A highly efficient procedure for purifying the ribosome-inactivating proteins alpha- and beta-momorcharins from Momordica charantia seeds, N-terminal sequence comparison and establishment of their N-glycosidase activity.

A new purification scheme, involving two successive ion exchange chromatographic steps on DEAE-cellulose and Mono-S FPLC, was developed for the isolation of the ribosome-inactivating proteins, alpha- and beta-momorcharins, from the Chinese herb Kuquazi (seeds of Momordica charantia). This simple and rapid procedure yielded 3.1 and 1.7 mg of alpha- and beta-momorcharins, respectively, from 2.5 g of decorticated seeds in only two days. The N-terminal amino acid sequence of beta-momorcharin was found to be DVNFDLSTATAKTYTKFIED. It differed from that of alpha-momorcharin (DVSFRLSGADPRSYGMFIKD) in 10 out of the 20 positions investigated. Like other ribosome-inactivating proteins, the purified momorcharins showed specific N-glycosidase activity at nanomolar concentrations, when rRNA from rabbit reticulocyte lysate was used as substrate. The N-glycosidase activity of both momorcharins was optimal at pH7, not inhibited by K+ and not appreciably affected by NH4+. The activity of alpha-momorcharin was not drastically altered by Mn2+ but (1-10mM) Mn2+ inhibited the activity of beta-momorcharin by about 40%.

Demonstration of ribonuclease activity in the plant ribosome-inactivating proteins alpha- and beta-momorcharins.

Alpha- and beta-momorcharins, ribosome-inactivating proteins from Momordica charantia seeds, were utilized in this investigation. Ribonucleolytic cleavage was observed after naked rRNA was incubated with either momorcharin. Beta-momorcharin, and to a lesser extent alpha-momorcharin, also acted on tRNA to release acid-soluble UV-absorbing products. Such activity was optimal at pH around 5.5. Using polyhomoribonucleotides as substrate, it was found that the momorcharins preferentially acted on polyU, but exerted negligible effects on polyA, polyC and polyG. Chromatographic analysis of the reaction product indicated that mono and/or oligo-ribonucleotides, but not free base, were generated from polyU, suggesting that the enzymatic action involved ribonucleolytic cleavage. Similar to the results obtained with tRNA as substrate, beta-momorcharin was about 15-fold more active than alpha-momorcharin on polyU.

Creatine kinase is modified by 2-chloromercuri-4-nitrophenol at the active site thiols with complete inactivation.

Creatine kinase modified by mercurials has been reported to be fully reactive as the native enzyme. This was ascribed to the modification of a second class of thiol groups instead of the reactive thiols at the active site (Laue, M.C. and Quiocho, F.A. (1977) Biochemistry 16, 3838-3845). It has now been shown by spectrophotometric titration and fluorescence studies that 2-chloromercuri-4-nitrophenol (MNP) reacts preferentially with the active-site thiol. Moreover, if the activity of the modified enzyme is determined in the absence of added bovine serum albumin or other enzymes, as usually employed in coupled activity assay systems for creatine kinase, the modified enzyme is completely inactive. Addition of an excess of bovine serum albumin or rabbit muscle glyceraldehyde-3-phosphate dehydrogenase restores the activity of the enzyme to over 80% of its original level. It appears that the active thiol groups at the active site of creatine kinase are after all modified by MNP with complete inactivation.

C-terminal angiogenin peptides inhibit the biological and enzymatic activities of angiogenin.

Synthetic peptides corresponding to the C-terminal region of angiogenin (Ang) inhibit the enzymatic and biological activities of the molecule while peptides from the N-terminal region do not affect either activity. The peptide Ang(108-121) transiently abolishes the inhibition of cell-free protein synthesis caused by angiogenin coincidentally with its cleavage of reticulocyte RNA. Several C-terminal peptides also inhibit nuclease activity of angiogenin when tRNA is the substrate. Furthermore, peptide Ang(108-123) significantly decreases neovascularization elicited by angiogenin in the chick chorioallantoic membrane assay.

Angiogenin mRNA in human tumor and normal cells.

Angiogenin mRNA was characterized in HT-29 human colon adenocarcinoma cells and its distribution in other human cell types was studied. Several RNA species ranging from 800 to 6000 nucleotides hybridized to the angiogenin probes with the smallest being the major poly(A)-containing transcript. This transcript was detected in tumor cells of diverse cellular origin. Expression of angiogenin mRNA is not limited to neoplastic cells and is detected in normal epithelial cells, fibroblasts and peripheral blood cells. The latter, when induced by mitogens, expressed more mRNA than did unstimulated or HT-29 cells. Transformed fibroblasts did not contain higher levels of angiogenin mRNA than their normal counterparts, demonstrating that increased angiogenin mRNA expression does not necessarily occur upon neoplastic transformation. This study demonstrates that angiogenin mRNA is expressed in a wide spectrum of cells and is not correlated to a particular cell phenotype.

Changes in circular dichroism and exposure of buried thiol groups during denaturation of rabbit muscle creatine kinase.

The denaturation of creatine kinase in guanidine solutions has been followed by both CD changes and the increase in rapid reacting SH groups. The rates of the exposure of SH groups are in general agreement with the ultraviolet absorbance and fluorescence changes reported previously whereas changes in the ellipticity of the enzyme molecule can be detected at low guanidine concentrations before significant changes in the exposure of the aromatic residues could be observed. On the other hand, the rates of changes in the mean residue ellipticity at 220 nm are clearly slower than the changes in ultraviolet absorbance, fluorescence and exposure of SH groups. It is suggested that the secondary structure of the external regions of the peptide chains is affected at low guanidine concentrations followed by gross changes in the tertiary structure of the molecule resulting in the exposure of the buried aromatic residues. The destruction of ordered secondary structure of the peptide chain is a slower process than the opening up of the folded tertiary structure of the molecule.

Comparison of the rates of inactivation and conformational changes of creatine kinase during urea denaturation.

The denaturation of creatine kinase in urea solutions of different concentrations has been studied by following the changes in the ultraviolet absorbance and intrinsic fluorescence as well as by the exposure of hidden SH groups. In concentrated urea solutions, the denaturation of the enzyme results in negative peaks at 285 nm with shoulders at 280 and 290 nm and positive peaks at 244 and 302 nm in the denatured minus native enzyme difference spectrum. The fluorescence emission maximum of the enzyme red shifts with increasing intensity in urea solutions of increasing concentrations. At least part of these changes can be attributed to direct effects of urea on the exposed Tyr and Trp residues as shown by experiments with model compounds. The inactivation of this enzyme has been followed and compared with the conformational changes observed during urea denaturation. A marked decrease in enzyme activity is already evident at low urea concentrations before significant conformational changes can be detected by the exposure of hidden SH groups or by ultraviolet absorbance and fluorescence changes. At higher urea concentrations, the enzyme is inactivated at rates 3 orders of magnitude faster than the rates of conformational changes. The above results are in accord with those reported previously for guanidine denaturation of this enzyme [Yao, Q., Hou, L., Zhou, H., & Tsou, C.-L. (1982) Sci. Sin. (Engl. Ed.) 25, 1186-1193] and can best be explained by assuming that the active site of this enzyme is situated near the surface of the enzyme molecule and is sensitive to very slight conformational changes.

A comparison of denaturation and inactivation rates of creatine kinase in guanidine solutions.

Both the denaturation, as followed by UV absorbance and fluorescence changes, and inactivation of creatine kinase in guanidine solutions have been found to be first order reactions. In 3 M guanidine, at 30 degrees C, the inactivation rate constant was found to be 5.9 sec-1 and the denaturation rate constant 1.9 sec-1. At lower guanidine concentrations, the inactivation rate constants were only little affected whereas the denaturation rate constants decreased markedly, being of the order of 0.04 in 1 M and 0.004 in 0.5 M guanidine. The kinetics of the inactivation reaction in 0.5 M guanidine was found to be in agreement with a combination of two first order reactions. The enzyme lost activity first by a fast reaction with a rate constant only slightly lower than the rate constant in 3 M guanidine followed by a slower reaction with a rate constant of 0.003 sec-1. In 0.3 M guanidine, very little change in either UV absorbance or in fluorescence was observed, but, in sharp contrast, the enzyme lost considerable activity by a fast reaction and this was followed by a slower reaction of inactivation. Even after prolonged denaturation in 0.5 and 0.3 M guanidine, residual activities of 3.4% and 30% remained respectively. The above results suggest a very fragile active site although dissociation of the dimer and reversible guanidine inhibition may also contribute to the initial rapid inactivation. It is also to be noted that the multiphasic courses of inactivation at lower guanidine concentrations seem to suggest the presence of partly active intermediates during denaturation.

Conformational changes of creatine kinase during guanidine denaturation.

The conformational changes of creatine kinase during denaturation by different concentrations of guanidine hydrochloride have been studied by fluorescence and ultraviolet difference spectroscopic methods. At low concentrations of guanidine, less than 1 M, the denatured minus native difference spectra showed two negative peaks at 281 and 287 nm, whereas the fluorescence emission increased markedly with its maximum red-shifted from 337 to 345 nm. Control experiments showed that guanidine also increased the emission of ionized tyrosine at 345 nm. With the increase of concentrations of guanidine, both negative peaks at 281 and 287 nm increased in magnitude to reach maximal values at 3 M guanidine and at this time a small peak appeared at 292 nm. The fluorescence maximum was further red-shifted to 355 nm, whereas the emission intensity of the main peak decreased and a small shoulder appeared at 310 nm when the guanidine concentration increased from 1 to 3 M. Further increase in guanidine concentration produced little further change either in UV absorption or in fluorescence. From the above results, it seems that, in the native enzyme. Trp residues are partly buried and partly exposed and some of the Tyr residues are in ionized state. Guanidine below 1 M does not expose the buried Trp residues nor affects significantly the microenvironments of the ionized Tyr residues. At 3 M guanidine, Trp residues are exposed and the ionization state of Tyr residues is also affected. At this concentration, the peptide chain seems to be fully unfolded as evidenced by the fact that 5 M guanidine produces little further change in both UV absorption and fluorescence.