Relative deficiency of acidic isoforms of osteopontin from stone former urine. Urol Res 2012 Oct;40(5):447-54
Date
02/11/2012Pubmed ID
22322528Pubmed Central ID
PMC4978133DOI
10.1007/s00240-012-0459-1Scopus ID
2-s2.0-84867899105 (requires institutional sign-in at Scopus site) 16 CitationsAbstract
We have tested the relative electrophoretic mobility of osteopontin (OPN) isolated from urine obtained from normal individuals (NU) against similar samples derived from the urine of stone formers (SFU) using high-resolution isoelectric focusing (isoelectric point, pI range 3.5-4.5) in 2D electrophoresis, with Western blot detection. We also report the results from competitive ELISA analyses of these samples. We demonstrated that human urinary OPN has a discrete four band separation pattern that conforms to four previously documented OPN isoforms. The lower two M(r) isoforms migrate to a greater degree toward the acidic end of the gel than do the higher two M(r) isoforms. Densitometry of the signal reveals significant difference in the migration pattern of OPN from SFU as compared to that from NU based on an analysis of the spot intensities grouped in 0.1 pI unit increments. A novel method for the calculation of a weight-averaged pI based on the relative signal strength in an OPN 2D Western blot was developed. The analysis revealed a significantly increased weight-averaged pI values for the higher M(r) forms of OPN in the stone former compared to normal population. Additionally, alkaline phosphatase-treated NU samples resulted in a significant average pI shift of 0.05 units in the alkaline direction, suggesting that a decrease in the average degree of phosphorylation could be responsible for the difference between NU and SFU pI.
Author List
Kolbach AM, Afzal O, Halligan B, Sorokina E, Kleinman JG, Wesson JAAuthor
Jeffrey A. Wesson MD, PhD Professor in the Medicine department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AdultAged
Blotting, Western
Densitometry
Enzyme-Linked Immunosorbent Assay
Female
Humans
Male
Middle Aged
Osteopontin
Phosphorylation
Protein Isoforms
Urinary Calculi
