Classification of humans as rapid or slow acetylators is based on hereditary differences in rates of N-acetylation of therapeutic and carcinogenic agents, but N-acetylation of certain arylamine drugs displays no genetic variation. Two highly homologous human genes for N-acetyltransferase (NAT; arylamine acetyltransferase, acetylCoA:arylamine N-acetyltransferase, EC 2.3.1.5), NAT] and NAT2, presumably code for the genetically invariant and variant NAT proteins, respectively. In the present investigation, 1.9-kilobase human genomic EcoRI fragments encoding NAT2 were generated by the polymerase chain reaction with liver and leukocyte DNA from seven subjects phenotyped as homozygous and heterozygous acetylators. Hepatic cytosolic N-acetyltransferase (NAT; arylamine acetyltransferase, acetyl CoA:arylamine N-acetyltransferase, EC 2.3.1.5) participates in the detoxification of a plethora of hydrazine and arylamine drugs, as well as in activation pathways of occupational carcinogens (1, 2). A reactive cysteinyl thiolate is part of the active site of NAT (3), which displays a ping-pong reaction mechanism with a covalent acetyl-cysteinyl-NAT species as the catalytic intermediate (3, 4). Hereditary differences in N-acetylation activity among individuals and in populations of diverse raciogeographic origin have led to a phenotypic classification of humans as rapid or slow acetylators. This genetic heterogeneity in NAT activity, widely referred to as the N-acetylation polymorphism, is determined in people and several animal species by a single autosomal gene with two major alleles expressed codominantly (2, 5). On the other hand, rates of elimination in vivo and acetylation in vitro of certain drugs (e.g., p-aminosalicylic acid) do not differ appreciably among rapid and slow acetylators, indicating that metabolism of these substrates takes place without genetic variation. The term monomorphic has been coined for genetically invariant N-acetylation activity (ref. 2, pp. 137-138). The molecular mechanisms for monomorphic and polymorphic N-acetylation in humans are not yet fully understood.The amino acid composition reported for several tryptic peptides of electrophoretically homogeneous liver NAT from homozygous rapid acetylator rabbits (4) proved pivotal in the design of oligonucleotide screening probes and isolation of rabbit NAT cDNAs (6, 7), which eventually enabled identification of human NAT cDNA and genomic clones (7,8). The libraries screened for human NAT cDNAs were made with mRNA from two livers of undetermined acetylator phenotype (7). It was inferred from the substrate selectivity profiles of the cDNA products expressed in Chinese hamster ovary cells that two of the isolated cDNA clones coded for the genetically variant NAT and one coded for the monomorphic protein (7). Three NAT gene loci, corresponding to EcoRI fragments of 1.3 kilobases (kb) (NATJ), 1.9 kb (NAT2), and 4.7 kb (NA TP), were identified upon screening of a library constructed with leukocyte DNA from a heterozygous person. Indirect evidence f...

Tyrosine phosphorylation plays a central role in the control of neuronal cell development and function. Yet, few neuronal protein tyrosine phosphatases (PTPs) have been identified. We examined rat olfactory neuroepithelium for expression of novel PTPs potentially important in neuronal development and regeneration. Using the polymerase chain reaction with degenerate DNA oligomers directed to the conserved tyrosine phosphatase domain, we identified 6 novel tyrosine phosphatases. One of these, PTP NE-3, is a receptor-type PTP expressed selectively in both rat brain and olfactory neuroepithelium. In the olfactory neuroepithelium, PTP NE-3 expression is restricted to neurons and describes a novel pattern of expression with a high level in the immature neurons and a lower level in mature olfactory sensory neurons.

A novel protein tyrosine phosphatase [homologue of vaccinia virus H1 phosphatase gene clone 5 (hVH‐5)] was cloned; it shared sequence similarity with a subset of protein tyrosine phosphatases that regulate mitogen‐activated protein kinase. The catalytic region of hVH‐5 was expressed as a fusion protein and was shown to hydrolyze p‐nitrophenylphosphate and inactivate mitogen‐activated protein kinase, thus proving that hVH‐5 possessed phosphatase activity. A unique proline‐rich region distinguished hVH‐5 from other closely related protein tyrosine phosphatases. Another feature that distinguished hVH‐5 from related phosphatases was that hVH‐5 was expressed predominantly in the adult brain, heart, and skeletal muscle. In addition, in situ hybridization histochemistry of mouse embryo revealed high levels of expression and a wide distribution in the central and peripheral nervous system. Some specific areas of abundant hVH‐5 expression included the olfactory bulb, retina, layers of the cerebral cortex, and cranial and spinal ganglia. hVH‐5 was induced in PC12 cells upon nerve growth factor and insulin treatment in a manner characteristic of an immediate‐early gene, suggesting a possible role in the signal transduction cascade.

The vaccinia virus VH1 gene product is a dual specificity protein phosphatase with activity against both phosphoserine-and phosphotyrosine-containing substrates. We investigated the potential presence of VH1 analogs in other viruses. Hybridization and sequence data indicated that a phosphatase related to the VH1 phosphatase is highly conserved in the genomes of smallpox variola virus and other orthopoxviruses. The open reading frames from the raccoonpox virus and the smallpox variola virus Bangladesh major strain genomes encoding the VH1 analogs were sequenced and found to be highly conserved with the vaccinia virus VH1. An open reading frame from the baculovirus Autographa californica has sequence similarity to the VH1 phosphatase. The viral proteins appear to be structurally related to the cell cycle control protein p8Ocdc2-. A recombinant phosphatase expressed from the baculovirus gene was found to share with the VH1 phosphatase the ability to hydrolyze substrates that contained both phosphoserine and phosphotyrosine.

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