Tryptic peptide analysis of normal and mutant forms of hypoxanthine phosphoribosyltransferase from HeLa cells.

Abstract

Hypoxanthine phosphoribosyltransferase (HPRT, atmosphere of 5% C02/95% air. Methods for growing cells on plastic petri plates and for the selection of HPRT-deficient mutants or revertants have been previously described (12). When large quantities of cells are desired, they are grown in suspension at a density of S to 100 X 104/ml in Joklik's modified minimal essential medium (Gibco, 13.3 g of powder medium per liter) containing 5% calf serum (Irvine Scientific).Radioisotope-labeled amino acids are incorporated into HeLa proteins as previously described (12) buffer (10 mM MgCl2, 30 mM KC1, 0.1 mM dithiothreitol, 0.5% Triton X-100, and 10 mM Tris-HCl, pH 7.4) and centrifuged for 2 hr at 96,000 X g in a Spinco type 50 rotor. The high-speed supernatant fraction is removed, and to 2 ml supernatant are added 0.6 ml enzyme buffer (20 mM KC1, 6 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol, and 20 mM Tris-HCl, pH 7.8), 0.4 ml of a solution containing 10% Triton X-10(0 by volume and 10% sodium deoxycholate by weight, and 1.0 ml of 4 M NaCI. The sample is applied to 0.12 ml of

Human hypoxanthine phosphoribosyltransferase. Purification and properties

“…The enzyme appears to have a blocked N terminus. been extended to the human enzyme because our current genetic studies of HeLa hypoxanthine phosphoribosyltransferase (Milman et al, 1976(Milman et al, , 1977 and Lesch-Nyhan erythrocytes (Ghangas and Milman, 1975) require a knowledge of the characteristics of the normal human enzyme. We have adapted our procedures for purification of hypoxanthine phosphoribosyltransferase from Chinese hamster brain and liver to the purification of the enzyme from human erythrocytes.…”

Methotrexate-resistant Chinese hamster ovary cells contain a dihydrofolate reductase with an altered affinity for methotrexate

“…The studies described in this paper and those described by others with different markers in various systems (Ingles et al, 1976;Milman et al, 1977;Steinberg et al, 1977;Boersma et…”