Evidence for two promoters upstream of the pts operon: regulation by the cAMP receptor protein regulatory complex.

Several potential target sites for multiple regulatory mechanisms were previously identified in the 5' flanking region of the pts operon. We have investigated the in vitro interactions of the cAMP receptor protein (CRP).cAMP regulatory complex with two DNA binding sites, by gel mobility-shift assays, and report the analysis of the functional role of each of the binding sites in vivo.

Site-directed mutagenesis of the phosphocarrier protein. IIIGlc, a major signal-transducing protein in Escherichia coli.

The glucose-specific phosphocarrier protein (IIIGlc) of the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) is a major signal transducer that mediates the intricate interplay among extracellular signals (PTS and non-PTS sugars), cytoplasmic and membrane proteins (PTS and non-PTS transporters), and adenylate cyclase. To further define the central role of IIIGlc in these multiplex signaling mechanisms, we have used site-directed mutagenesis to construct three mutant IIIGlc proteins containing single amino acid changes; Phe-3 was replaced with tryptophan [( Trp3]IIIGlc), and His-75 and the active-site His-90 were replaced with glutamine [( Gln75]IIIGlc and [Gln90]IIIGlc, respectively). [Trp3]IIIGlc resembles the wild-type protein in most properties and should be valuable for spectrophotometric experiments.

Sugar transport by the bacterial phosphotransferase system. Molecular cloning and structural analysis of the Escherichia coli ptsH, ptsI, and crr genes.

Specialized lambda-transducing phages that carry the Escherichia coli genes ptsH, ptsI, crr, cysM, and cysA have been isolated, and the genes were subcloned in plasmid pBR322. Subcloning and restriction mapping data gave the following clockwise order of genes located at about 52 min on the E. coli genetic map: lig, cysK, ptsH, ptsI, crr, cysM, cysA.

Bovine angiotensin-converting enzyme: amino-terminal sequence analysis and preliminary characterization of a hybridization-selected primary translation product.

Bovine lung angiotensin-converting enzyme was isolated in pure form and the sequence of the first twenty-two NH2-terminal amino acids determined. Oligonucleotides, complementary to a selected portion of the NH2-terminal amino acid sequence of the bovine glycoprotein (Mr 145,000), were synthesized and used for hybridization selection of angiotensin-converting enzyme mRNA. The hybridization-selected mRNA programmed the in vitro synthesis of a single polypeptide (Mr 130,000) that was specifically immunoadsorbed by anti-bovine enzyme antibodies.

Purification and kinetic studies of an alpha-L-fucosidase of Venus mercenaria.

An alpha-L-fucosidase activity has been isolated from the liver (hepatopancreas) of the common edible clam, Venus mercenaria, and has been purified approximately 300-fold (11% yield) by affinity chromatography on agarose-epsilon-amino-caproylfucosamine. Isoelectric focusing profiles were heterogeneous, revealing several isoenzymes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of a single subunit of Mr 50,000.