NAD+ Glycohydrolase of the Plasma Membrane Prepared from Glial and Neuronal Cells

NAD+ glycohydrolase (EC 3.2.2.5) activity was detected in the plasma membrane prepared from the primary culture of rat astrocytes. The enzyme has a broad optimum pH range. From the kinetic analysis, a Michaelis constant of 91.2 microM and a maximum velocity of 0.785 mumol/min/mg protein were obtained. ADPribose exhibited a competitive inhibition with respect to NAD. The inhibition by nicotinamide was shown to be of a non-competitive type. ATP and GTP were found to be competitive inhibitors. NAD+ glycohydrolase activity was not detected in the plasma membrane prepared from the primary culture of neuronal cells of chick embryos.     

Enzyme Immunoassay for Cholecystokinin Octapeptide Sulfate and Its Application

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for cholecystokinin octapeptide sulfate (CCK-8S) has been developed using N-terminal specific antibody for CCK-8S. In this assay CCK-8S coupled with poly-L-Glu (CCK-poly-Glu), which is adsorbed on a solid phase, competes with CCK-8S for the binding sites of rabbit anti-CCK antibody, and the complex of the immobilized antibody and CCK-poly-Glu is measured using goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase. The total time for completion of the assay is less than 24 h. Near 50% bound levels, the intraassay coefficient of variation is 5.2-6.2% and the interassay coefficient of variation is 5.9-8.5%. This assay is sensitive enough to detect 9 pg of CCK-8S, and the data from rat brain regions using this ELISA are very similar to the data from those using radioimmunoassay (RIA). Therefore, this ELISA is simpler and more rapid in comparison with conventional RIA. In the preliminary experiments, we applied this method for determination of CCK content in the brain regions of adult rats treated with 6-hydroxy-dopamine or in newborn rats subjected to anoxia, and showed that this system is applicable to detection of changes of endogenous CCK content.     

The bialaphos biosynthetic genes ofStreptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster

Summary We have isolated and studied the organization ofStreptomyces hygroscopicus genes responsible for the biosynthesis of the antibiotic herbicide bialaphos. Bialaphos production genes were cloned from genomic DNA using a plasmid vector (pIJ702). Three plasmids were isolated which restored productivity toS. hygroscopicus mutants blocked at different steps of the biosynthetic pathway. Subcloning experiments using other nonproducing mutants showed that four additional bialaphos production genes were also contained on these plasmids. A gene conferring resistance to bialaphos, which was independently cloned using the plasmid vector pIJ61, and an antibiotic-sensitive host (S. lividans), was also linked to the production genes. Cosmids were isolated which defined the location of these genes in a 16 kb cluster.     

Purification of natural human interferon-gamma by antibody affinity chromatography: analysis of constituent protein species in the dimers

A simple procedure for purifying human interferon-gamma from leukocytes was established, based on monoclonal antibody affinity chromatography. The recovery of interferon activity was essentially quantitative, and the specific activity of the product was (4-12) x 10(7) international units/mg protein. SDS-polyacrylamide gel electrophoresis reproducibly revealed four components associated with interferon activity (and no other proteins): two major ones with molecular weights (MW) of 24,000-25,000 (25K) and 19,000-20,000 (20K), a minor one with MW 14,000-15,000 (15K) (these three bands were doublets), and a still less prominent one(s) with MV 40,000-48,000. Gel filtration in neutral solution indicated that all the 25K, 20K, and 15K species exist as oligomers, probably dimers. By means of experiments using a cleavable crosslinking reagent, the dimers were shown to comprise both homo-and heterodimers. Gel filtration in alkali (the condition used during purification) indicated that the molecules are largely in a monomeric state. Thus, the molecules once dissociated in alkali appear to reassociate at random upon neutralization; this process takes place without being accompanied by inactivation.     

Purified protein kinase C phosphorylates microtubule-associated protein 2

We have investigated actions of purified protein kinase C on microtubule- and microfilament-related proteins. Among the cytoskeletal proteins examined, microtubule-associated protein 2 (MAP2) was found to serve as a good substrate. Other cytoskeletal proteins, tubulin, fodrin, cofilin, tropomyosin, and 53,000-Da protein, were very poorly phosphorylated. The amino acid residues of MAP2 that were phosphorylated by the protein kinase C were almost exclusively serine. The peptide mapping analysis indicated that protein kinase C and cAMP-dependent protein kinase phosphorylate MAP2 differently. The ability of MAP2 to interact with actin was markedly reduced by this protein kinase C-mediated phosphorylation. These data raise the possibility that phosphorylation of MAP2 by activated protein kinase C may be involved in cell-surface signal transduction.     

A New Myelin-Deficient Mutant Hamster: Biochemical and Morphological Studies

Biochemical and morphological studies were done on a new trembling mutant hamster CBB. The yield of myelin from the mutant was 30 and 40% of the control at 46 and 140 days of age, respectively, but myelin composition and 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase) activity were normal. Morphologically, about 18% of the axons were myelinated in the mutant optic nerve at 46 days of age, in which the myelinated fibers were those with larger diameters (more than 0.6 micron), while the control had a peak at 0.4 micron in diameter. The ultrastructure and thickness of compact myelin lamellae in the mutant were normal. Myelination and the structure of peripheral nerve myelin appeared normal. The results indicate that the essential defect is the delay and arrest of myelination in the CNS, which is probably caused by either a decreased rate of synthesis of myelin components in oligodendrocytes or a defect in the oligodendrocyte-axon recognition in smaller axons.     

Structural organization of the human kininogen gene and a model for its evolution

The entire human kininogen gene has been isolated as a set of overlapping genomic DNA fragments, and the 11 exons encompassing approximately 27 kilobase pairs have been mapped by restriction enzyme analysis and nucleotide sequence determination. The nine 5'-terminal exons encode the 5'-untranslated region and the protein-coding region for the signal peptide and the heavy chain, which are common for high molecular weight (HMW) and low molecular weight (LMW) prekininogen mRNAs. Exon 10 consists of the common sequence for bradykinin and the immediately following unique sequence for HMW prekininogen mRNA. Exon 11 is then located following a 90-nucleotide sequence downstream from exon 10 and precisely specifies the sequence unique to LMW prekininogen mRNA. This, together with the hybridization analysis of total human cellular DNA, leads us to conclude that human HMW and LMW prekininogen mRNAs are produced from a single gene as a consequence of alternative RNA processing events. The structural analysis of the kininogen gene also shows that each of the nine 5'-terminal exons discretely specifies the nine protein domains observed in the amino-terminal portion of the kininogens. Furthermore, these nine genetic domains can be characterized by a thrice repeated pattern of three genetic segments, and two sets of these three domains, encompassing exons 3-5 and exons 6-8, are most closely related to each other. Therefore, we have proposed two successive duplication mechanisms as a model for the generation of the structure of the kininogen gene.     

Nucleotide sequence of the Syrian hamster intracisternal A-particle gene: close evolutionary relationship of type A particle gene to types B and D oncovirus genes.

We determined the complete nucleotide sequence of the intracisternal A-particle gene, IAP-H18, cloned from the normal Syrian hamster liver DNA. IAP-H18 was 7,951 base pairs in length with two identical long terminal repeats of 376 base pairs at both ends. On the coding strand, imperfect open reading frames corresponding to gag and pol of the retrovirus genome were observed, whereas many stop codons were present in the region corresponding to env. The putative H18 gag gene (809 amino acids) had a sequence homologous to the N-terminal half of the mouse mammary tumor virus gag gene and locally to the Rous sarcoma virus gag gene. The putative H18 pol gene (900 residues) was homologous to the Rous sarcoma virus pol gene almost throughout the entire region. Two conserved regions among the retrovirus pol genes have been reported. One presumably corresponds to the DNA polymerase and the RNase H domain, and the other corresponds to the DNA endonuclease domain of the multifunctional protein pol. By the comparison of the deduced amino acid sequences of the putative endonuclease domain of six representative oncovirus genomes, a phylogenetic tree of the oncovirus genomes was constructed, and the intracisternal A-particle (type A) genome was found to be more closely related to the mouse mammary tumor virus (type B) and squirrel monkey retrovirus (type D) genomes.     

Characteristics of a Urate-Degrading Diamine Oxidase-Peroxidase Enzyme System in Soybean Radicles

The cadaverine content of soybean radicles showed a maximumpeak 3–4 days after planting. The variation coincidedwith radicle uricase activity during seed germination. The uricase activity could not be fractionate when the bufferpH for the extraction was at 6.0. The addition of 1 M KCl orNaCl to the buffer allowed the extraction of the uricase activity,but an addition of 1 M MgCl₂ or BaCl₂ inhibited this enzyme'sactivity. The urate-degrading enzyme system was purified 248-fold permilligram of protein from soybean radicles. The respective K_mvalues of the diamine oxidase activity for cadaverine and ofthe urate-degrading activity for hydrogen peroxide and uratewere 1.25, 2.93 and 50.3 µM. Analysis by gel electrophoresisof the partially purified enzyme fraction revealed that theurate-degrading enzyme system consisted of a peroxidase thatdegrades urate with hydrogen peroxide and a diamine oxidasethat releases hydrogen peroxide. These data are evidence that a urate-degrading diamine oxidaseand peroxidase system exists in soybean radicles and that thereaction rate of urate-degradation is controlled by the concentrationof cadaverine. (Received November 28, 1984; Accepted April 8, 1985)