Background: Low molecular mass hyaluronan (LMHA) is proinflammatory, but the role of the N-acetyl moieties is unknown. Results: Chemical reacetylation of LMHA results in maximal proinflammatory cytokine production by human macrophages, compared with other N-acylations. Partial N-butyrylation blocks cytokine stimulation. Conclusion:The N-acetyl moieties of glucosamine are critical for LMHA proinflammatory properties. Significance: N-Acetylation and butyrylation of LMHA modulate proinflammatory cytokine production.

Aldehyde reductase is an enzyme capable of metabolizing a wide variety of aldehydes to their corresponding alcohols. The tertiary structures of aldehyde reductase and aldose reductase are similar and consist of an alpha/beta-barrel with the active site located at the carboxy terminus of the strands of the barrel. We have determined the X-ray crystal structure of porcine aldehyde reductase holoenzyme in complex with an aldose reductase inhibitor, tolrestat, at 2.4 A resolution to obtain a picture of the binding conformation of inhibitors to aldehyde reductase. Tolrestat binds in the active site pocket of aldehyde reductase and interacts through van der Waals contacts with Arg 312 and Asp 313. The carboxylate group of tolrestat is within hydrogen bonding distance with His 113 and Trp 114. Mutation of Arg 312 to alanine in porcine aldehyde reductase alters the potency of inhibition of the enzyme by aldose reductase inhibitors. Our results indicate that the structure of the inhibitor-binding site of aldehyde reductase differs from that of aldose reductase due to the participation of nonconserved residues in its formation. A major difference is the participation of Arg 312 and Asp 313 in lining the inhibitor-binding site in aldehyde reductase but not in aldose reductase.

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The free amino group of GlcN seems responsible for inhibition of chondrocyte proliferation and PG synthesis. These effects were greater under higher concentrations of GlcN in AD vs AI conditions. GlcN.HCl behaves similarly to GlcN.S, but differential effects with GlcN-X,Y,Z(SO(4))(n) isomers were observed. Acetylation or sulfation of the GlcN amino group reverses or partially reverses, respectively, anti-proliferative effects of GlcN. Sulfation of GlcN, at positions 3 and 6 results in complex effects on AC proliferation and PG synthesis.

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Mg" ions, essential for the catalytic activity of mammalian inositol monophosphatase, increase the ellipticity in the near-ultraviolet region of the CD spectrum of the enzyme. These spectral changes are not affected by the additional presence of substrate and are reversed if EDTA is added to the solution of enzyme and metal ions. Titration of the spectral perturbation at 275 nm shows that this binding occurs with a dissociation constant ( K J around 275 pM, 292 pM and 302 pM for the wild-type, [Gln217]inositol monophosphatase and [Phe219]inositol monophosphatase enzymes respectively. The source of the spectroscopic change at 275 nm is not Trp219.The addition of Mgz+ also causes a decrease in ellipticity over most of the far-ultraviolet region of the spectrum (between 205-240 nm). The Kd values describing the binding of Mg" ions are 3.9 mM, 6.8 mM and 29.1 mM for the wild-type, [Gln217]inositol monophosphatase and [Phe219]inositol monophosphatase enzymes, respectively, each showing an approximate 12 % change in ellipticity. In the additional presence of 10 mM P,, there is a fourfold increase in the affinity of wild-type enzyme for Mg".It is concluded that CD spectral changes at wavelengths around 275 nm are indicative of metal ions interacting with a high-affinity metal-binding site (site 3 ) . The spectral changes around 225 nm are associated with interactions at a lower-affinity site normally occupied by the Mg2' ion which is reflected by the K,, value for this metal ion.Other metal ions such as Ca2+ and Tb" (but not Mn'+ or Znz+) also perturb the CD spectrum of the enzyme in both regions of the spectrum. The amplitudes of these signal changes are greater for Mg" or Tb" (25%) ions than for Ca2+ (8.5%), although two Caz+-binding sites with Kd values of 20 pM and 100 pM have been identified.The uncompetitive inhibitor Li' causes little change in the near-ultraviolet spectrum in the absence or presence of either substrate or P,. However, in contrast to other metal ions, Li' ions elicit a 10% increase in ellipticity at 220 nm with a Kd of 0.8 mM.Keywords: inositol monophosphatase ; circular dichroism; lithium ; magnesium.Inositol monophosphatase is a homodimeric enzyme of subunit molecular mass 30 kDa that catalyses the dephosphorylation of Ins( 1)P, Ins(3)P and Ins(4)P (inositol monophosphates) to liberate inositol and P, (inorganic phosphate), the former of which may be used to replenish stores of membrane-bound phosphatidylinositol 4,5-bisphosphate [l]. The pivotal role in the phosphoinositol lipid signalling pathway played by this enzyme is of greater significance in the brain due to the inability of dietary inositol to cross the blood-brain barrier effectively 121, although inositol may be synthesised de novo from glucose 6-phosphate via the action of inositol 1-synthase [3]. A high level of sequence similarity between the human, rat and bovine forms of the enzyme has been demonstrated using cDNA cloning and sequencing techniques [4, 51. Mg2' ions are absolutely required for activity [6], although concentratio...

Bovine brain inositol monophosphatase is inactivated when trypsin catalyses the cleavage of a single peptrde bond between Lys-36 and Ser-37. This proteolysis is closely followed by cleavage at two other sites in the protein between Lys-78 and Ser-79 and between Lys-156 and Ser-157 suggesting that all of these sites are exposed in the native conformation of the protein. All of these residues are predicted to lie at the ends of a helices. The most susceptible bond (Lys-36Ser-37) is predicted to lie in a highly flexible region of the protein. Circular dichroism studies suggest that approximately 40% of the secondary structure of this protein is helical which is similar to that predtcted by the algorithm of Gamier et al. [(1978) J. Mol. Biol. 120, 97-1201.