The amino acid sequence of ferredoxin from the alga Mastigocladuslaminosus

Ferredoxin was purified from the thermophilic blue-green alga, Mastigocladuslaminosus. The physicochemical properties of this ferredoxin are similar to those of other [2Fe-2S] plant ferredoxins except for its unusual thermal stability. The primary structure of the protein was determined and consists of 98 amino acid residues, 5 of which are cysteines. The positions of 4 cysteines which bind the iron atoms of the active centre are identical to those in other ferredoxins. The primary structure of the ferredoxin does not reveal any special features to account for its high thermal stability.     

Hydroxylamine inhibition of the nitrate reductase complex from Amaranthus

Nitrate reductase from Amaranthus viridis is similar to nitrate reductase from other plant sources. NH₂OH inhibits nitrate reduction from NADH by the nitrate reductase complex, but it does not inhibit either the NADH-dehydrogenase activity or nitrate reduction from reduced flavin mononucleotides. The inhibition observed was non-competitive with nitrate when the enzyme was pre-incubated with NH₂OH and NADH, and competitive with nitrate without pre-incubation. The K_i values for NH₂OH were 5 μM and 30 μM with or without pre-incubation respectively.     

NMR studies of the variant-3 neurotoxin from Centruroides sculpturatus Ewing

We report a preliminary high-resolution proton nuclear magnetic resonance characterization of the variant-3 toxin from the scorpion Centruroides sculpturatus Ewing (range Southwestern USA). This toxin assumes a well defined folded conformation in aqueous solutions at room temperature and undergoes reversible thermal denaturation. A number of amide hydrogens exhibit exchange life times varying from several minutes to several hours. A few tentative assignments of the low field aromatic CH resonances has been made on the basis of 2D-COSY and NOE experiments. The upfield shifts exhibited by Trp-47 suggest a unique microenvironment for this residue. The NMR data suggest that there is some degree of correlation between the solution structure of the variant-3 toxin and its crystallographic structure. Our studies provide a basis for a detailed elucidation of the structure-function relationships of these interesting scorpion toxins which bind to the sodium channels of excitable membranes and delay sodium current inactivation.     

Biosynthesis of phosphatidylinositol glycan-anchored membrane proteins. Design of a simple protein substrate to characterize the enzyme that cleaves the COOH-terminal signal peptide

Many nascent proteins that are destined to be anchored to plasma membranes by a phosphatidylinositol glycan (PI-G) are in the range of 50-70 kDa so that changes of 2-3 kDa between precursors and products during processing are not easily detected. Furthermore, PI-G-anchored proteins are generally glycosylated so that changes between the nascent (prepro) proteins and the mature products are not due simply to the loss of signal peptides. These problems have made it difficult to monitor the processing of the prepro form of wild type human placental alkaline phosphatase (PLAP) in a cell-free system. We have designed a smaller and simpler substrate of PI-G "transamidase" derived by deletion of approximately 60% of the internal sequence of preproPLAP 513. This engineered protein, preprominiPLAP 208, retains the NH2- and COOH-terminal signal peptides of PLAP as well as all the epitopes for site-directed antibodies of the latter, but is devoid of glycosylation sites, the active site, and most of the cysteine residues. With preprominiPLAP, it has been possible to demonstrate, in a cell-free system, step by step conversion to the pro form and then to the mature form, with the concomitant loss of the appropriate signal peptides. These changes were shown to be time- and enzyme concentration-dependent. Studies with Asp-179 site-directed mutants of preprominiPLAP showed the same specificity for amino acids with a monosubstituted beta carbon at the cleavage/attachment site that were found previously with wild type PLAP.     

Phosphatidylinositol glycan (PI-G) anchored membrane proteins. Amino acid requirements adjacent to the site of cleavage and PI-G attachment in the COOH-terminal signal peptide.

Secreted proteins are processed from a nascent form that contains an NH2-terminal signal peptide. During processing, the latter is cleaved by a specific NH2-terminal signal peptidase. The nascent form of phosphatidylinositol glycan (PI-G) tailed proteins contain both an NH2- and a COOH-terminal signal peptide. The two signal peptides have much in common, such as size and hydrophobicity. The COOH-terminal peptide is also cleaved during processing. We propose that the amino acid in a nascent protein that ultimately combines with the PI-G moiety be designated the omega site. Amino acids adjacent and COOH-terminal to the omega site would then be omega + 1, omega + 2, etc. In previous studies, we showed that allowable substitutions at the omega site of an engineered form of placental alkaline phosphatase (miniPLAP) are limited to 6 small amino acids. In the present study, mutations were made at the omega + 1 and omega + 2 sites. At the omega + 1 site, processing to varying degrees was observed with 8 of the 9 amino acids substituted for alanine, the normal constituent. Only the proline mutant showed no processing. By contrast, the only substituents permitted at the omega + 2 site were glycine and alanine, with only trace activity observed with serine and cysteine. Thus, just as there is a -1, -3 rule for predicting cleavage by NH2-terminal signal peptidase, there appears to be a comparable omega, omega + 2 rule for predicting cleavage/PI-G addition by COOH-terminal signal transamidase.     

Synthesis of lysine-containing fragments of the Proteus mirabilis O27 O-specific polysaccharide and neoglycoconjugates therefrom.

Amide-linked lysine mono- and di-uronic acid fragments of the O-specific polysaccharide from P. mirabilis O27 have been synthesised. N epsilon-Boc-L-lysine tert-butyl ester was condensed with 2-azidoethyl glycosides of glucuronic acid and beta-D-GlcpNAc-(1----3)-beta-D-GlcpA. Transformation of the products into 2-acrylamidoethyl glycosides, followed by deprotection using trifluoroacetic acid, gave the target monomers that were converted into high-molecular-weight copolymer-type neoglycoconjugates.     

Identification and characterization of the enzymatic activity of zeta-crystallin from guinea pig lens. A novel NADPH:quinone oxidoreductase.

zeta-Crystallin is a major protein in the lens of certain mammals. In guinea pigs it comprises 10% of the total lens protein, and it has been shown that a mutation in the zeta-crystallin gene is associated with autosomal dominant congenital cataract. As with several other lens crystallins of limited phylogenetic distribution, zeta-crystallin has been characterized as an "enzyme/crystallin" based on its ability to reduce catalytically the electron acceptor 2,6-dichlorophenolindophenol. We report here that certain naturally occurring quinones are good substrates for the enzymatic activity of zeta-crystallin. Among the various quinones tested, the orthoquinones 1,2-naphthoquinone and 9,10-phenanthrenequinone were the best substrates whereas menadione, ubiquinone, 9,10-anthraquinone, vitamins K1 and K2 were inactive as substrates. This quinone reductase activity was NADPH specific and exhibited typical Michaelis-Menten kinetics. Activity was sensitive to heat and sulfhydryl reagents but was very stable on freezing. Dicumarol (Ki = 1.3 x 10(-5) M) and nitrofurantoin (Ki = 1.4 x 10(-5) M) inhibited the activity competitively with respect to the electron acceptor, quinone. NADPH protected the enzyme against inactivation caused by heat, N-ethylmaleimide, or H2O2. Electron paramagnetic resonance spectroscopy of the reaction products showed formation of a semiquinone radical. The enzyme activity was associated with O2 consumption, generation of O2- and H2O2, and reduction of ferricytochrome c. These properties indicate that the enzyme acts through a one-electron transfer process. The substrate specificity, reaction characteristics, and physicochemical properties of zeta-crystallin demonstrate that it is an active NADPH:quinone oxidoreductase distinct from quinone reductases described previously.     

Identification of nitroxide radioprotectors.

The nitroxide Tempol, a stable free radical, has recently been shown to protect mammalian cells against several forms of oxidative stress including radiation-induced cytotoxicity. To extend this observation, six additional water-soluble nitroxides with different structural features were evaluated for potential radioprotective properties using Chinese hamster V79 cells and clonogenic assays. Nitroxides (10 mM) were added 10 min prior to radiation exposure and full radiation dose-response curves were determined. In addition to Tempol, five of the six nitroxides afforded in vitro radioprotection. The best protectors were found to be the positively charged nitroxides, Tempamine and 3-aminomethyl-PROXYL, with protection factors of 2.3 and 2.4, respectively, compared with Tempol, which had a protection factor of 1.3. 3-Carboxy-PROXYL, a negatively charged nitroxide, provided minimal protection. DNA binding characteristics as studied by nonequilibrium dialysis of DNA with each of the nitroxides demonstrated that Tempamine and 3-amino-methyl-PROXYL bound more strongly to DNA than did Tempol. Since DNA is assumed to be the target of radiation-induced cytotoxicity, differences in protection may be explained by variabilities in affinity of the protector for the target. This study establishes nitroxides as a general class of new nonthiol radioprotectors and suggests other parameters that may be exploited to find even better nitroxide-induced radioprotection.     

Nuclear magnetic resonance and molecular modeling studies on O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-se rine, a carbohydrate-protein linkage region fragment from connective tissue proteoglycans

The solution conformation of O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-ser ine (GXS), a carbohydrate-protein linkage region fragment from connective tissue proteoglycans, was investigated by two-dimensional NMR spectroscopy and molecular modeling calculations. Specifically, the 1H and 13C resonances were assigned by 2D-COSY and by 1H-13C heteronuclear correlation spectroscopy methods. 2D-NOESY was used to generate distance constraints between the galactose and xylose and between the xylose and serine residues. The 1H vicinal coupling constants for the sugars and the serine were also determined. A general molecular modeling methodology suitable for complex carbohydrates was developed. This methodology employed molecular dynamics and energy minimization procedures together with the application of inter-residue spatial constraints across the linkages derived from 2D-NOESY. The first step in this methodology is the generation of a wide variety of starting conformations that span the (phi, psi) space for each linkage. In the present study, nine such conformations were constructed for each linkage using the torsion angles phi and psi corresponding to the gauche+, gauche-, and trans configurations across each of the two bonds constituting the linkage. These conformations were subjected to a combined molecular dynamics/energy minimization refinement using the NOESY derived constraints as pseudoenergy functions. Families of conformations for the whole molecule were then constructed from the structures derived for each linkage. Characterization of GXS using this methodology identified a single family of conformations that are consistent with the solution phase NMR data on this molecule.     

Monoclonal antibodies to a nitroxide lipid hapten

The isolation and characterization of a hybridoma cell line producing a monoclonal IgG₁ antibody against a spin-label nitroxide group is described. The antibody recognizes a synthetic hapten containing linked dinitrophenyl and 2,2,6,6-tetramethylpiperidinyl 1-oxy groups, having an affinity of 3.6±1.0·10⁶ M^?1 for the soluble hapten at 25°C. The antibody binds to phospholipid vesicles containing 2 mol% of spin label-derivitized lipid (lipid hapten) with an affinity of 1.5±0.2·10⁸ M^?1. This monoclonal IgG₁ mediates the binding of hapten-bearing lipid vesicles to mouse macrophage RAW264 cells bearing Fc receptors. The cellular responses to this binding are similar to those observed previously using polyclonal rabbit anti-hapten IgG. As with the heterogeneous antibodies, the monoclonal IgG₁ is more efficient in mediating cellular uptake when the vesicles are in the ‘fluid’ physical state (dimyristoylphosphatidylcholine at 37°C) compared to ‘solid’ (dipalmitoylphosphatidylcholine at 37°C). Despite the enhanced binding of ‘fluid’ phospholipid vesicles to cells, only the ‘solid’ vesicles triggered a significant respiratory burst in RAW264 macrophages.