Cyclohydrolase preparation

To prepare lysates from (nonstimulated) fibroblasts, cells from one confluent 78-cm2 plate are suspended in 0.15 ml lysis buffer (see below) and lysed by freezing and thawing six times and subsequent centrifugation at 13,000 x for 5 min. An aliquot of 0.05 ml of the supernatant is directly used for the enzyme assay. The preparation of tissue homogenate is described in section 6.1.4.1. GTP cyclohydrolase I, subheading Specimen . 

The conversion of 5-formyl-tetrahydrofolate to methenyl-tetrahydrofolate, catalyzed by 5-formyl-tetrahydrofolate cyclohydrolase, is important. Although 5-formyl-tetrahydrofolate is the most commonly used pharmaceutical preparation of the vitamin, a relatively large proportion of orally administered... 

Fig. 6.5. A schematic presentation of the Ci cycle (prepared on the basis of Rouviere and Wolfe, 1988 Thauer and Bonacker, 1994 Vaupel and Thauer, 1998). Circled numbers 1, formylmetha-nofuran dehydrogenase (contains Fe/S, and Mo, or W) 2, formylmethanofuran-tetrahydrometha-nopterin A-formyltransferase 3, methenyltetrahydromethanopterin cyclohydrolase 4, methylenetetrahydromethanopterin dehydrogenase 5, methylenetetrahydromethanopterin reductase 6, tetrahydromethanopterin S-methyltransferase (contains Fe/S and vitamin B12) 7, methyl-CoM reductase (contains F430) 8, H2 heterodisulfide oxidoreductase...

The formylation of GAR to produce FGAR is catalyzed by glycinamide ribonucleotide transformylase. The immediate formyl donors are derivatives of tetrahydrofolic acid (FIR) (96, 107, 108). With purified enzyme preparations, iV, Ar -anhydroformyltetrahydrofolic acid alone donates its formyl group to GAR (/09). Because of their rapid interconversion by the enzyme cyclohydrolase (110), both JV -formyltetrahydrofolic acid and W, Ar -arjhydroformyltetrahydrofolio acid were reactive in crude enzyme... 

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