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We present the first description of KmDNA, KdDNA, Kcat, and Kmethylation for a mammalian DNA methyltransferase. Homogeneous, 190 000 MTDNA (cytosine-5-)-methyltransferase isolated from mouse erythroleukemia cells has turnover constants of 0.15-0.59 h-1 with single-stranded and unmethylated double-stranded oligonucleotides containing a single CpG dinucleotide. These substrates were designed to mimic DNA transcriptional cis elements previously reported to have cytosine C-5-methylated regulation. The rate-limiting step for these substrates is the methylation step itself. In contrast, hemimethylated double-stranded substrates show burst kinetics, consistent with a rapid methylation event (3 h-1) followed by a slower step which determines steady-state Kcat. Hemimethylated and unmethylated double-stranded DNA shows similar binding affinities; these results reveal the molecular basis for the enzyme's preference for hemimethylated DNA to be the methyl transfer step. Substrates with multiple recognition sites do not show burst kinetics and have turnover rate constants of 6 h-1. Catalytic turnover for the mammalian enzyme is thus approximately 10-fold slower than that for the related bacterial enzymes. Our combined results show quantitatively that one enzyme is certainly capable of both maintenance and de novo methylation and that maintenance of the genomic methylation pattern is preferred over the de novo establishment of new patterns. Direct comparison of the mammalian enzyme with the bacterial DNA cytosine-C5 methyltransferase, M.SssI, indicates dramatic differences in preferences for single-stranded, double-stranded, and hemimethylated double-stranded substrates. Moreover, the specificity hierarchy shown for the M.SssI is derived from very different changes in K(m) and catalysis than those observed for the mammalian DCMTase. These results demonstrate that the M.SssI, and perhaps other DNA cytosine methyltransferases from bacteria, is functionally dissimilar to the mammalian enzyme.

Cytosine methylation within DNA has been implicated in genetic imprinting, X-chromosome inactivation, regulation of tissue-specific gene expression, aging, and cancer. Unfortunately, DNA (cytosine-5)-methyltransfersases (EC 2.1.1.37) from various mammalian sources have been difficult to isolate and stabilize, precluding investigations of these critical enzymes. We describe a novel FPLC purification of the 190,000 Mr DNA methyltransferase from mouse Friend erythroleukemia cells. The homogeneous 190 kD Mr form of the enzyme is the only polypeptide detected at various stages of cell growth and has not undergone detectable N-terminal proteolysis.

The murine C-5 cytosine DNA methyltransferase (MTase, E.C.2.1.1.37) containing a hexahistidine affinity leader peptide has been expressed at levels which are at least 50-fold higher than previously reported. The recombinant enzyme has activity levels similar to the wild-type enzyme. The recombinant polypeptide binds to and elutes from a nickel affinity resin (IMAC resin). No dramatic differences in post-translational modification between the wild-type and recombinant enzyme were observed. The recombinant system will be useful in performing site-directed mutagenesis and will facilitate enzymological and biological investigations of this enzyme.

The primary structure of bovine rhodopsin kinase (RK), which phosphorylates light-activated rhodopsin (Rho*), terminates with the amino acid sequence Cys558-Val-Leu-Ser561, a motif that has been shown to direct the isoprenylation and alpha-carboxyl methylation of many proteins (e.g. p21Ha-ras). Transient expression of RK in COS-7 cells revealed the presence of two immunoreactive protein species. Consistent with RK being modified by isoprenylation, interconversion of these two species was dependent upon isoprenoid biosynthesis in the cells. Moreover, a serine substitution for Cys558 resulted in a single RK species whose migration on sodium dodecyl sulfate-polyacrylamide gels was identical to that of RK from cells treated with mevinolin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and, thus, of isoprenoid biosynthesis. This finding indicates that isoprenylation of RK requires Cys558. The electrophoretic mobility of isoprenylated RK synthesized in COS-7 cells was identical to that of RK from bovine rod outer segments, suggesting that RK is isoprenylated in vivo. RK was determined to be modified by a farnesyl moiety and alpha-carboxyl-methylated. A time course of Rho* phosphorylation revealed that non-processed RK is approximately 4-fold less active than wild-type RK. This is the first demonstration of isoprenylation/alpha-carboxyl methylation of a protein kinase, and suggests that these modifications markedly influence enzymatic activity in vivo.

This report describes the integration of laser-scanning fluorometric cytometry and nonseparation ligand-binding techniques to provide new assay methods adaptable to miniaturization and high-throughput screening. Receptor-bound, cyanine dye-labeled ligands, [Cy]ligands, were discriminated from those free in solution by measuring the accumulated fluorescence associated with a receptor-containing particle. To illustrate the various binding formats accommodated by this technique, saturation- and competition-binding analyses were performed with [Cy]ligands and their cognate receptors expressed in CHO cells or as fusion proteins coated on polystyrene microspheres. We have successfully applied this technique to the analysis of G protein-coupled receptors, cytokine receptors, and SH2 domains. Multiparameter readouts from ligands labeled separately with Cy5 and Cy5.5 demonstrate the simultaneous analysis of two target receptors in a single well. In addition, laser-scanning cytometry has been used to assay enzymes such as phosphatases and in the development of single-step fluorescent immunoassays.

DNA methylation is essential for normal embryonic development in mice. An understanding of how DNA methylation is controlled is largely dependent upon the isolation and characterization of the cellular components of the DNA methylation system. The enzyme which methylates DNA in eukaryotic cells is a C-5 cytosine DNA methyltransferase. Historically, the characterization of this enzyme has been limited by its availability and purity. Here, we present a single-step purification of 4 mg of baculovirus-expressed mouse DNA methyltransferase containing a nickel-affinity leader peptide. The recombinant DNA methyltransferase copurified with inhibitory RNA which was removed by treatment with ribonuclease A. Like its non-recombinant counterpart, the recombinant enzyme is activated by hemi-methylation. A direct steady-state kinetic comparison between the recombinant baculovirus-expressed enzyme with its MEL cell-derived counterpart is presented.

The antibiotic azatyrosine [DL-3-(5-hydroxy-2-pyridyl)alanine] suppressed meiotic maturation in oocytes induced by progesterone or the combination of [Val12]p21Ha-ras microinjection and insulin-like growth factor I. The suppression was dose-dependent in the range of 20-250 microM azatyrosine. In addition, azatyrosine blocked the tyrosine phosphorylation of Xp42, a member of the mitogen-activated protein kinase family, after progesterone or [Val12]p21Ha-ras/insulin-like growth factor I stimulation. Activation of maturation-promoting factor, as shown by a decrease in the tyrosine phosphorylation of the Xenopus homolog of p34cdc2, was also suppressed by azatyrosine. Azatyrosine had no effect in vivo or in vitro on the growth factor-induced autophosphorylation of the oocyte insulin-like growth factor I receptor. Azatyrosine has been shown by others [Shindo-Okada, N., Makabe, O., Nagahara, H. & Nishimura, S. (1989) Mol. Carcinog. 2, 159-167] to inhibit the growth of ras-transformed cells without affecting that of nontransformed cells. In oocytes, the antibiotic exerts an inhibitory action on both a ras-dependent and a ras-independent pathway. Lack of an effect of azatyrosine on germinal vesicle breakdown induced by the microinjection of an extract from mature oocytes, however, suggests that azatryosine is acting upstream of maturation-promoting factor activation.