O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme which catalyzes the final step in the biosynthesis of L-cysteine in Salmonella, viz., the conversion of O-acetyl-L-serine (OAS) and sulfide to L-cysteine and acetate. UV-visible spectra of OASS exhibit absorbance maxima at 280 and 412 nm with pH-independent extinction coefficients over the range 5.5-10.8. Addition of OAS to enzyme results in a shift in the absorbance maximum from 412 to 470 nm, indicating the formation of an alpha-aminoacrylate Schiff base intermediate [Cook, P. F., & Wedding, R. T. (1976) J. Biol. Chem. 251, 2023]. The spectrum of the intermediate is also pH independent from 5.5 to 9.2. The observed changes in absorbance at 470 nm at different concentrations of OAS were used to calculate a Kd of 3 microM for OAS at pH 6.9. As the pH decreases, the Kd increases an order of magnitude per pH unit. The 31P NMR signal of the bound PLP has a pH-independent chemical shift of 5.2 ppm in the presence and absence of OAS. These results indicate that the phosphate group is present as the dianion possibly salt-bridged to positively charged groups of the protein. In agreement with this, the resonance at 5.2 ppm has a line width of 20.5 Hz, suggesting that the cofactor is tightly bound to the protein. The sulfhydrylase was also shown to catalyze an OAS deacetylase activity in which OAS is degraded to pyruvate, ammonia, and acetate. The activity was detected by a time-dependent disappearance of the 470-nm absorbance reflecting the alpha-aminoacrylate intermediate. The rate of disappearance of the intermediate was measured at pH values from 7 to 9.5 using equal concentrations of OAS and OASS. The rate constant for disappearance of the intermediate decreases below a pK of 8.1 +/- 0.1, reflecting the deprotonation of the active-site lysine that originally formed the Schiff base with PLP in free enzyme. A possible mechanism for the deacetylase activity is presented where the lysine displaces alpha-aminoacrylate which decomposes to pyruvate and ammonia.

During in vitro aging, deamidation of recombinant human stem cell factor produced in Escherichia. coli was detected by HPLC analysis and by the release of soluble ammonia. The deamidation rate is very slow in buffers at low pH or at low temperatures; however, the rate is significantly accelerated in alkaline buffers such as sodium bicarbonate in combination with elevated temperatures. HPLC isolation of various deamidated forms followed by peptide mapping and mass spectrometric analyses revealed that the deamidation involves Asn10 in the sequence -T9NNV- near the N-terminus of the protein. Following peptide mapping analysis, significant amounts of aspartyl and isoaspartyl peptides were identified, indicating the conversion of asparagine into both aspartate and isoaspartate residues. As a result of spontaneous association-dissociation of stem cell factor dimer, a total of five deamidated forms, including two homodimers and three heterodimers, were detected and isolated. Cell proliferation assays showed that two rhSCF heterodimeric species, derived from dimerization between isoaspartyl and other stem cell factor monomers, retain only approximately half of the biological activity. The homodimer with isoaspartic acid in place of Asn10 is 50-fold less potent, while the aspartyl homodimer, either isolated during deamidation experiments or recombinantly prepared by site-directed mutagenesis (e.g., N10D and N10D/N11D variants), exhibits higher activity than the standard molecule. In comparison, synthetic N10A and N10E variants, though missing the deamidation site, are significantly less active. All these variants lacking the Asn10 deamidation site are relatively more stable than those containing the asparagine residue. The results indicate that the biological function and chemical stability of stem cell factor are influenced by the nature of the residue at position 10.

The absorption spectrum of Ti3+:Al2O3 crystals grown in a reducing atmosphere consists of the main blue-green absorption band and a weak infrared band. This infrared absorption decreases in intensity on annealing the as-grown samples in a reducing atmosphere. The analysis of electron spin resonance spectra of the as-grown samples, in which the infrared absorption band is observed, indicates that the center associated with the band is a cluster involving Ti3+ and Ti4+ ions with a neighboring, charge compensating Al3+ vacancy. The coexistence of a Ti4+ ion and an Al3+ vacancy in the neighborhood of the Ti3+ ion weakens the crystal field at this ion more than a single Ti4+ ion, giving rise to a red shift of the Ti3+ absorption.

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Expression vectors constructed from human and rat pro-neu differentiation factor (NDF) cDNAs were transfected in Chinese hamster ovary cells for expression of recombinant NDF molecules. Soluble NDF forms were released into culture medium after post-translational processing of the membrane-bound pro-NDF forms. Different human and rat NDF isoforms, after being purified from the culture medium, were subjected to structural and biochemical characterizations. The isolated human and rat NDF isoforms have been proteolytically processed at a specific site at the N terminus, which is different from that observed for the processing of rat or human NDF molecule prepared from natural origins. The processing of each recombinant NDF isoform at its C terminus was heterogeneous but consistently occurred at nearby peptide bonds. Specific N- and C-terminal processing by Chinese hamster ovary cells has resulted in the production of two types (alpha and beta) of recombinant NDFs containing 222-225 amino acid residues. Both human and rat NDF molecules are heavily glycosylated at two of the three potential Asn-linked glycosylation sites and contain O-linked sugars at 11 of the Thr/Ser sites. Glycosylation occurs at a short, Ser/Thr-rich spacer region that connects the N-terminal immunoglobulin homology unit to the epidermal growth factor domain. Cellular phosphorylation assay indicated that these secreted forms contain similar biological activity in receptor tyrosine autophosphorylation of mammary tumor cells.