Attachment of hyaluronic acid to polypropylene, polystyrene, and polytetrafluoroethylene.

Surfaces of polypropylene (PP), polystyrene (PS) and polytetrafluoroethylene (PTFE) were activated with radio frequency plasmas Ar and NH3 to aminate the polymer surface and were subsequently reacted with hyaluronic acid (HA) in one of the three different attachment schemes. Results show that ammonia plasma treated polymers were more reactive toward HA attachment. The three chemistry schemes consisted of two distinct approaches: (1) direct attachment of the HA to the aminated surface, and (2) extending the reactive group away from the surface with succinic anhydride and then reacting the newly formed carboxylic acid group with an adipic dihydrazide modified HA (HA-ADH). The latter scheme proved to be more effective, suggesting that steric effects were involved with the reactivity of the HA with surface groups. These HA-coated polymers are a candidate for cell attachment and growth.

Anti-pterins as tools to characterize the function of tetrahydrobiopterin in NO synthase.

Nitric oxide synthases (NOS) are homodimeric enzymes that NADPH-dependently convert L-arginine to nitric oxide and L-citrulline. Interestingly, all NOS also require (6R)-5,6,7, 8-tetrahydro-L-biopterin (H4Bip) for maximal activity although the mechanism is not fully understood. Basal NOS activity, i.e. that in the absence of exogenous H4Bip, has been attributed to enzyme-associated H4Bip. To elucidate further H4Bip function in purified NOS, we developed two types of pterin-based NOS inhibitors, termed anti-pterins. In contrast to type II anti-pterins, type I anti-pterins specifically displaced enzyme-associated H4Bip and inhibited H4Bip-stimulated NOS activity in a fully competitive manner but, surprisingly, had no effect on basal NOS activity. Moreover, for a number of different NOS preparations basal activity (percent of Vmax) was frequently higher than the percentage of pterin saturation and was not affected by preincubation of enzyme with H4Bip. Thus, basal NOS activity appeared to be independent of enzyme-associated H4Bip. The lack of intrinsic 4a-pterincarbinolamine dehydratase activity argued against classical H4Bip redox cycling in NOS. Rather, H4Bip was required for both maximal activity and stability of NOS by binding to the oxygenase/dimerization domain and preventing monomerization and inactivation during L-arginine turnover. Since anti-pterins were also effective in intact cells, they may become useful in modulating states of pathologically high nitric oxide formation.

Controlled chemical modification of hyaluronic acid: synthesis, applications, and biodegradation of hydrazide derivatives.

Controlled modification of the carboxylic acid moieties of hyaluronic acid with mono- and polyfunctional hydrazides leads to biochemical probes, biopolymers with altered physical and chemical properties, tethered drugs for controlled release, and crosslinked hydrogels as biocompatible scaffoldings for tissue engineering. Methods for polyhydrazide synthesis, for prodrug preparation, for hydrogel crosslinking, and for monitoring biodegradation are described.

Two subunits of heptaprenyl diphosphate synthase of Bacillus subtilis form a catalytically active complex.

Heptaprenyl diphosphate synthase of Bacillus subtilis, which participates in the biosynthesis of the side chain of menaquinone-7, is composed of two dissociable subunits, component I and component II, which are encoded by two cistrons in a novel gene cluster of gerC operon [Zhang, Y.-W., et al. (1997) J. Bacteriol. 179, 1417-1419]. This enzyme essentially requires the coexistence of both subunits for its catalysis. Expression vector systems for the two structural genes, gerC1 and gerC3, were constructed separately, and the two components were overproduced in Escherichia coli cells. After purification, their dynamic interactions in forming a catalytically active complex were investigated by gel filtration and immunoblotting analyses. When a mixture of the two components that had been preincubated in the presence of Mg2+ and farnesyl diphosphate was subjected to Superdex 200 gel filtration, a significant elution peak appeared in a region earlier than those observed when they were chromatographed individually. This fraction contained both components I and II, and it corresponded to a molecular mass that is in accord with the sum of the values of the two components. Cross-linking studies indicate that the two essential subunits, farnesyl diphosphate, and Mg2+ form a ternary complex which seems to represent a catalytically active state of the heptaprenyl diphosphate synthase. On the other hand, no complex was formed in the presence of isopentenyl diphosphate or inorganic pyrophosphate and Mg2+. A photoaffinity analogue of farnesyl diphosphate was shown to preferentially label the component I protein, suggesting that component I possesses a specific affinity for the allylic substrate. Furthermore, the photoaffinity labeling of component I significantly increased in the presence of component II. The mechanism of catalysis of this unique heteromeric enzyme is understood by assuming that association and dissociation of the two subunits facilitate turnover of catalysis for the synthesis of the amphipathic product from soluble substrates.

Synthesis and in vitro degradation of new polyvalent hydrazide cross-linked hydrogels of hyaluronic acid.

New polyvalent hydrazide cross-linkers were synthesized, characterized, and used to prepare hydrazide cross-linked hydrogels derived from hyaluronic acid (HA). First, the chemical synthesis and characterization of the di-, tri-, tetra-, penta-, and hexahydrazides are presented. Second, HA concentration, buffer type and concentration, and ratio of HA to carbodiimide to cross-linker were varied to obtain HA-hydrogels with different chemical and physical properties. Third, two new assays are described to monitor the stability of HA-hydrogels toward hyaluronidase (HAse) and other media. These assays were used to evaluate the stability of cross-linked HA-hydrogels to HAse solutions and different pH values. Hydrophobic cross-linkers gave the most stable gels, and the susceptibility of the gels to HAse was independent of cross-linker concentration. HAse does not significantly penetrate the HA-hydrogels and acts primarily at the gel-solution interface. The HA-hydrogels are stable in acid environments and dissolve gradually above pH 7.0.

Benzophenone photoprobes for phosphoinositides, peptides and drugs.

Benzophenones (BP) and related aryl ketone photophores have become established as the photoactivatable group of choice for high-efficiency covalent modification of hydrophobic regions of binding proteins, including enzymes and receptors that recognize peptide hormones, (oligo) nucleotides and nucleosides, phosphoinositides, inositol polyphosphates and a wide variety of therapeutic molecules. This review presents the advantages of BP as photoaffinity labels and provides specific examples from the last 3 years of applications of BP-containing ligands used in biochemistry.