Hidenori Yamada

Unfolding rates of globular proteins determined by kinetics of proteolysis

A convenient method for the determination of unfolding rates of small globular proteins under physiological conditions was developed using digestion with proteases. The apparent first-order rate constants for digestion of lysozyme with thermolysin and with Pronase at pH 8 and 50 degrees C were shown to be saturated with increases of concentrations of these proteases. The maximum rate constants extrapolated were identical in digestions with two different proteases, and were found to be equal to the unfolding rate constant of lysozyme. Similarly, the unfolding rate constant of RNase A at pH 8 and 50 degrees C, and those of lysozyme, RNase A and beta-lactoglobulin at pH 8 and 40 degrees C, were determined by the digestion method. Thus, it was shown that digestion by proteases proceeds mainly via the unfolded state of proteins.

A convenient S-2-aminoethylation of cysteinyl residues in reduced proteins

The cysteinyl residues in proteins had been S-2-aminoethylated with ethylenimine quantitatively. However, ethylenimine is now listed as a carcinogen and is no longer commercially available. A method of converting cysteinyl residues to S-2-aminoethyl derivatives quantitatively using 2-bromoethylamine under mild conditions was developed here.

A convenient protein substrate for the determination of protease specificity: reduced and S-3-(trimethylated amino)propylated lysozyme

Reduced lysozyme was alkylated with 3-bromopropyltrimethylammonium bromide to give reduced and S-3-(trimethylated amino)propylated lysozyme. It was soluble in a wide range of pH and is suitable as the protein substrate to determine protease specificity.

Chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozyme

Preparation of chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozymes and its application to separation of N-bromosuccinimide-oxidized lysozymes are described. By pH gradient elution, two diastereomers of oxindolealanine-62-lysozyme, delta 1-acetoxytryptophan-62-lysozyme (intermediate product in the reaction in acetate buffer), and native lysozyme were all separated within 40 min.

Characterization of a Low-Molecular-Weight Growth Inhibitor Formed by Density-inhibited, Tumorigenic V79 Chinese Hamster Cells

A novel type of low-molecular-weight growth-inhibitory factor responsible for the density inhibition of tumorigenic V79 Chinese hamster cells has been purified, if not homogenously, by a series of reverse-phase and gel filtration high-performance liquid chromatography. The factor is an acid-stable, heat-labile substance distinct from antiproliferative nucleoside analogues or polyamines and has a molecular weight of approximately 2000. The biological activity of this inhibitor was enhanced nearly 5-fold by trypsin treatment, thereby suggesting that the inhibitor may be a precursor peptide which becomes an oligopeptide with intense biological activity by proteolysis, or that trypsin treatment allows resultant small molecules to efficiently transfer across the cytoplasmic membrane. This inhibitor reversibly inhibits the growth of a broad spectrum of cell types from neoplastic and nonneoplastic cells from various species. These data suggest that this inhibitor is primarily a growth-regulatory molecule common to mammalian cells and may play an important role in regulating growth of both normal and neoplastic cells.

A new electrophoretic variant of hemoglobin (Ogi) in which a leucine residue is replaced by an arginine residue at position 34 of the α-chain

Hemoglobin Ogi, in which an arginine is substituted for a leucine residue at position 34 of the alpha-chain, was detected in a Japanese family. Although slightly increased oxygen affinity is associated with this amino acid substitution in the alpha 1 beta 1 contact, it is without obvious deleterious effect on the hematological parameters of the individuals heterozygous for this variant.

Modifications of stability and function of lysozyme

Approaches to improving the functionality of lysozyme are presented. Lysozyme was variously modified and the stabilities of the derivatives were determined by thermal denaturation experiments. Contributions of salt bridge(s), hydrophobic interactions(s), and cross-linkage(s) were evaluated. The stabilities against proteolysis were also considered. For the latter stability, it might be important to depress the rate of unfolding, i.e., to stabilize the native conformation. As a rule, salt bridges and hydrophobic interactions stabilize the native conformation and cross-linkages destabilize the denatured conformation. However, cross-linkages are apt to introduce strains in the native conformation and only suitable lengths of cross-linkages can stabilize the protein. The stabilization was shown to be generally effective in improving the functionality of proteins. Catalytic groups in lysozyme (Glu-35 and Asp-52) were variously modified and finally converted to the respective amides. The participation of these groups in the catalytic function was confirmed. The specificity of lysozyme was modified. Asp-101, which lies on the top of the active site cleft of lysozyme, was variously modified and the effects on the hydrolysis patterns of a hexamer of N-acetylglucosamine were analyzed. Some approaches to endowing lysozyme with altered functions are also presented. In order to give higher esterase activity to lysozyme, the complementarity of enzyme and substrate was investigated by modifying substrate and the active site cleft of lysozyme. An attempt was made to convert lysozyme into a transaminase by introducing pyridoxamine to the active site cleft of lysozyme. Finally, we have started to apply genetic engineering to this kind of investigation and would like to see how far we can go with protein engineering to improve the nature of proteins.

A new electrophoretic variant of Hemoglobin (Munakata) in which a lysine residue is replaced by a methionine residue at position 90 of the α-chain

Abstract Hemoglobin Munakata, in which a methionine residue is substituted for a lysine residue at position FG2(90) of the α-chain, was detected in a Japanese family. Slightly increased oxygen affinity is associated with this amino acid substitution at the α 1 β 2 contact site in the FG nonhelical segment of the α-chain.

Evidence for the involvement of endogenous thymidine in the density-inhibition of tumorigenic Chinese hamster V79 cells

Two distinct low-molecular-weight growth inhibitory activities were isolated from supernatants of a density-inhibited, tumorigenic V79 Chinese hamster cell line. By chromatographic analyses, one of these was purified to homogeneity and eventually proved to be thymidine (dThd). In order to investigate the biological role of dThd in a density-inhibited culture of these cells, a dThd-kinase deficient (TK-) clone resistant to the excess of dThd was isolated from V79 cells and the effect of the supernatants on growth of these TK- or TK-proficient (TK+) cells was examined. As a result, the growth of TK- cells was not inhibited but enhanced by the supernatant at the concentrations which significantly inhibited the growth of TK+ cells. Such TK-dependent differential responses to supernatants suggest the presence of deoxyribonucleosides including a high level of dThd in the supernatants. Since it is unlikely that dThd might derive from denatured DNA of dead cells, an accumulation of endogenous dThd in confluent culture appears to be responsible for dThd triphosphates which are synthesized de novo, degraded and excreted into the medium rather than incorporated into DNA as a consequence of aberrant growth in the presence of certain growth inhibitors produced by density-inhibited V79 cells.

Isolation and chemical properties of a haptenic substance from buckwheat dialysate

A haptenic substance was isolated from the dialysate of the aqueous extract of buckwheat. This substance, BWD II 22-3, which was composed of Asp(1), Thr(1), Ser(1), Gly(1), Gly(4), Ala(1), Val(1), Leu(1), Orn(2), Lys(1), Arg(1), Cys(1) and glucose was demonstrated to be homogeneous by gel filtration and paper electrophoretic analyses. The molecular weight was estimated to be 1600 by gel filtration on Sephadex G-50. The haptenic substance apparently has-SH group determinant caused about 50% inhibition at a concentration of 100 micrograms/disc in the RAST procedure using human serum sensitive to buckwheat. An active component, BWD II 22-3, might prove effective in the hyposensitization therapy of buckwheat-sensitive patients.