Abstract
Three monoclonal antibodies, 1F11, 1G11, and 2F5, were developed toward rabbit microsomal cytochrome P-450 1. Each was linked to Sepharose in order to indirectly immunoprecipitate microsomal proteins. All three antibodies bind polypeptides from solubilized microsomes that exhibit the same electrophoretic mobility as P-450 1. However, 2F5 recognizes an additional microsomal protein that exhibits a relative electrophoretic mobility between that of P-450 1 and 3b that does not appear to be P-450 2. 1G11 also reacts with several microsomal proteins that include both P-450 1 and P-450 3b. These results indicate that at least three electrophoretically distinct forms of microsomal P-450 share one or more antigenic determinants with P-450 1. Binding studies demonstrate that each antibody can react independently with P-450 1, indicating that the three antigenic determinants recognized by the respective monoclonal antibodies are spatially distinct and nonoverlapping. Reconstituted P-450 1 isolated from rabbit liver microsomes that exhibit 10-fold higher progesterone 21-hydroxylase than most preparations of liver microsomes obtained from outbred New Zealand White rabbits catalyzes this reaction with relatively high rates compared to that exhibited by microsomes or to that catalyzed by five electrophoretically distinct forms of P-450. Both the 1F11 and 2F5 antibodies extensively inhibit the liver microsomal 21-hydroxylation of progesterone. When the 1F11 antibody is used to indirectly immunoprecipitate the antigens it recognizes in microsomes displaying a high rate of progesterone 21-hydroxylase activity, a single electrophoretic band corresponding in mobility to P-450 1 was observed.
Highlights
From the Departmentof Basic and Clinical Research, Division of Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California 92037
Thesethreeantibodies were chosen duringinitialtyping because they exhibited distinct patterns of reactivity; l G l l reacted with P-450 36 in addition to P-450 1, l F l l displayed differencesinbinding to microsomes that exhibit high 21hydroxylase activity when compared to those that exhibilot w activity, and 2F5 recognized kidney microsomes whereas the Microsomes and individual forms of cytochrome P-450 were prepared as described previously [9, 10] with minor modifications
The results presentedhere indicate that thel F l l antibody extensively inhibits the progesterone21-hydroxylase activity of rabbit liver microsomes
Summary
From the Departmentof Basic and Clinical Research, Division of Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California 92037. Ll forms of P-450 revealed very little cross-reactivity among three antibodies bind polypeptides from solubilimzeid- heterologous cytochromes as judged by double immunodiffucrosomes that exhibit the same electrophoreticmobil- sion analysis [1].immunological cross-reactivityhas ity as P-4501.2F5 recognizes an additional been demonstrated for rat P-450 b and e as well as rat P-450 microsomal protein that exhibitas relative electropho- c and d [2,3,4]. This together with recent evidence indicating tnmr4reo5oittc0ipcraho3pmosbporo.eembTatiirahcllieattpysloleryobdrtbePeeietssi-wtnui4esnlt5ecstn0thft2iaohn.trad1mtiiGncosacfltllPeua-dtole4hsfao5btm0or1atiecthaParnloc-edtsa4so3s5mtbw0att1hilhtrhaaPente-eddsl4eoePv5ecs--e0raltidlnihemhariietvtreeeeddlnetpactnrdritomiifpbfahoircodyuryleattwmyichiaonelfonlyduaesdcfeiiidsdntisifnneogqrcuttftehohnerecmiemsspm(e5ocu-ifnf8oi)Pcci-uht4yne5dm0oeifrcmsacalaosycerehrssuahmrtaah-rceeshare one or more antigenic determinants withP-450 terization of structurally related yet dissimilar forms of P1.Binding studies demonstratethat each antibody can 450. Immunological reagents can contributegreatly to the characterizationof this isozyme system, and their utilityreflects their specificity
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