Abstract
Injury to rat blood vessels in vivo was found to release intracellular pools of protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT) into the extracellular milieu, where it becomes trapped. This trapped cohort of PIMT is able to utilize radiolabeled S-adenosyl-L-methionine (AdoMet) introduced into the circulation to methylate blood vessel proteins containing altered aspartyl residues. As further shown in this study, methylated substrates are detected only at the specific site of injury. In vitro studies more fully characterized this endogenous PIMT activity in thoracic aorta and inferior vena cava. Methylation kinetics, immunoblotting, and the lability of methylated substrates at mild alkaline pH were used to demonstrate that both types of blood vessel contain an endogeneous protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT). At least 50% of the PIMT activity is resistant to nonionic detergent extraction, suggesting that the enzyme activity becomes trapped within or behind the extracellular matrix (ECM). Quantities of lactate dehydrogenase (LDH), another soluble enzyme of presumed intracellular origin, were found to be similarly trapped in the extracellular space of blood vessels.
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REFERENCES
Ahn, S. S., Arca, M. J., et al. (1990). Histological and morphologic effects of rotary atherectomy on human cadaver arteries, Ann. Vasc. Surg. 4, 563–569.
Aswad, D. (1995). Purification and properties of protein L-isoaspartyl methyltransferase, in Deamidation and Isoaspartate Formation in Peptides and Proteins (Aswad, D. W., ed.), CRC Press, Boca Raton, Florida, pp. 32–45.
Aswad, D. W., and Deight, E. A. (1983). Endogenous substrates for protein carboxyl methyltransferase in cytosolic fractions of bovine brain, J. Neurochem. 31, 1702–1709.
Ben, B. G., Paz, A., et al. (1993). The uniquely distributed isoprenylated protein methyltransferase activity in the rat brain is highly expressed in the cerebellum, Biochem. Biophys. Res. Commun. 195, 282–288.
Bert, J. L., Pearce, R. H., et al. (1980). Characterization of collagenous meshworks by volume exclusion of dextrans, Biochem. J. 191, 761–768.
Boivin, D., Bilodeau, D., and Beliveau, R. (1995). Immunochemical characterization of L-isoaspartyl-protein carboxyl methyltransferase from mammalian tissues, Biochem. J. 309, 993–998.
Campbell, E., Pierce, J., et al. (1991). Evaluation of extracellular matrix turnover, Chest 99, 49S.
Campbell, J., Kocher, O., et al. (1989). Cytodifferentiation and expression of alpha-smooth muscle actin mRNA and protein during primary culture of aortic smooth muscle cells, Arteriosclerosis 9, 633–643.
Chameley-Campbell, J., Cambell, G. R., and Ross, R. (1979). The smooth muscle cell in culture, Physiol. Rev. 59, 2–61.
Clarke, S. (1985). Protein carboxyl methyltransferases: Two distinct classes of enzymes, Annu. Rev. Biochem. 54, 479–50.
Decker, T., and Lohmann-Matthes, M. (1988). A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity, J. Immunol. Meth. 15, 61–69.
Ghomashchi, F., Zhang, X., et al. (1995). Binding of prenylated and polybasic peptides to membranes: Affinities and intervesicle exchange, Biochemistry 34, 11910–11918.
Gilbert, J. M., Fowler, A., et al. (1988). Purification of homologous protein carboxyl methyltransferase isozymes from human and bovine erythrocytes, Biochemistry 27, 5227–5233.
Gingras, D., Menard, P., and Beliveau, R. (1991). Protein carboxyl methylation in kidney brush-border membranes, Biochim. Biophys. Acta 1066, 261–267.
Gingras, D., Boivin, D., and Beliveau, R. (1994). Asymmetrical distribution of L-isoaspartyl protein carboxyl methyltransferases in the plasma membranes of rat kidney cortex, Biochem. J. 297, 145–150.
Giulidori, P., and Stramentinoli, G. (1984). A radioenzymatic method for S-adenosyl-L-methionine determination in biological fluids, Anal. Biochem. 137, 217–220.
Harlow, E., and Lane, D. eds. (1988). Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Springer Harbor, New York.
Heimark, R. L., and Schwartz, S. M. (1988). Cellular organization of blood vessels in development and disease, in Endothelial Cells (Ryan, U.S., ed.), CRC Press, Boca-Raton, Florida, Vol. 2, pp. 104–114.
Johnson, D. J., LaBourene, J., et al. (1993). Relative efficiency of incorporation of newly synthesized elastin and collagen into aorta, pulmonary artery and pulmonary vein of growing pigs, Connect. Tiss. Res. 29, 213–221.
Kimzey, A. L., and McFadden, P. N. (1994). Spontaneous methylation of hemoglobin by S-adenosylmethionine by a specific and saturable mechanism, J. Protein Chem. 13, 537–546.
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227, 680–685.
Lindquist, J. A., Barofsky, E., and McFadden, P. N. (1996). Determination of two sites of automethylation in bovine erythrocyte (D-aspartyl/L-isoaspartyl) carboxyl methyltransferase, J. Protein Chem. 15, 115–122.
Lowenson, J. D., and Clarke, S. (1992). Recognition of D-aspartyl residues in polypeptides by the erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase. Implications for the repair hypothesis, J. Biol. Chem. 267, 5985–5995.
McFadden, P. N., and Clarke, S. (1982). Methylation at D-aspartyl residues in erythrocytes: Possible step in the repair of aged membrane proteins, Proc. Natl. Acad. Sci. USA 79, 2460–2464.
McFadden, P. N., Horwitz, J., and Clarke, S. (1983). Protein carboxyl methyltransferase from cow eye lens, Biochem. Biophys. Res. Commun. 113, 418–424.
Meyer, F. A., Koblentz, M., and Silberberg, A. (1977). Structural investigation of loose connective tissue by using a series of dextran fractions as non-interacting macromolecular probes, Biochem. J. 161, 285–291.
Monos, E., Berczi, V., and Nadasy, G. (1995). Local control of veins: Biomechanical, metabolic and humoral aspects, Physiol. Rev. 75, 611–666.
O'Conner, C. M., Aswad, D. W., and Clarke, S. (1984). Mammalian brain and erythrocyte carboxyl methyltranserases are similar enzymes that recognize both D-aspartyl and L-isoaspartyl, Proc. Natl. Acad. Sci. USA 81, 7757.
Owens, G. K. (1995). Regulation of differentiation of vascular smooth muscle cells, Physiol. Rev. 75, 487–517.
Paik, W. K., and Kim, S., eds. (1990). Protein Methylation, CRC Press, Boca Raton, Florida.
Pearce, R. H., and Laurent, T. C. (1977). Exclusion of dextrans by meshworks of collagenous fibres, Biochem. J. 163, 617–625.
Pema, A. F., Ingrosso, D., et al. (1993). Enzymatic methyl esterification of erythrocyte membrane proteins is impaired in chronic renal failure. Evidence for high levels of the natural inhibitor S-adenosylhomocysteine, J. Clin. Invest. 91, 2497–2503.
Robert, L., and Labat-Robert, J. (1995). Extracellular matrix, in Molecular Basis of Aging (Macieira-Coelho, ed.), CRC Press, Boca Raton, Florida, pp. 459–488.
Takemoto, L. J. (1995). Degradation of aspartyl and asparaginyl residues of lens protein in vivo, in Deamidation and Isoaspartate Formation in Proteins and Peptides (Aswad, D. W., ed.), CRC Press, Boca Raton, pp. 157–166.
Weber, D. J., and McFadden, P. M. (1997). Injury-induced enzymatic methylation of aging collagen in the extracellular matrix of blood vessels, J. Protein Chem., 269–281.
Xie, H., and Clarke, S. (1994). An enzymatic activity in bovine brain that catalyzes the reversal of the C-terminal methyl esterification of protein phosphatase 2A, Biochem. Biophys. Res. Commun. 203, 1710–1715.
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Weber, D.J., McFadden, P.N. Detection and Characterization of a Protein Isoaspartyl Methyltransferase Which Becomes Trapped in the Extracellular Space During Blood Vessel Injury. J Protein Chem 16, 257–267 (1997). https://doi.org/10.1023/A:1026300924908
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DOI: https://doi.org/10.1023/A:1026300924908