IDENTIFICATION OF ALPHA-2-MACROGLOBULIN CONFORMATIONAL INTERMEDIATES BY ELECTRON-MICROSCOPY AND IMAGE-ANALYSIS - COMPARISON OF ALPHA-2-MACROGLOBULIN-THROMBIN AND ALPHA-2-MACROGLOBULIN REACTED WITH CIS-DICHLORODIAMMINEPLATINUM(II) AND TRYPSIN

被引:0
作者
MARSHALL, LB
FIGLER, NL
GONIAS, SL
机构
[1] UNIV VIRGINIA,HLTH SCI CTR,DEPT PATHOL,BOX 214,CHARLOTTESVILLE,VA 22908
[2] UNIV VIRGINIA,HLTH SCI CTR,DEPT BIOCHEM,CHARLOTTESVILLE,VA 22908
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中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Human alpha-2-macroglobulin (alpha-2M) exists in two well defined, highly distinct conformations and in less well described intermediate conformations. In this study, previously characterized reactions were used to partially or completely transform the conformation of alpha-2M. Electron micrographs of each preparation were subjected to image analysis. TernarY alpha-2M-trypsin (2 mol of trypsin/mol of alpha-2M) was analyzed as a control for the fully transformed state. Correspondence analysis (CORAN) and hierarchical ascendant classification (HAC) generated five image clusters from 330 aligned alpha-2M-trypsin complexes. Average images of each cluster resembled the letter "H" with four nearly equivalent lateral arms. Abnormally shaped lateral arms were not demonstrated by HAC, using a variety of factor sets. In a native polyacrylamide gel electrophoresis system, alpha-2M-thrombin migrated in a diffuse band partially behind alpha-2M-trypsin, suggesting conformational heterogeneity. CORAN and HAC of 733 alpha-2M-thrombin complexes identified two neighboring clusters, the average images of which showed an H-like structure in which one arm was replaced by a globular stain-excluding body. The two alpha-2M-thrombin clusters included 125 images (17.1 % of image population). The complete absence of atypical lateral arm structure in the alpha-2M-trypsin clusters suggests that this variation is not the result of orientation or staining artifact. Native alpha-2M was reacted with cis-dichlorodiammineplatinum(II) and then with trypsin to form alpha-2M-Pt-trypsin, a preparation that includes partially transformed alpha-2M structures. CORAN and HAC of 580 alpha-2M-Pt-trypsin complexes generated five clusters, the average images of which showed atypical lateral arm structure equivalent to that demonstrated with alpha-2M-thrombin. The five alpha-2M-Pt-trypsin clusters accounted for 15.2% of the image population. These studies suggest that alpha-2M conformational change intermediates demonstrate common structural characteristics, permitting an elucidation of the steps involved in this complex transformation.
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页码:6347 / 6352
页数:6
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