[Modification of L-asparaginase with colominic acid and the new characteristics of the modified enzyme].

The colominic acid was covalently coupled to L-asparaginase molecule by reductive amination. Depending on the molar ratios of colominic acid-asparaginase (30:1, 50:1 and 100:1), a modified enzyme molecule contained 4.7, 7.2 and 12 colominic acid molecule, they retained 58%, 56% and 33.2% of the initial asparaginase activity, respectively. In comparison with the native enzyme, modified enzyme had lower immunogenicity and antigenicity, longer half-life time (in vitro), more resistance ability to trypsin proteolysis, and similar Km value for L-asparagine.

Inter-tryptophan distances in rat cellular retinol binding protein II by solid-state NMR.

Structural constraints for the tryptophans in rat cellular retinol binding protein II (CRBP II) have been obtained by rotational-echo double-resonance (REDOR) solid-state NMR. CRBP II was labeled with L-[6-19F]tryptophan and L-[2-13C]tryptophan. The 13C-19F dipolar coupling was determined for various possible tryptophan geometries. The allowed distance between the closest two of the four tryptophans in CRBP II was obtained for each geometry. The minimum possible distance between these two tryptophans in CRBP II is 7 A, and the maximum possible distance is 11 A.

Alteration of the binding specificity of cellular retinol-binding protein II by site-directed mutagenesis.

Rat cellular retinol-binding protein II (CRBP II) is an abundant 134-residue intestinal protein that binds all-trans-retinol and all-trans-retinal. It belongs to a family of homologous, 15-kDa cytoplasmic proteins that bind hydrophobic ligands in a noncovalent fashion. These binding proteins include a number of proteins that bind long chain fatty acids. X-ray analyses of the structure of two family members, rat intestinal fatty acid-binding protein and bovine myelin P2 protein, indicate that they have a high degree of conformational similarity and that the carboxylate group of their bound fatty acid interacts with a delta-guanidium group of at least 1 of 2 "buried" arginine residues. These 2 Arg residues are conserved in other family members that bind long chain fatty acids and in cellular retinoic acid-binding protein, but are replaced by Gln109 and Gln129 in CRBP II. We have genetically engineered two amino acid substitutions in CRBP II: 1) Gln109 to Arg and 2) Gln129 to Arg. The purified Escherichia coli-derived CRBP II mutant proteins were analyzed by fluorescence and nuclear magnetic resonance spectroscopy. Both mutants exhibit markedly decreased binding of all-trans-retinol and all-trans-retinaldehyde, but no increased binding of all-trans-retinoic acid. Arg substitution for Gln109 but not for Gln129 produces a dramatic increase in palmitate binding activity. Analysis of the endogenous fatty acids associated with the purified E. coli-derived proteins revealed that E. coli-derived intestinal fatty acid binding protein and the Arg109 CRBP II mutant are complexed with endogenous fatty acids in a qualitatively and quantitatively similar manner. These results provide evidence that this internal Arg may play an important role in the binding of long chain fatty acids by members of this protein family.

Fluorine nuclear magnetic resonance analysis of the ligand binding properties of two homologous rat cellular retinol-binding proteins expressed in Escherichia coli.

Comparative 19F NMR studies were performed on rat cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBPII) to better understand their role in intracellular retinol metabolism within the polarized absorptive epithelial cells (enterocytes) of the intestine. Efficient incorporation of 6-fluorotryptophan (6-FTrp) into these homologous proteins was achieved by growing a tryptophan auxotroph of Escherichia coli, harboring prokaryotic expression vectors with either a full-length rat CRBPII or CRBP cDNA on defined medium supplemented with the analog. It is possible to easily distinguish resonances corresponding to 6-FTrp-apoCRBP, 6-FTrp-CRBP-retinol (or retinal), 6-FTrp-apoCRBPII, and 6-FTrp-CRBPII-retinol (or retinal). We were thus able to use 19F NMR spectroscopy to monitor transfer of all-trans-retinol and all-trans-retinal between CRBPII and CRBP in vitro. Retinol complexed to CRBPII is readily transferred to CRBP, whereas retinol complexed to CRBP is not readily transferred to CRBPII. We estimated that the Kd for CRBP-retinol is approximately 100-fold less than the Kd for CRBPII-retinol. Transfer of all-trans-retinal occurs readily from CRBPII to CRBP and from CRBP to CRBPII. Results from competitive binding studies with retinol and retinal indicated that there is a much larger difference between the affinities of CRBP for retinol and retinal than between the affinities of CRBPII for these two ligands. However, the differences in binding specificities reflect differences in how the two proteins interact with retinol, rather than with retinal. 19F NMR analysis of recombinant isotopically labeled proteins represents a sensitive new and useful method for monitoring retinoid flux between the CRBPs in vitro.

19F nuclear magnetic resonance studies of 6-fluorotryptophan-substituted rat cellular retinol binding protein II produced in Escherichia coli. An analysis of four tryptophan substitution mutants and their interactions with all-trans-retinol.

Rat cellular retinol binding protein (CRBP II) is a 134-amino acid intracellular protein synthesized in the polarized absorptive cells of the intestine. We have previously used 19F nuclear magnetic resonance (NMR) spectroscopy to survey the structural effects of ligand binding on the apoprotein. For these studies, all 4 Trp residues of rat CRBP II were efficiently labeled with 6-fluorotryptophan (6-F-Trp) by inducing its expression in a tryptophan auxotroph of Escherichia coli. Resonances corresponding to 2 of its Trp residues underwent large downfield shifts upon binding of all-trans-retinol and retinal, while resonances corresponding to the other 2 Trp residues underwent only minor perturbations in chemical shifts. To identify which Trp residues undergo changes in their environment upon ligand binding, we have constructed four CRBP II mutants where Trp9, Trp89, Trp107, or Trp110 have been replaced by another hydrophobic amino acid. By comparing the 19F NMR spectrum of each 6-F-Trp-labeled mutant with that of wild type 6-F-Trp CRBP II, we demonstrate that the 19F resonance corresponding to Trp107 undergoes the largest change in chemical shift upon ligand binding (2.0 ppm downfield). This is consistent with the position of this residue predicted from molecular modeling studies. The 19F resonance corresponding to Trp9 also undergoes a downfield change in chemical shift of 0.5 ppm associated with retinol binding even though it is predicted to be removed from the ligand binding site. By contrast, the resonances assigned to Trp89 and Trp110 undergo only minor perturbations in chemical shifts. These results have allowed us to identify residue-specific probes for evaluating the interactions of all-trans-retinol (and other retinoids) with this intracellular binding protein.