The transition state in the folding-unfolding reaction of four species of three-disulfide variant of hen lysozyme: the role of each disulfide bridge.

The effects of lacking a specific disulfide bridge on the transition state in folding were examined in order to explore the folding-unfolding mechanism of lysozyme. Four species of three-disulfide variant of hen lysozyme (3SS-lysozyme) were prepared by replacing two Cys residues with Ala or Ser: C6S/C127A, C30A/C115A, C64A/C80A and C76A/C94A. The recombinant hen lysozyme was studied as the standard reference containing four authentic disulfide bridges and the extra N-terminal Met: the recombinant hen lysozyme containing the extra N-terminal. Folding rates were measured by monitoring the change in fluorescence intensity associated with tri-N-acetyl-d-glucosamine binding to the active site of refolded lysozyme. It was confirmed that the folding rate of the recombinant hen lysozyme containing the extra N-terminal was the same as that of wild-type lysozyme, and that the folding rate was little affected by the presence of tri-N-acetyl-d-glucosamine (triNAG). The folding rate of C64A/C80A was found to be the fastest and almost the same as that of the recombinant hen lysozyme containing the extra N-terminal, and that of C30A/C115A the second, and that of C6S/C127A the third. The folding rate of C76A/C94A was particularly slow. On the other hand, the unfolding rates which were measured in the presence of triNAG showed the dependence on the concentration of triNAG. The intrinsic unfolding rate in the absence of triNAG was determined by extrapolation. Also in the unfolding rate, C76A/C94A was markedly slower than the others. It was found from the analysis of binding constants of triNAG to C64A/C80A during the unfolding process that the active site of C64A/C80A partly unfolds already prior to the unfolding transition. On the basis of these kinetic data, we suggest that C64A/C80A folding transition can occur with leaving the loop region around SS3 (C64-C80) flexible, while cross-linking by SS4 (C76-C94) is important for the promotion of folding, because it is an indispensable constraint on the way towards the folding transition state.

Effects of dietary fats and phytosterol on serum fatty acid composition and lipoprotein cholesterol in rats.

The effects of dietary fats and phytosterol on the fatty acid composition and lipoprotein cholesterol in serum were studied in female rats, with the following results. (1) The addition of 1% cholesterol to the 20% butter diet decreased the ratio of polyunsaturated fatty acid (PUFA) to saturated fatty acid (SFA) in serum. This phenomenon was negated when there was an intake of cod liver oil and wheat germ oil. (2) When cholesterol was added to the 20% butter diet, the serum total cholesterol increased 3.7-fold, due to an increase in the lower density lipoprotein (LDL + VLDL). (3) The addition of 5% phytosterol to the 10% butter-cholesterol diet reduced the total cholesterol level and increased the ratio of cholesterol in high density lipoprotein (HDL) to the cholesterol in LDL + VLDL. Although a 10% cod liver oil addition also reduced the total cholesterol level, the ratio of HDL/LDL + VLDL was similar to that of the 10% butter-cholesterol diet. (4) A direct relationship was found between the concentration of oleic acid (18:1) in serum and the total cholesterol level (r = 0.947) and also the level of LDL + VLDL-cholesterol (r = 0.935). These results show that cod liver oil, wheat germ oil, and phytosterol induce an increase in the PUFA/SFA ratio, promote hypocholesterolemia, and change lipoprotein concentration. However, there were indications that no relationship exists between the change in the total cholesterol level and the change in the ratio of HDL/LDL + VLDL, and that the increase of total cholesterol and LDL + VLDL-cholesterol was consistent with the increase of oleic acid in serum.

Forward mutation assay for screening carcinogens by alkaline phosphatase constitutive mutations in Escherichia coli K-12.

A new forward mutation assay was developed with Escherichia coli using alkaline phosphatase (APase) constitutive mutations as a genetic marker. Mutation in any one of the three regulator genes (phoR, T and S) is known to make the cell constitutive for APase synthesis and enable the mutants to form larger colonies on beta-glycerophosphate plate under the condition of excess inorganic phosphate. This property was used for qualitative and quantitative assay of chemical mutagens. Attempts were made to construct suitable strains for this assay by introduction of various genetic traits that might increase the sensitivity of mutation. Three known chemical mutagens (N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), and 4-nitroquinoline-1-oxide (NQNO)) were employed as reference compounds in the quantitative assay. Among the strains constructed, a tester strain with genetic markers tif-1, uvrA and pKM101 was the most sensitive to these compounds, judging from tests on concentration-dependent mutagenic activity. The merits and limitations of the present system are discussed.

Escherichia coli mutants deficient in the production of alkaline phosphatase isozymes.

Escherichia coli K-12 mutants showing an altered isozyme pattern of alkaline phosphatase were isolated. Whereas wild-type strains synthesized all three isozymes in a synthetic medium supplemented with Casamino Acids or arginine but synthesized only isozyme 3 in a medium without supplement, the mutant strains synthesized isozyme 1 and a small amount (if any) of isozyme 2, but no isozyme 3, under all growth conditions. The mutation responsible for the altered isozyme pattern, designated iap, was mapped by P1 transduction in the interval between cysC and srl (at about 58.5 min on the E. coli genetic map). It was cotransducible with cysC and srl at frequencies of 0.54 and 0.08, respectively. The order of the genes in this region was srl-iap-cysC-argA-thyA-lysA. Three more independent mutations were also mapped in the same locus. We purified isozymes 1' and 3' from iap and iap+ strains and analyzed the sequences of four amino acids from the amino terminus of each polypeptide. They were Arg-Thr-Pro-Glu (or Gln) in isozyme 1' and Thr-Pro-Glu (or gln)-Met in isozyme 3', which were identical with those of corresponding isozymes produced by the wild-type phoA+ strain (P.M. Kelley, P.A. Neumann, K. Schriefer, F. Cancedda, M.J. Schlesinger, and R.A. Bradshaw, Biochemistry 12:3499-3503, 1973; M.J. Schlesinger, W. Bloch, and P.M. Kelley, p. 333-342, in Isozymes, Academic Press Inc., 1975). These results indicate that the different mobilities of isozymes 1, 2, and 3 are determined by the presence or absence of amino-terminal arginine residues in polypeptides.

Factors affecting the formation of alkaline phosphatase isozymes in Escherichia coli K-12.

Physiological and genetic factors affecting the formation of isozymes of alkaline phosphatase in Escherichia coli K-12 were studied. Our results are compatible with the hypothesis proposed by Schlesinger and his co-workers (Schlesinger et al., 1975) that the multiple forms of the enzyme are produced by converting a newly synthesized one (the least negatively charged one) into less negatively charged forms. Neither energy source nor de novo synthesis of the enzyme was necessary for the conversion. It is also confirmed that the conversion is effectively inhibited by externally added arginine (Piggott et al., 1972) but only partially by canavanine (arginine analog) or casamino acids. We isolated a mutant strain which did not form isozyme(s), if any, under the condition in which the wild type strain formed isozymes. The mutation(s) was proved to be mapped in the locus (or loci) other than alkaline phosphatase structure gene in the E. coli genetic map. We tentatively proposed to designate this the iap gene(s), an abbreviation for isozyme of alkaline phosphatase, which plays a role in isozyme formation.