Immunochemical characterization of hypoxanthine guanine phosphoribosyl transferase (HGPRT) mutants

C. T. Caskey, C. S. Chiang, K. H. Astrin, R. G. Fenwick

Research output: Contribution to journalArticlepeer-review

Abstract

Complement fixation (CF) and radioimmune precipitation were used to study HGPRT structural gene mutations in cultured Chinese hamster cell lines. Monospecific immunoglobulins to native Chinese hamster HGPRT were used in the studies. CF is 10-fold more sensitive for detection of HGPRT crossreacting material (CRM) than previous methods. A number of mutants (E36, N10, N17, S76, U53) were HGPRT- and CRM-. A class of HGPRT+ mutants and revertants had altered CF optimum titers (M) and extents of fixation (e), indicating altered enzymes and structural gene mutations. If wild type M and E are defined as 1, then clone N3 had M of 0.28 and E of 0.72, revertants S76U5, M = 0.41, E = 0.98; N10S3, M = 0.61, E = 0.72; U53U2, M = 0.23, E = 0.12. Another HGPRT-, CRM+ mutant, E39, had M = 2.1 and E = 0.43. Urea denatured wild type extract had much reduced CF activity when tested similarly. Autoradiographic analysis, after SDS polyacrylamide slab gel electrophoresis of in vivo labeled (35S)HGPRT purified by immunoabsorption to anti-HGPRT sepharose gave CRM results identical to that of CF. The migration of N3 HGPRT subunit in SDS-polyacrylamide gels indicated a molecular weight 5.5% less than wild type. In order to further investigate HGPRT structural gene mutations that prevented the oligomerization of HGPRT subunits, antiserum to urea denatured HGPRT has recently been obtained. Preliminary tests showed that it reacted with urea denatured HGPRT on immunodiffusion plates and by complement fixation inhibition. These immunologic methods provide sensitive means of detecting and classifying structural alterations in animal cell mutants for a protein which occurs at a low level (0.029%).

Original languageEnglish
Pages (from-to)No.54
JournalUnknown Journal
VolumeNo. 397
StatePublished - 1976

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