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Adenocarcinomas of glandular tissues produce a hypoglycosylated form of a normal glycoprotein (mucin) that elicits an immune response. A tumor-specific epitope of mucin occurs in a 20-amino-acid, tandemly repeated domain of human MUC1 mucin. A synthetic gene encoding five tandem repeats of the tumor-specific epitope of human mucin (m5tr) was designed for efficient cloning and expression in Escherichia coli for subsequent use in preparing reagent quantities of the mucin 5 tandem repeat (mtr5) polypeptide. The synthetic gene was cloned in the correct reading frame into the maltose-binding protein (MBP) fusion expression vector pMAL-p2. Bacterial clones containing the mucin synthetic gene (m5tr) were shown to produce the intended recombinant fusion protein, MBP–mtr5. The fusion protein represents a significant fraction of the cell protein, 50% or more of which is secreted into the periplasm. The MBP–mtr5 protein is largely intact and easily prepared in sufficient quantity and purity for preliminary structure–function studies.

EctoATPases are extracellular membrane-bound enzymes that catalyze the hydrolysis of the gamma phosphate from ATP. EctoATPase is expressed by activated and immortalized Epstein-Barr virus-transformed human peripheral blood B lymphocytes and murine B cell hybridomas. By contrast, ectoATPase activity is not expressed on nontransformed human peripheral blood B lymphocytes, murine spleen cells, or murine myeloma cells. The K(m) for ATP for the B cell ectoATPases ranged from 5 to 77 microM; the Vmax ranged from 48 to 129 pmol/ min/10(4) cells. The enzyme required Mg2+ for maximal activity with little dependence on Ca2+. ADP and purine and pyrimidine nucleoside triphosphates were competitive inhibitors of the catalytic reaction. A putative ectoATPase protein has been identified by Western blot analysis of membrane proteins from the immortalized B cells. Under reducing conditions, antiectoATPase antibodies cross-reacted with a 66-kDa protein from murine B cell hybridoma membranes. By contrast a 200-kDa protein from the B cell hybridoma membranes cross-reacted with the antibodies under nonreducing conditions, suggesting a disulfide-linked trimer. The antibodies also cross-reacted with a 66-kDa protein from human B cell membranes under reducing conditions, but did not cross-react with membrane proteins under nonreducing conditions. This suggests that the antibody epitope(s) recognized on the reduced human protein is masked under nonreducing conditions. Thus, this work demonstrates: (1) that ectoATPase may serve as a marker for B cell activation; and (2) mammalian and avian ectoATPases have conserved interspecies immunological epitopes and kinetic properties.

A new bifunctional affinity label, 5'-p-(fluorosulfonyl)benzoyl-8-azidoadenosine (5'-FSBAzA), has been synthesized by condensation of p-(fluorosulfonyl)benzoyl chloride with 8-azidoadenosine. 5'-FSBAzA has been characterized by elemental analysis, thin-layer chromatography, and ultraviolet and 1H NMR spectroscopy. The affinity label contains both an electrophilic fluorosulfonyl moiety and a photoactivatable azido group which are capable of reacting with several classes of amino acids found in enzymes. 5'-FSBAzA reacts with bovine liver glutamate dehydrogenase in a two-step process: a dark reaction yielding about 0.5 mol of the sulfonylbenzoyl-8-azidoadenosine (SBAzA) group bound/mol enzyme subunit by reaction of the enzyme at the fluorosulfonyl group, followed by photolysis in which 25% of the covalently bound SBAzA becomes crosslinked to the enzyme. 5'-FSBAzA-modified glutamate dehydrogenase, both before and after photolysis, retains full catalytic activity but is less sensitive to allosteric inhibition by GTP, to activation by ADP, and to inhibition by 1 mM NADH. These results suggest the modification in the dark reaction of a regulatory nucleotide binding site. Photoactivation of the covalently bound reagent may have general applicability in relating modified amino acids which are close to each other in the region of the purine nucleotide binding sites of glutamate dehydrogenase and other proteins.