Melanocyte cultures from the normally pigmented skin of patients with neurofibromatosis 1 (NF 1) have a higher melanin content than those from the skin of healthy donors. An additional increase in the amount of melanin per cell was found in 5 out of 6 lines of melanocytes derived from café au lait macules of NF 1 patients. Omission of the tumor promoter phorbol-12-myristate-13-acetate from the culture medium brings about a comparable increase in the melanin content in all three kinds of melanocyte cultures. Cultures of NF 1 melanocytes show a higher tyrosine hydroxylase activity than those of control melanocytes, and incorporate larger amounts of dihydroxyphenylalanine than the latter. We conclude that melanogenesis in epidermis melanocytes is affected by defective alleles of the NF 1 gene. Our findings do not contradict the hypothesis that the defect underlying NF 1 impairs the inhibition of a wild-type RAS oncogene by interfering with the GTPase-activating function of the NF 1 gene product.

The Kms for esculetin and S-adenosyl-L-methionine for catechol O-methyltransferase from the yeast Candida tropicalis were 6.2 and 40 ,uM, respectively. S-Adenosyl-L-homocysteine was a very potent competitive inhibitor with respect to S-adenosyl-L-methionine, with a Ki of 6.9 ,uM. Of the catechol-related inhibitors, purpurogallin, with a Ki of 0.07 ,uM, showed the greatest inhibitory effect. Sulfhydryl group-blocking reagents, such as thiol-oxidizing 2-iodosobenzoic acid and mercaptide-forming p-chloromercuribenzoic acid, provided evidence for sulfhydryl groups in the active site of the enzyme. Yeast catechol O-methyltransferase is a metal-dependent enzyme and requires Mg2' for full activity. Zn2+ and Mn2' but not Ca2+ were able to substitute for Mg2+.Mn2+ showed optimal enzyme activation at concentrations 50-to 100-fold lower than those of Mg2+.Numerous investigations have shown that catechol 0-methyltransferase (S-adenosyl-L-methionine:catechol 0-methyltransferase; catechol methyltransferase; EC 2.1

In an effort to investigate catechol methyltransferase activity in sources other than mammalian tissues and cells, a high level of enzyme activity was found in the yeast fungus Candida tropicalis CBS 94. Partial purification of the enzyme (approx. 550 fold with a recovery of 7%) could be achieved by using ion-exchange and gel filtration techniques. The molecular weight was estimated at 32,000 ± 2,000 by gel filtration on Sephadex G-100. In isoelectric focusing experiments on Sephadex G-75 the enzyme exhibited a pl-value o f 5.0 ± 0.1. In contrast to catechol methyltransferase from various mammalian tissues the enzyme activity was prepared from the pH 5-sediment. The substrate specifity is comparable to other catechol methyltransferases.

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The low-molecular-mass form of two distinct catechol 0-methyltransferase activities (S-adenosyl-Lmethionine : catechol 0-methyltransferase, COMT, EC 2.1.1.6) has been purified to homogeneity from rat liver using 40 -70% ammonium sulfate precipitation, gel filtration on Sephadex G-100, adsorption on hydroxyapatite C and ion-exchange chromatography on DEAE-Sepharose CL-6B. The relative molecular mass M,, determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis is 22400 f 500. Irradiation of the enzyme in the presence of 8-a~ido-[methyl-~HIAdoMet results in the specific labeling of the catalytic site of the enzyme. Photolabeling was successful with crude COMT preparations and with the isolated enzyme. Immunocytocheniical studies present new information about the localization of the low-molecular-mass form in the liver parenchyma. Subcellularly C O W immunoreactivity could be attributed exclusively to the compartment with glycogen granules. Nucleus, mitochondria and endoplasmic reticulum showed no immunostaining.The transfer of the methyl group from AdoMet to a variety of methyl acceptors such as catechol compounds [l, 21, phospholipids [3, 41, nucleic acids 151, neuropeptides and proteins [6] is catalysed by methyltransferases. Moreover, these enzymes have a regulatory function in the modulation of the concentration of AdoMet. The catecholic compounds are methylated by the enzyme S-adenosyl-L-methionine: catechol 0-methyltransferase (COMT). Numerous investigations have demonstrated COMT in a variety of animal tissues and cells, with the highest levels present in liver and kidney [7]. Metabolic studies have clearly shown that the primary site of 0-methylation of circulating catecholamines is the liver. The fact that COMT is found in high quantity in the 100000 x g supernatant of liver extracts does not indicate the precise location within the cell, whether it is free in the cytoplasm or loosely bound to membranes and released during homogenization. In 1974 [S] a form of COMT which is firmly bound to membranes was solubilized and partially purified from liver microsomes of the rat, but the microsomal COMT represented only about 0.1% of the total activity and was not released in the presence of saturated sodium chloride. The membraneassociated form exhibited biochemical and immunological properties similar to those of the soluble COMT.Correspondence to J. Veser, Abteilung Physiologische Chemie, Abbreviations. AdoMet, S-adenosyl-L-methionine; COMT, catEnzyme. S-Adenosyl-L-methionine : catechol 0-methyltransferase