Detection of sequence-specific tyrosine nitration of manganese SOD and SERCA in cardiovascular disease and aging.
Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies toward site-specific nY-modified proteins and to use histochemistry and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies toward peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with ANG II with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum with known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite, which chemically reduces nitrotyrosine to aminotyrosine, and the antigenic nY-peptide blocked staining for each respective nY site but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites.
Xu
Shanqin
S
Vascular Biology Unit X708, Whitaker Cardiovascular Unit, Evans Department of Medicine, Boston University Medical Center, 650 Albany St., Boston, MA 02118, USA.
Ying
Jia
J
Jiang
Bingbing
B
Guo
Wei
W
Adachi
Takeshi
T
Sharov
Viktor
V
Lazar
Harold
H
Menzoian
James
J
Knyushko
Tatyana V
TV
Bigelow
Diana
D
Schöneich
Christian
C
Cohen
Richard A
RA
D000375:Aging / Q000378:metabolism*; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002318:Cardiovascular Diseases / Q000378:metabolism*; D004227:Dithionite / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology; D030421:Peroxynitrous Acid / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013482:Superoxide Dismutase / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism
C506948:Atp2a2 protein, rat; D030421:Peroxynitrous Acid; D004227:Dithionite; C002744:3-nitrotyrosine; D014443:Tyrosine; D013482:Superoxide Dismutase; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506946:ATP2A2 protein, human; D000252:Calcium-Transporting ATPases