Intramolecular crosslinking of gamma-glutamyl transpeptidase

gamma-Glutamyl transpeptidase (rat kidney) is a heterodimeric glycoprotein (subunit molecular weights 52,000 and 25,000). In addition to its single-chain biosynthetic precursor (Mr 78,000), glycosylated high molecular weight forms (Mr 85,000-95,000) have been reported in various rat tissues as well as during in vitro translation of its mRNA. Studies reported here suggest that these might be attributed to the anomalous behavior of intramolecularly crosslinked species. Thus, chemical crosslinking of the purified enzyme (as well as enzyme on the renal brush border membranes) by bifunctional reagents such as dimethyl suberimidate and by an active site-directed reagent, diazotized p-amino-hippurate, produces stable heterodimers which exhibit molecular weights identical to that of the native enzyme when subjected to gel filtration. However, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the crosslinked species exhibit apparent Mr values of 85,000 to 110,000, depending upon the crosslinking agent used. Protein glycosylation alone does not account for such anomalous electrophoretic behavior; the extent and the regions of the enzyme involved in formation of crosslinks appear to exert considerable constraints upon their conformation even in denaturing media.     

Observation of inverted cubic phase in hydrated dioleoylphosphatidylethanolamine membranes

We report the observation of an inverted cubic phase in aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) by small-angle X-ray diffraction. DOPE is a paradigm in the study of nonlamellar phases in biological systems: it exhibits a well-known phase transition from the lamellar (L alpha) to the inverted hexagonal phase (HII) as the temperature is raised. The transition is observed to occur rapidly when a DOPE dispersion is heated from 2 degrees C, where the L alpha phase is stable, to 15 degrees C, where the HII phase is stable. We report on the induction of a crystallographically well-defined cubic lattice that is slowly formed when the lipid dispersion is rapidly cycled between -5 and 15 degrees C hundreds of times. Once formed, the cubic lattice is stable at 4 degrees C for several weeks and exhibits the same remarkable metastability that characterizes other cubic phases in lipid-water systems. X-ray diffraction indicates that the cubic lattice is most consistent with either the Pn3m or Pn3 space group. Tests of lipid purity after induction of the cubic indicate the lipid is at least 98% pure. The cubic lattice can be destroyed and the system reset by cycling the specimen several times between -30 and 2 degrees C. The kinetics of the formation of the cubic are dependent on the thermal history of the sample, overall water concentration, and the extreme temperatures of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Naloxone inhibits superoxide release from human neutrophils

Using the superoxide dismutase inhibitable reduction of cytochrome c assay, we studied, the effect of (-) naloxone on N-formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated superoxide (O2-) release from human neutrophils. Neutrophils were pre-incubated with the range of concentrations of (-) naloxone that is administered in models of experimental sepsis (10(-6) - 10(-4.5) M). (-) Naloxone inhibited O2- release in a dose dependent manner. 02- produced by a cell-free xanthine-xanthine oxidase system was not inhibited by (-) naloxone, indicating that (-) naloxone was not scavanging O2-. There was no difference between the effect of (-) and (+) naloxone suggesting that the inhibition of O2- was not specific for an opiate receptor. Another opiate antagonist, nalorphine, as well as the opiate agonist, morphine, also inhibited O2- release in the same concentration range. There was no difference between the effect of naloxone and morphine.     

Virulence properties of strains of agrobacterium on the apical and Basal surfaces of carrot root discs

Most pathogenic strains of Agrobacterium are able to induce crown gall or hairy root on both the apical surface (facing the root tip) and the basal surface (facing the shoot) of carrot (Daucus carota L.) root discs. Tumorigenic strains carrying mutations in the shoot inhibition region of the T-DNA (TL-DNA genes 1 and 2) are markedly attenuated on the basal surface but remain virulent on the apical surface. Coinoculation of two attenuated tumorigenic strains, with mutations in gene 1 and gene 2, respectively, resulted in restoration of virulence on the basal surface. Wild type hairy root-inducing strains can be divided into two groups: those that are virulent on both apical and basal surfaces and those that are virulent only on the apical surface. α-Naphthalene acetic acid stimulated virulence of hairy root strain TR7, belonging to the latter group, on the basal surface. Attenuated virulence on the basal surface can be explained in terms of an auxin deficiency in the basal tissues and unidirectional auxin transport to the apical surface.     

Cloning of the Escherichia coli release factor 2 gene.

The protein release factor 2 (RF2) participates in Escherichia coli polypeptide chain termination with codon specificity (UAA or UGA). A colicin E1 recombinant identified in the Carbon and Clarke E. coli bank contains the protein release factor 2 gene. A 1.7-kilobase E. coli fragment has been subcloned into the plasmid pUC9 vector. Bacterial cells, containing the plasmid recombinant, produce elevated levels of protein release factor 2 as detected by an immune precipitation assay and in vitro measurement of UGA-directed peptide chain termination and [3H]UGA codon recognition.     

Agrocinopine A, a tumor-inducing plasmid-coded enzyme product, is a phosphodiester of sucrose and L-arabinose

Opines are unusual compounds found specifically in plant crown gall tumors. Genes for their synthesis and catabolism reside in agrobacteria as tumor-inducing (Ti) plasmid DNA. Only a small Ti-plasmid segment (24 kilobase pairs), the T-DNA, is transferred to the plant cell where it commonly codes for enzymes involved in the biosynthesis of nitrogenous opines such as nopaline (N2-(1,3-D-dicarboxypropyl)-L-arginine) as well as the tumor phenotype. Ellis and Murphy, (Ellis, J.G., and Murphy, P.J. (1981) Mol. Gen. Genet. 181, 36-43) reported the existence of the phosphorylated opines, agrocinopines A and B in tumors containing nopaline. Pure agrocinopine A has now been isolated in a yield of 0.05-0.06 g/100 g, fresh weight, from such tumors. Physical, chemical, and biological data establish the structure of agrocinopine A as an unusual non-nitrogenous opine of sucrose and L-arabinose with a phosphodiester linkage from the 2-hydroxyl of the arabinose to the 4-hydroxyl of the fructose moiety in sucrose. Agrocinopine B is the corresponding phosphodiester, in which the glucose has been hydrolyzed from the sucrose portion of agrocinopine A. Borohydride reduction of the free L-arabinose anomeric carbon of agrocinopine A, to the corresponding arabinitol derivative eliminates the characteristic inhibition zone enhancement produced by both agrocinopines A and B in the agrocin 84 (a fraudulent adenine nucleotide) bioassay. Because of the limited number of genes in the T-DNA, a generalization is proposed, whereby all opines will be found to comprise two common plant cell constituents linked in an uncommon manner by the minimum number of enzymes.     

The importance of the Escherichia coli ribosomal protein L16 for the reconstitution of the peptidyl-tRNA hydrolysis activity of peptide chain termination

The incubation of the 50 S ribosomal subunit of Escherichia coli with 1.5 M LiCl yields 1.5c core particles inactive in the peptidyl-tRNA hydrolysis activity of in vitro termination. The omission of L16 alone from reconstitutions of the proteins into the core results in inactive ribosomes. The single omission of a number of other proteins, in particular L7/L12, L10, L25, L27, and L15, gives ribosomes with intermediate activity. L16 alone is unable to restore significant activity to 1.5c cores, but together L16 and the above "stimulating" proteins produce particles as active as those reconstituted with the full complement of proteins. The ribosomal proteins important for the expression of peptidyl-tRNA hydrolysis and peptidyl transferase activities are very similar. However, ribosomes lacking both L11 and L16, but not L16 alone, surprisingly can catalyze codon- and release factor 2-dependent peptidyl-tRNA hydrolysis. The addition of L16 dramatically increases the activity. L16 is, therefore, important but not essential for the expression of the release factor 2-dependent peptidyl-tRNA hydrolysis.