Graduate Thesis Or Dissertation
 

Protein methylation at sites of blood vessel injury

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  • Blood vessel injury was found to release intracellular pools of protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT) into the extracellular milieu, where it became trapped. Trapped PIMT was able to utilize radiolabeled S-adenosyl-L-methionine (AdoMet) introduced into the circulation to methylate blood vessel proteins containing altered aspartyl residues specifically at the site of injury. In vitro studies more fully characterized this endogenous PIMT activity in thoracic aorta and inferior vena cava. At least 50% of the PIMT activity released during injury, was resistant to non-ionic detergent extraction, suggesting that the enzyme activity can become trapped within or behind the extracellular matrix (ECM). Analysis of inferior vena cava, found that 90% of the altered aspartyl residues in blood vessels are inaccessible to methylation by intracellular PIMT under physiological conditions. Subfractionation of inferior vena cava on the basis of solubility found that at least 40% of the altered aspartyl containing proteins in blood vessels are insoluble in non-ionic detergent containing buffers and are highly resistant to extraction by protein denaturants. Analysis of peptides revealed that the majority of the altered aspartyl groups in blood vessels are located extracellularly. Digestion of these extracellular matrix proteins with cyanogen bromide (CNBr), followed by methylation with (PIMT), found that about 60% of the altered aspartyl residues in the ECM are solubilized by this treatment. The presence of hydroxyproline in amino acid hydrosolates of this fraction and acidic pH gel electrophoresis of methylated peptides, allowed the identification of collagen as the major PIMT substrate in the CNBr-soluble material. CNBr peptides derived from both type I and type III collagen were found to methylated. It is estimated that one centimeter of blood vessel contains on the order of 5 x 10¹⁴ altered aspartyl residues involving 1% to 5% of the total extracellular protein.
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