Amino acid sequence of the active site of human pseudocholinesterase

The usualE ₁ ^u and atypicalE ₁ ^a human pseudocholinesterases (acylocholine acylhydrolase, EC 3.1.1.8) were purified to homogeneity. The active-site serine residue was conjugated with diisopropyl fluorophosphate and digested with trypsin. The tryptic peptide containing the active site was isolated by gel filtration followed by two-dimensional paper chromatography and electrophoresis. The amino acid sequence of the active site peptide obtained from the usualE ₁ ^u enzyme was found to be Gly-Glu-Ser-Ala-Gly-Ala-Ser-Ala-Val-Ser-Leu. A remarkable structural homology exists between the human and the horse enzymes in their active sites. From the difference in electrophoretic mobility of the active-site peptides obtained from the usual and atypical enzymes, the probable structure of the atypical human enzyme was deduced as Gly-His-Ser-Ala-Gly-Ala-Ser-Ala-Val-Ser-Leu.     

Isolation of microbodies from plant tissues

Specialized microbodies have previously been isolated and characterized from fatty seedling tissues (glyoxysomes) and leaves (leaf peroxisomes). We have now examined 11 other plant tissues, including tubers, fruits, roots, shoots, and petals, and find that all contain particulate catalase, a distinctive common enzyme component of microbodies. On linear sucrose gradients the catalase activity peaks sharply at a higher equilibrium density (1.20 to 1.25 gram per cm³ in the various tissues) than the mitochondria (1.17 to 1.20). Only small amounts of protein are recovered in the fractions containing catalase, although a definite band is visible in preparations from some tissues, e.g., potato. As in the preparations from castor bean endosperm and spinach leaves for which comparable data are provided, the distribution of glycolate oxidase and uricase follows closely that of catalase on the gradients. The preparations from potato lack glyoxylate reductase and the transaminases, typical enzymes of leaf peroxisomes, and the distinctive enzymes of glyoxysomes are missing. Nonspecialized microbodies with limited enzyme composition can thus be isolated from a variety of plant tissues.     

Genetic Analysis of Mutant Strains of Escherichia coli Requiring p-Aminobenzoic Acid for Growth

Mutant strains of Escherichia coli K-12 which required p-aminobenzoic acid for growth were isolated. The mutations were mapped by conjugation and by transduction, and two genes concerned with the biosynthesis of p-aminobenzoic acid were identified.     

Type I protein kinase C isozyme in the visual-information-processing pathway of monkey brain

Previously using PKC isozyme-specific antibodies for immunoblot analysis, we demonstrated the heterogeneous distribution of PKC isozymes in various regions of monkey and rat brains and that type I PKC was most abundant in cerebellum, hippocampus, amygdala, and cerebral cortex (Huang et al.: J Biol Chem 262:15714-15720, 1987). Using these antibodies, we have also demonstrated that type I, II, and III PKC are products of PKC genes gamma, beta, and alpha, respectively (Huang et al.: Biochem Biophys Res Commun 149:946-952, 1987). By immunocytochemical analysis, type I PKC-specific antibody showed strong reactivity in various types of neuron in hippocampal formation, amygdala, cerebellum, and neocortex. In hippocampal formation, granule cells of dentate gyrus and pyramidal cells of hippocampus were heavily stained. By immunoblot analysis, relative levels of PKC isozymes in several areas of monkey cerebral cortex involved in the visual information processing and storage were determined. Both type II and III PKCs appeared to be evenly distributed and at moderate levels, type I PKC formed a gradient of increasing concentration rostral along the cerebral cortex of occipital to temporal and then to the limbic areas. Neurobehavioral studies have demonstrated that the neocortical and limbic areas of the anterior and medial temporal regions participate more directly than the striate, prestriate, and posterior temporal regions in the storage of visual representations and that both hippocampus and amygdala are important in the memory formation. As type I PKC is present at high levels in hippocampus, amygdala, and anterior temporal lobe, we predict that the type I protein kinase C may participate in the plastic changes important for mnemonic function.     

N-cyanoimidazole and diimidazole imine: water-soluble condensing agents for the formation of the phosphodiester bond

The reaction of BrCN with imidazole results in the formation of N-cyanoimidazole and diimidazole imine. These compounds were shown to be useful condensing agents for the formation of the phosphodiester bound in aqueous solution.     

Interactions between a lymphoma membrane-associated guanosine 5'-triphosphate-binding protein and the cytoskeleton during receptor patching and capping

In this study we have used several complementary techniques to isolate and characterize a lymphoma membrane-associated 41-kDa protein that shares a number of structural and functional similarities with the alpha i subunit of the guanosine 5'-triphosphate (GTP)-binding protein (e.g., Gi alpha-like protein). In addition, using permeabilized lymphoma cells, we have found that: 1) GTP or GTP-tau-S augments, and pertussis toxin inhibits, phospholipase C (PLC) activity and receptor capping; and 2) the addition of lymphoma 41-kDa Gi alpha-like protein stimulates PLC activity and receptor patching/capping, and reverses the inhibitory effect of pertussis toxin on both activity and receptor patching/capping. Additional cytochemical and biochemical data indicate that the lymphoma 41-kDa protein is closely associated with several cytoskeletal proteins (e.g., actin, myosin, and fodrin) all of which colocalize under receptor cap structures. Furthermore, both the 41-kDa-mediated phospholipase C activity and receptor patching/capping are inhibited by cytochalasin D (a microfilament disrupting drug) and W-7 drug (a calmodulin inhibitor). Together, these data provide strong evidence for a functional association between the lymphoma membrane cytoskeleton and the 41-kDa (Gi alpha-like) protein. Specifically, this association appears to be required for the activation of phospholipase C that results in inositol triphosphate production, subsequent internal Ca2+ release, and finally surface receptor patching and capping.     

Combined in-beam electron impact-B/E-linked scan mass spectrometry of oxazoline derivatives for the structure determination of long-chain unsaturated fatty acids

Long-chain unsaturated fatty acids (UFA) having up to six double bonds are derivatized to 2-substituted 4,4-dimethyloxazolines (DMOX) and then analyzed by combined in-beam electron impact (IBEI)-B/E-linked scan mass spectrometry. This technique provides highly characteristic mass spectra and may serve as an auxiliary means for direct structure determination of individual UFA in mixtures.