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Ribonucleotide reductase activity is markedly elevated in cell lines selected for resistance to hydroxyurea, a cytotoxic drug known specifically to inhibit ribonucleotide reductase. From a cDNA library constructed from a highly hydroxyurea-resistant hamster lung cell line, 600H in which the activity is elevated more than 80-fold, we have isolated a full length cDNA for the small subunit of the reductase. The cDNA is 3.48 kb long with an open reading frame of 1158 nucleotides and a long 3' flanking region of 2169 nucleotides from the termination codon. The derived polypeptide sequence is closely similar to the small subunit of the mouse, differing from it in 20 amino acid positions. Most of these replacements occur in the N-terminal segment of the protein. The hamster subunit does not contain 4 amino acid residues found in the mouse small subunit near the C-terminal end. RNA blots probed with the cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells. 相似文献
15.
Trypsin-subtilisin inhibitor from marine turtle eggwhite refolded quantitatively from its fully reduced state atpH 8.5 in the presence of reduced and oxidized glutathione. The refolding process was studied by following the accompanying changes in inhibitory activity, fluorescence, sulfhydryl group titer, and hydrodynamic volume. The refolding process followed second-order kinetics with rate constants of 4.80×102 M–1 sec–1 for trypsin-inhibiting domain and 0.77× 102 M–1 sec–1 for subtilisin-inhibiting domain of the inhibitor at 30°C and their respective activation energies of the refolding process were 15.9 and 21.6 kcal/mol. Fluorescence intensity of the reduced inhibitor decreased with time of refolding until it corresponded to the intensity of the native inhibitor. The inhibitor contained 1–2%-helix, 40–42%-sheet, and 57–58% random coil structure. Refolded inhibitor gave a circular dichroic spectrum identical to that of the native inhibitor. A number of principal intermediates were detected as a function of the refolding time. Size-exclusion chromatography separated the intermediates differing in hydrodynamic volume (Stokes radius). The Stokes radius ranged from 23 Å (fully reduced inhibitor) to 18.8 Å (native inhibitor). Results indicated the independent refolding of two domains of the inhibitor and multiple pathways of folding were followed rather than an ordered sequential pathway. 相似文献
16.
A new method to improve refolding yields and to increase the concentration of refolded proteins in a single operation has been developed. The method uses size-exclusion chromatography matrices to perform buffer exchange, aggregate removal, and the folding reaction. The reduced diffusion of proteins in gel-filtration media has been shown to suppress the nonspecific interactions of partially folded molecules, thus reducing aggregation. Hen egg white lysozyme (HEWL) and bovine carbonic anhydrase (CAB) were successfully refolded from initial protein concentrations of up to 80 mg/mL using Sephacryl S-100 (HR). The aggregation reaction for lysozyme was reduced and was only detected at the highest protein concentration used. The average recovery of lysozyme was 63%, with an average specific activity of 104%. Carbonic anhydrase experiments also showed that aggregation was suppressed and the average protein recovery from the column was 56%, with a specific activity of 81%. This process enables refolding and the purification of active species to be achieved in a single step. (c) 1996 John Wiley & Sons, Inc. 相似文献
17.
Summary The recovery of proteins solubilised inside reversed micelles is generally a low yielding process. We have studied three methods
for recovery of lysozyme. The protein laden organic phase was contacted with solutions of varying pH. Greater than 95% recovery
was achieved at pH values close to the isoelectric point (10.9). The use of 2.5 M KCl gave close to 100% recovery of lysozyme.
The organic phase was passed onto a ion exchange matrix. This resulted in disruption of the reversed micelle releasing 60–80%
of the protein into the eluent. 相似文献
18.
The production of cellulases in batch culture was studied using a mutant strain of Trichoderma reesei C-5 growing on lactose. Growth kinetic parameters on 2% lactose were studied and the comparative results for growth and enzyme productivities at two different lactose levels are discussed. The cellulase synthesis rate depended on the lactose concentration in the medium. Although growth was favoured at a higher lactose level, the volumetric enzyme productivity did not increase in proportion and the specific enzyme productivity decreased to a certain extent, indicating that partial catabolic inhibition at higher lactose concentrations may be possible. However, it was noted that the mutant strain was highly depressed and capable of synthesising active cellulases on lactose. 相似文献
19.
Studies on the batch extraction of lactic acid using an emulsion liquid membrane system are reported. The membrane phase consists of the tertiary amine carrier Alamine 336 and the surfactant Span 80 dissolved in n-heptane/paraffin and aqueous solutions of sodium carbonate in the internal phase. The effects of internal phase reagent, extraction temperature, and initial external phase pH on the extraction efficiency and the emulsion swelling are examined. A statistical factorial experiment on extraction from clarified lactic acid fermentation broth was carried out to obtain knowledge of the performance of the extraction system from a broth. The extraction efficiency from the fermentation broth is found to be lower as compared to aqueous solutions of pure lactic acid. The effect of pH and the presence of other ionic species on selectivity are discussed. (c) 1993 John Wiley & Sons, Inc. 相似文献
20.
A basic trypsin-subtilisin inhibitor has been isolated from the egg white of marine turtle (Caretta caretta Linn.) and purified to homogeneity by gel filtration followed by ion-exchange chromatography. It has a single polypeptide chain of 117 amino acid residues, having a molecular weight of 13,600. It lacks methionine and tryptophan. Its isoelectric point is atpH 10.0 and the sedimentation coefficient (s20,w) value of 1.62 S is independent of protein concentration. It has a Stokes radius of 18.8 Å, an intrinsic viscosity of 0.048 dl g–1 and a diffusion coefficient of 10.17×10–7 cm2 sec–1. Its fluorescence emission spectrum is similar to that of free tyrosine and the bimolecular quencing rate constant of its tyrosine residues with acrylamide is 3.15×109 M–1 sec–1. The inhibitor strongly inhibits both trypsin and subtilisin by forming enzyme-inhibitor complexes at a molar ratio of unity. The nature of inhibition toward both enzymes is not temporary. It has independent binding sites for inhibition of trypsin and subtilisin. Chemical modification with tetranitromethane suggests the presence of three tyrosine residues on the surface of the inhibitor molecule. 相似文献