Interaction of hemorrhagic metalloproteinases with human alpha 2-macroglobulin.
The interaction between four Crotalus atrox hemorrhagic metalloproteinases and human alpha 2-macroglobulin was investigated. The proteolytic activity of the hemorrhagic toxins Ht-c, -d, and -e against the large molecular weight protein substrates, gelatin type I and collagen type IV, was completely inhibited by alpha 2-macroglobulin. The proteolytic activity of Ht-a against the same substrates was not significantly inhibited. Each mole of alpha 2-macroglobulin bound maximally 2 mol of Ht-e and 1.1 mol of Ht-c and Ht-d. These proteinases interacted with alpha 2-macroglobulin rapidly at 22 degrees C. Rate constants based on intrinsic fluorescence measurements were 0.62 X 10(5) M-1 s-1 for interaction of alpha 2-macroglobulin with Ht-c and -d and 2.3 X 10(5) M-1 s-1 for the interaction of alpha 2-macroglobulin with Ht-e. Ht-a interacted with alpha 2-macroglobulin very slowly at 22 degrees C. Increasing the temperature to 37 degrees C and prolonging the time of interaction with alpha 2-macroglobulin resulted in the formation of Mr 90,000 fragments and high molecular weight complexes (Mr greater than 180,000), in which Ht-a is covalently bound to the carboxy-terminal fragment of alpha 2-M. The identification of the sites of specific proteolysis of alpha 2-macroglobulin shows that the cleavage sites for the four metalloproteinases are within the bait region of alpha 2-macroglobulin. Ht-c and -d cleave only at one site, the Arg696-Leu697 peptide bond, which is also the site of cleavage for plasmin, thrombin, trypsin, and thermolysin. Ht-a cleaves alpha 2-macroglobulin primarily at the same site, but a secondary cleavage site at the His694-Ala695 peptide bond was also identified.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Microbiology, University of Virginia Medical School, Charlottesville 22908.
E N
EN
Baramova
J D
JD
Shannon
J B
JB
Bjarnason
S L
SL
Gonias
J W
JW
Fox
D000595:Amino Acid Sequence; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003094:Collagen / Q000378:metabolism; D003435:Crotalid Venoms / Q000378:metabolism; D005780:Gelatin / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000378:metabolism*
D003435:Crotalid Venoms; D000511:alpha-Macroglobulins; C032814:hemorrhagin components (Crotalid); D005780:Gelatin; D003094:Collagen; D008666:Metalloendopeptidases; D015032:Zinc