Biomedical subjects
Publications and source records attributed to M Bucciantini.
Single crystals of a low molecular weight phosphotyrosine protein phosphatase from bovine liver have been grown. The crystals belong to space group P2(1)2(1)2(1), have cell dimensions a = 46.3 A, b = 62.2 A, c = 62.7 A and diffract to better than 2.0 A resolution. The crystals are well suited for high resolution X-ray studies.
Three peptides complementary to exposed regions of two low-M(r) phosphotyrosine protein phosphatase isoenzymes and of the acylphosphatase muscle isoenzyme have been synthesized. Each peptide was synthesized on two different types of resins; the peptides were anchored to the resins by amide linkages. The peptide resins were checked by amino acid analysis and Edman degradation and directly used for enzyme purification. Despite our attempts, none of the resins was able to bind significant amounts of the corresponding protein, indicating the lack of interaction between the three proteins and the corresponding complementary peptides. This result agrees with many other reports, confirming that the molecular-recognition theory has no general validity.
Five phosphotyrosine-containing peptides have been synthesized by FMOC solid-phase peptide synthesis. These peptides correspond to the 411-419 sequence of the Xenopus src oncogene, to the 1191-1220 sequence of the human EGF receptor precursor, to the 1146-1158 sequence of the human insulin receptor, to the 856-865 sequence of the human beta-PDGF receptor, and to the 5-16 sequence of the erythrocyte human band 3. The peptides were used as substrates for activity assay of two isoforms (AcP1 and AcP2) of a low molecular weight cytosolic PTPase. The assay, performed in microtiter EIA plates using Malachite green to determine the released phosphate, was rapid, reproducible, and sensitive. Both PTPase isoforms were able to hydrolyze all synthesized peptides, though with different affinity and rate. The main kinetic parameters were compared and discussed with respect to the role of the two enzymes in the cell.
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