Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thrombospondin is a 450-kDa glycoprotein secreted by a variety of cells including endothelial cells, fibroblasts and platelets. The aim of this study was to compare the structural and immunological properties of human endothelial, fibroblast and platelet thrombospondins. All three thrombospondins were purified, digested with thermolysin, and the subsequent thermolysin-generated fragments isolated on a Superose 12 gel-permeation column using non-denaturating conditions. Each isolated proteolytic fragment of thrombospondins was then detected using either a radioimmunoassay with a polyclonal antibody or an enzyme-linked immunosorbent assay with three monoclonal antibodies (P10, MA-I, MA-II) directed against different epitopes of whole platelet thrombospondin. The fragmentation pattern of human endothelial thrombospondin consists of six major thermolysin-generated fragments (135-110, 98-82, 54-47, 25-20, 18-15 and 10 kDa) having molecular masses very similar to those observed with human fibroblast thrombospondin (115-100, 92-80, 54-49, 27-21, 17-13 and 12-10 kDa). Treatment of platelet thrombospondin with thermolysin only generated four proteolytic fragments having molecular masses of 110, 50, 25 and 12/10 kDa respectively. All these proteolytic fragments of endothelial, fibroblast and platelet thrombospondins were recognized by a polyclonal antibody. Monoclonal antibodies MA-I and P10 essentially recognized two proteolytic fragments (135-110, 98-82 kDa) of endothelial and fibroblast (115-100, 92-80 kDa) thrombospondins, and the 110-kDa fragment of platelet thrombospondin. Monoclonal antibody MA-II recognized three proteolytic fragments (54-47, 25-20, 18-15 kDa) of endothelial and fibroblast (54-49, 27-21, 17-13 kDa) thrombospondins, and two fragments (50, 25 kDa) of platelet thrombospondin, different from those detected by P10 an MA-I. The results clearly demonstrate that, under non-denaturating conditions, endothelial and fibroblast thrombospondins are structurally different from platelet thrombospondin since two fragments of endothelial thrombospondin (98-82, 18-15 kDa), equivalent to those of fibroblast thrombospondin (92-80, 17-13 kDa), are not released from platelet thrombospondin after thermolysin treatment. These three forms of thrombospondin are, however, immunologically indistinguishable. To investigate further the structural differences observed between platelet and the two other forms of thrombospondin, their degree of polymerization was compared. Prior to thermolysin treatment, the three forms of thrombospondin were separated into several oligomers ranging from 450 kDa to 3300 kDa when injected onto a Superose 6 gel-permeation column.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structural and immunological comparison of human thrombospondins isolated from platelets and from culture supernatants of endothelial cells and fibroblasts. Evidence for a thrombospondin polymorphism. 375 79

Amine oxidases (EC 1.4.3.6) from Aspergillus niger, AO-I (2 x 75 kDa) and AO-II (80 kDa), were examined to determine the cofactor structure. Inactivated with p-nitrophenylhydrazine, they showed absorption and fluorescence spectra similar to those published for other copper amine oxidases and to topa hydantoin p-nitrophenylhydrazone. After digestion by thermolysin and pronase, cofactor peptides were purified by HPLC and sequenced. For thermolytic peptides, a typical topa consensus sequence, Asn-X-Glu-Tyr, was obtained for AO-II, although in case of AO-I it overlapped with Val-Val-Ile-Glu-Pro-Tyr-Gly. For pronase peptides of AO-I, only the latter sequence was obtained. NMR and mass spectroscopy confirmed the residue X as topa p-nitrophenylhydrazone in AO-II and revealed the presence of a residue Z attached to the Glu in the peptide Val-Val-Ile-Glu(Z)-Pro of AO-I. This residue was separated from the peptide by hydrolysis and identified as a product derived from topa quinone. The data, together with amino-acid sequence of AO-I, confer strong evidence for topa quinone as the cofactor, bound in the typical consensus sequence. Raman spectra of the p-nitrophenylhydrazone derivative of AO-I and its pronase peptide showed essentially the same peaks matching to a model compound for topa p-nitrophenylhydrazone. However, there may exist an unusual ester link between the topa-404 and Glu-145 in the native enzyme.
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PMID:Two amine oxidases from Aspergillus niger AKU 3302 contain topa quinone as the cofactor: unusual cofactor link to the glutamyl residue occurs only at one of the enzymes. 867 75