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NAD:arginine ADP-ribosyltransferases catalyze the ADP-ribosylation of arginine residues in proteins. Coding region nucleic acid and deduced amino acid sequences of a human skeletal muscle ADP-ribosyltransferase cDNA were, respectively, 80.8% and 81.3% identical to those of the rabbit skeletal muscle transferase. A human transferase-specific cDNA probe detected major mRNA of 1.2 kb (mouse and rat), 3.0 kb (rabbit), 3.8 kb (monkey), and 5.7 kb (human) upon Northern analysis. Polyclonal anti-rabbit ADP-ribosyltransferase antibodies reacted with 36,000 M(r) proteins in partially purified transferase preparations from bovine, dog, and rabbit heart muscle and a 40,000 M(r) protein from human skeletal muscle. The human muscle ADP-ribosyltransferase cDNA, like the previously cloned rabbit muscle transferase, predicts predominantly hydrophobic amino- and carboxy-terminal amino acid sequences, which is characteristic of glycosylphosphatidylinositol (GPI)-anchored proteins. On immunoblots of partially purified rabbit and human skeletal muscle ADP-ribosyltransferases, anti-cross-reacting determinant antibodies detected at 36,000 and 40,000 M(r), respectively, phosphatidylinositol-specific, phospholipase C-sensitive, GPI-anchored proteins. These data are consistent with the conclusion that GPI-anchored skeletal and cardiac muscle ADP-ribosyltransferases are conserved across mammalian species.

A cDNA clone corresponding to a portion of the catalytic subunit of calmodulin (CaM)-dependent phosphoprotein phosphatase (calcineurin) was isolated from a murine brain library by expression vector immunoscreening. A (3-galactosidase fusion protein that reacted on Western blots with anti-calcineurin antibodies and biotinylated CaM was purified in preparative amounts using CaM-Sepharose affinity chromatography. Partial digestion of the hybrid protein with Staphylococcus aureus V-8 protease produced several immunoreactive peptides that appeared identical to fragments generated from authentic brain calcineurin. The 1111-base-pair (bp) EcoRI insert contained an open reading frame encoding a protein of 35 kDa followed by a 190-bp 3' noncoding region; seven peptides obtained by partial amino acid sequencing of the bovine brain enzyme were found in the deduced sequence. A domain %12 kDa from the carboxyl terminus was deduced to be the CaM-binding site based on consensus structural features and a sequence of seven amino acids highly related to smooth muscle myosin light-chain kinase. Two regions with identity to protein phosphatases 1 and 2A were found in the amino half of the cloned sequence; however, the intervening sequence contained apparent insertions, suggesting splicing of subdomains. Thus, the structure of calcineurin is chimeric, consisting of conserved catalytic elements and a regulatory CaM-binding domain.Regulation of phosphoprotein metabolism by specific classes of protein kinases and phosphatases is important for a wide spectrum of biological activities (1-3). The classical studies on the modulation of kinases involved in control ofglycolytic enzymes point to a central role for protein phosphorylation in maintenance of intermediary metabolism (for a review, see ref. 4). More recent observations regarding the role of phosphorylation in hormone receptor function (5) and in the expression of oncogenes (6, 7) suggest that specific biochemical transduction pathways utilize phosphorylation events to alter signaling responses. A role for Ca2+-regulated phosphoproteins in neurotransmission has been implicated by the high concentrations of the calmodulin (CaM)-dependent protein kinase in synaptic densities (8,9) and the phosphorylation of specific neuronotypic substrates under conditions of increased synaptic activity (10).In general, much less information is available regarding the role of phosphoprotein phosphatases in regulation of cellular responses. The major subgroups of phosphatases, distinguished by their substrate specificity and regulation by peptide inhibitors (11), are present in various proportions in different tissues. One of these is a CaM-dependent form (12) also called calcineurin (13) or phosphatase 2B (14) that is present in highest concentration in nervous tissue. Calcineurin is composed of both catalytic and regulatory subunits (60 and 18 kDa, respectively), the latter itself a calcium-binding peptide with structural similarities to CaM (13, 15). The activity of this phosphatase is hig...

ADP-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that enhance the enzymatic activities of cholera toxin. Two ARF cDNAs, ARF1 and ARF3, were cloned from a human cerebellum library. Based on deduced amino acid sequences and patterns of hybridization of cDNA and oligonucleotide probes with mammalian brain poly(A)+ RNA, human ARF1 is the homologue of bovine ARF1. Human ARF3, which differs from bovine ARF1 and bovine ARF2, appears to represent a newly identified third type of ARF. Hybridization patterns of human ARF cDNA and clone-specific oligonucleotides with poly(A)+ RNA are consistent with the presence of at least two, and perhaps four, separate ARF messages in human brain. In vitro translation of ARF1, ARF2, and ARF3 produced proteins that behaved, by SDS/PAGE, similar to a purified soluble brain ARF. Deduced amino acid sequences of human ARF1 and ARF3 contain regions, similar to those in other G proteins, that are believed to be involved in GTP binding and hydrolysis. ARFs also exhibit a modest degree of homology with a bovine phospholipase C. The observations reported here support the conclusion that the ARFs are members of a multigene family of small guanine nucleotide-binding proteins. Definition of the regulation of ARF mRNAs and of function(s) of recombinant ARF proteins will aid in the elucidation of the physiologic role(s) of ARFs.

Three (two soluble and one membrane) guanine nucleotide-binding proteins (G proteins) that enhance ADP-ribosylation ofthe G. stimulatory subunit ofthe adenylyl cyclase (EC 4.6.1.1) complex by choleragen have recently been purified from bovine brain. To further define the structure and function of these ADP-ribosylation factors (ARFs), we isolated a cDNA clone (AARF2B) from a bovine retinal library by screening with a mixed heptadecanucleotide probe whose sequence was based on the partial amino acid sequence of one of the soluble ARFs from bovine brain. Comparison of the deduced amino acid sequence of AARF2B with sequences of peptides from the ARF protein (total of 60 amino acids) revealed only two differences. Whether these are cloning artifacts or reflect the existence of more than one ARF protein remains to be determined. Deduced amino acid sequences of ARF, G. (the a subunit of a G protein that may be involved in regulation of ion fluxes), and c-Ha-ras gene product p21 show similarities in regions believed to be involved in guanine nucleotide binding and GTP hydrolysis. ARF apparently lacks a site analogous to that ADP-ribosylated by choleragen in G-protein a subunits. Although both the ARF proteins and the a subunits bind guanine nucleotides and serve as choleragen substrates, they must interact with the toxin Al peptide in different ways. In addition to serving as an ADP-ribose acceptor, ARF interacts with the toxin in a manner that modifies its catalytic properties.

Certain polycyclic aromatic hydrocarbons, ubiquitous environmental pollutants, destroy oocytes. Oocyte destruction by polycyclic hydrocarbons requires distribution of the parent hydrocarbon to the ovary where ovarian enzymes metabolize the compound to reactive intermediates responsible for ovotoxicity. Descriptive assays of polycyclic hydrocarbon metabolic activation such as the aryl hydrocarbon (benzo(a)pyrene) hydroxylase assay (AHH) are not good predictors of strain or species differences in sensitivity to polycyclic hydrocarbon ovotoxicity. Using benzo(a)pyrene as a probe of ovarian metabolic processing suggests that the rate of formation of metabolites along the metabolic pathway to the 7,8-dihydrodiol-9,10-epoxide may be the appropriate measure of the role of metabolic activation in strain or species differences in sensitivity to oocyte destruction. The multistep metabolic pathway involved in ovarian metabolic processing of benzo(a)pyrene may represent a useful model for exploring the roles of metabolic activation, detoxification, intrinsic sensitivity, and repair in reproductive toxicity.

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