Old yellow enzyme: Reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate

The reaction of the old yellow enzyme and reduced flavins with organic nitrate esters has been studied. Reduced flavins have been found to react readily with glycerin trinitrate (GTN ) (nitroglycerin) and propylene dinitrate, with rate constants at pH 7.0, 25°C of 145 M−1s−1 and 5.8 M−1s−1, respectively. With GTN, the secondary nitrate was removed reductively 6 times faster than the primary nitrate, with liberation of nitrite. With propylene dinitrate, on the other hand, the primary nitrate residue was 3 times more reactive than the secondary residue. In the old yellow enzyme-catalyzed NADPH-dependent reduction of GTN and propylene dinitrate, ping-pong kinetics are displayed, as found for all other substrates of the enzyme. Rapid-reaction studies of mixing reduced enzyme with the nitrate esters show that a reduced enzyme–substrate complex is formed before oxidation of the reduced flavin. The rate constants for these reactions and the apparent Kd values of the enzyme–substrate complexes have been determined and reveal that the rate-limiting step in catalysis is reduction of the enzyme by NADPH. Analysis of the products reveal that with the enzyme-catalyzed reactions, reduction of the primary nitrate in both GTN and propylene dinitrate is favored by comparison with the free-flavin reactions. This preferential positional reactivity can be rationalized by modeling of the substrates into the known crystal structure of the enzyme. In contrast to the facile reaction of free reduced flavins with GTN, reduced 5-deazaflavins have been found to react some 4–5 orders of magnitude slower. This finding implies that the chemical mechanism of the reaction is one involving radical transfers.

Split invertase polypeptides form functional complexes in the yeast periplasm in vivo.

The assembly of functional proteins from fragments in vivo has been recently described for several proteins, including the secreted maltose binding protein in Escherichia coli. Here we demonstrate for the first time that split gene products can function within the eukaryotic secretory system. Saccharomyces cerevisiae strains able to use sucrose produce the enzyme invertase, which is targeted by a signal peptide to the central secretory pathway and the periplasmic space. Using this enzyme as a model we find the following: (i) Polypeptide fragments of invertase, each containing a signal peptide, are independently translocated into the endoplasmic reticulum (ER) are modified by glycosylation, and travel the entire secretory pathway reaching the yeast periplasm. (ii) Simultaneous expression of independently translated and translocated overlapping fragments of invertase leads to the formation of an enzymatically active complex, whereas individually expressed fragments exhibit no activity. (iii) An active invertase complex is assembled in the ER, is targeted to the yeast periplasm, and is biologically functional, as judged by its ability to facilitate growth on sucrose as a single carbon source. These observation are discussed in relation to protein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.

Megalin-mediated endocytosis of transcobalamin-vitamin-B12 complexes suggests a role of the receptor in vitamin-B12 homeostasis.

Kidney cortex is a main target for circulating vitamin B12 (cobalamin) in complex with transcobalamin (TC). Ligand blotting of rabbit kidney cortex with rabbit 125I-TC-B12 and human TC-57Co-B12 revealed an exclusive binding to megalin, a 600-kDa endocytic receptor present in renal proximal tubule epithelium and other absorptive epithelia. The binding was Ca2+ dependent and inhibited by receptor-associated protein (RAP). Surface plasmon resonance analysis demonstrated a high-affinity interaction between purified rabbit megalin and rabbit TC-B12 but no measurable affinity of the vitamin complex for the homologous alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor related protein (LRP). 125I-TC-B12 was efficiently endocytosed in a RAP-inhibitable manner in megalin-expressing rat yolk sac carcinoma cells and in vivo microperfused rat proximal tubules. The radioactivity in the tubules localized to the endocytic compartments and a similar apical distribution in the proximal tubules was demonstrated after intravenous injection of 125I-TC-B12. The TC-B12 binding sites in the proximal tubule epithelium colocalized with megalin as shown by ligand binding to cryosections of rat kidney cortex, and the binding was inhibited by anti-megalin polyclonal antibody, EDTA, and RAP. These data show a novel nutritional dimension of megalin as a receptor involved in the cellular uptake of vitamin B12. The expression of megalin in absorptive epithelia in the kidney and other tissues including yolk sac and placenta suggests a role of the receptor in vitamin B12 homeostasis and fetal vitamin B12 supply.

Heterodimer formation and activity in the human enzyme galactose-1-phosphate uridylyltransferase.

One of the fundamental questions concerning expression and function of dimeric enzymes involves the impact of naturally occurring mutations on subunit assembly and heterodimer activity. This question is of particular interest for the human enzyme galactose-l-phosphate uridylyl-transferase (GALT), impairment of which results in the inherited metabolic disorder galactosemia, because many if not most patients studied to date are compound heterozygotes rather than true molecular homozygotes. Furthermore, the broad range of phenotypic severity observed in these patients raises the possibility that allelic combination, not just allelic constitution, may play some role in determining outcome. In the work described herein, we have selected two distinct naturally occurring null mutations of GALT, Q188R and R333W, and asked the questions (i) what are the impacts of these mutations on subunit assembly, and (ii) if heterodimers do form, are they active? To answer these questions, we have established a yeast system for the coexpression of epitope-tagged alleles of human GALT and investigated both the extent of specific GALT subunit interactions and the activity of defined heterodimer pools. We have found that both homodimers and heterodimers do form involving each of the mutant subunits tested and that both heterodimer pools retain substantial enzymatic activity. These results are significant not only in terms of their implications for furthering our understanding of galactosemia and GALT holoenzyme structure-function relationships but also because the system described may serve as a model for similar studies of other complexes composed of multiple subunits.

Immunotargeting of antioxidant enzyme to the pulmonary endothelium.

Oxidative injury to the pulmonary endothelium has pathological significance for a spectrum of diseases. Administration of antioxidant enzymes, superoxide dismutase (SOD) and catalase (Cat), has been proposed as a method to protect endothelium. However, neither these enzymes nor their derivatives possess specific affinity to endothelium and do not accumulate in the lung. Previously we have described a monoclonal antibody to angiotensin-converting enzyme (ACE) that accumulates selectively in the lung after systemic injection in rats, hamsters, cats, monkeys, and humans. In the present work we describe a system for selective intrapulmonary delivery of CuZn-SOD and Cat conjugated with biotinylated anti-ACE antibody mAb 9B9 (b-mAb 9B9) by a streptavidin (SA)-biotin bridge. Both enzymes biotinylated with biotin ester at biotin/enzyme ratio 20 retain enzymatic activity and bind SA without loss of activity. We have constructed tri-molecular heteropolymer complexes consisting of b-mAb 9B9, SA, and biotinylated SOD or biotinylated Cat and have studied biodistribution and pulmonary uptake of these complexes in the rat after i.v. injection. Biodistribution of biotinylated enzymes was similar to that of nonmodified enzymes. Binding of SA markedly prolonged lifetime of biotinylated enzymes in the circulation. In contrast to enzymes conjugated with nonspecific IgG, other enzyme derivatives, and nonmodified enzymes, biotinylated enzymes conjugated with b-mAb 9B9 accumulated specifically in the rat lung (9% of injected SOD/g of lung tissue and 7.5% of injected Cat/g of lung tissue). Pulmonary uptake of nonmodified enzymes or derivatives with nonspecific IgG did not exceed 0.5% of injected dose/g. Both SOD and Cat conjugated with b-mAb 9B9 were retained in the rat lung for at least several hours. Trichloracetic acid-precipitable radiolabeled Cat was associated with microsomal and plasma membrane fractions of the lung tissue homogenate. Thus, modification of antioxidant enzymes with biotin and SA-mediated conjugation with b-mAb 9B9 prolongs the circulation of enzymes resulting in selective accumulation in the lung and intracellular delivery of enzymes to the pulmonary endothelium. These results provide the background for an approach to provide protection of pulmonary endothelium against oxidative insults.

A rigid trans-spanning dinitrile ligand.

A rigid dinitrile ligand was synthesized from two xanthene units condensed to a naphthalene-1,4,5,8-diimide spacer. The rigidity and C shape of the ligand gave exclusively trans complexes with Pd(II), Ag(I), and Au(I). Evidence for complexation, coordination geometry, and stoichiometry was provided by a combination of 1H NMR, 19F NMR, and IR spectroscopy. The AuBF4 and PdCl2 complexes were shown to have a 1:1 (metal-to-ligand) stoichiometry and the AgBF4 complex was shown to have a 1:2 stoichiometry in solution. The preorganization of the dinitrile ligand resulted in complexes much more stable than their monodentate counterparts.

Sequence specificity of triplex DNA formation: Analysis by a combinatorial approach, restriction endonuclease protection selection and amplification.

We have devised a combinatorial method, restriction endonuclease protection selection and amplification (REPSA), to identify consensus ligand binding sequences in DNA. In this technique, cleavage by a type IIS restriction endonuclease (an enzyme that cleaves DNA at a site distal from its recognition sequence) is prevented by a bound ligand while unbound DNA is cleaved. Since the selection step of REPSA is performed in solution under mild conditions, this approach is amenable to the investigation of ligand-DNA complexes that are either insufficiently stable or not readily separable by other methods. Here we report the use of REPSA to identify the consensus duplex DNA sequence recognized by a G/T-rich oligodeoxyribonucleotide under conditions favoring purine-motif triple-helix formation. Analysis of 47 sequences indicated that recognition between 13 bases on the oligonucleotide 3' end and the duplex DNA was sufficient for triplex formation and indicated the possible existence of a new base triplet, G.AT. This information should help identify appropriate target sequences for purine-motif triplex formation and demonstrates the power of REPSA for investigating ligand-DNA interactions.