allosteric regulation of mammalian ribonucleotide reductase. Functional consequences of ATP- and dATP-induced oligomerization of the large subunit.

Reduction of NDPs by murine ribonucleotide reductase (mRR) requires catalytic (mR1) and free radical-containing (mR2) subunits and is regulated by nucleoside triphosphate allosteric effectors. Here we present a new, comprehensive, and quantitative model for allosteric control of mRR enzymatic activity based on molecular mass, ligand binding, and enzyme activity studies. In this model, nucleotide binding to the specificity site (s-site) drives formation of an active R1(2)R2(2) dimer, ATP or dATP binding to the adenine-specific site (a-site) results in formation of an inactive tetramer, and ATP binding to the newly described hexamerization site (h-site) drives formation of active R1(6)R2(6) hexamer. In contrast, an earlier phenomenological model [Thelander, L., and Reichard, P. (1979) Annu. Rev. Biochem. 67, 71-98] (the "RT" model) ignores aggregation state changes and mistakenly rationalizes ATP activation versus dATP inhibition as reflecting different functional consequences of ATP versus dATP binding to the a-site. Our results suggest that the R1(6)R2(6) heterohexamer is the major active form of the enzyme in mammalian cells, and that the ATP concentration is the primary modulator of enzyme activity, coupling the rate of DNA biosynthesis with the energetic state of the cell. Using the crystal structure of the Escherichia coliR1 hexamer as a model for the mR1 hexamer, a scheme is presented that rationalizes the slow isomerization of the tetramer form and suggests an explanation for the low enzymatic activity of tetramers complexed with R2. The similar specific activities of R1(2)R2(2) and R1(6)R2(6) are inconsistent with a proposed model for R2(2) docking with R1(2) [Uhlin, U., and Eklund, H. (1994) Nature 370, 533-539], and an alternative is suggested.

Adenine↗

PubMed2002

Large-scale motions within ribosomal 50S subunits as demonstrated using photolabile oligonucleotides.

Photolabile oligonucleotides (PHONTs) bind to rRNA sequences to which they are complementary and, on photolysis, incorporate into neighboring ribosomal components. Here we report on photocrosslinking results obtained with PHONTs targeting 23S rRNA nucleotides 1882-1892, in the long lateral arm of the 50S subunit (PHONT 1892), and 1085-1093, in the L11 binding domain (PHONT 1093). Photolysis of the PHONT 1892.50S and PHONT 1093.50S complexes leads to formation of 'long-range' crosslinks from C1892 to U1094/A1095 and G1950, and from G1093 to U1712/1716 and U1926, that are clearly incompatible with published crystal structures of 50S subunits. These results provide strong evidence that within the 50S subunit (a) the L11 binding domain can extend in an arm-like fashion, accessing large areas of the ribosome, and (b) the lateral arm can bend about the noncanonical helix at its center. Such motions may have functional relevance in identifying regions that undergo major conformational change as the ribosome moves through its catalytic cycle.

Base Sequence↗