Contribution of a Salt Bridge Triad to the Thermostability of a Highly Alkaline Protease from an Alkaliphilic Bacillus Strain*

Summary Crystallographic analysis of the highly alkaline M-protease from an alkaliphilic Bacillus strain shows the occurrence of a unique salt bridge triad Arg19–Glu271–Arg275 (in subtilisin BPN′ numbering), which is not found in less alkaline true subtilisins BPN′ and Carlsberg from Bacillus amyloliquefaciens and Bacillus licheniformis, respectively. Because the corresponding residues are all Gln residue in the subtilisin BPN′, Gln residue was engineered into the position(s) 19, 271 and/or 275 in M-protease by site-directed mutagenesis. Disruptions of the salt bridge caused the reduction of the thermostability of the mutant proteins at alkaline pH with the following decreasing order of thermal inactivation rate; the wild-type > Arg275 → Gln > Glu271 → Gln > Arg19 → Gln/Glu271 → Gln/Arg275 → Gln > Arg19 → Gln. This result provides the evidence that the salt bridge triad contributes to the thermostability and structural rigidity of the highly alkaline M-protease.     

Ultrastructural visualization of elastic fibres with a tannate-metal salt method

Summary A modification of the tannic acid-metal salt method was applied as an ultrastructural stain for elastin. Thin sections of glutaraldehyde-fixed, embedded rat aorta and rabbit elastic cartilage, with and without osmication, were examined. Raising the pH of the tannic acid solution from 2.7 to 9.0 progressively increased the electron-density of elastic fibres and collagen fibrils in osmicated and unosmicated specimens. The maximum tannic acid staining of elastic fibres was observed in the pH range 7.0–9.0. Collagen staining, although less intense than that of elastic fibres, was also greatest in this pH range. Elastic fibres in osmicated specimens demonstrated the strongest tannic acid staining with a minimal increase in density of collagen and cell nuclei when compared to the unosmicated specimens. Sequential treatments of osmicated specimens with tannic acid pH 7.0–9.0, and uranyl acetate, pH 4.1, enhanced the density of the elastin intensely, increased collagen staining moderately, but hardly increased the density of nuclei and microfibrils. In elastase-digested osmicated specimens, all tannic acid (pH 7.0)-uranyl acetate-reactive elastin was selectively removed. These results demonstrate that all the neutral and alkaline tannic acid-uranyl acetate methods can be used as a postembedment stain for elastin specimens fixed in glutaraldehyde and osmium tetroxide.     

Structure and development of rabbit pepsinogens. Stage-specific zymogens, nucleotide sequences of cDNAs, molecular evolution, and gene expression during development

In order to clarify the structure and development of rabbit pepsinogens, purification and molecular cloning of these proteins were performed at various developmental stages. Several pepsinogens were isolated, and they were classified as pepsinogens F and M, and into pepsinogen groups I, II, and III. The relative levels and specific activities of the various pepsinogens changed significantly during development. Pepsinogens F and M were present only at the early postnatal stage, and their level was higher than those of other pepsinogens at this stage. Pepsinogens in groups I, II, and III were the predominant zymogens at the late postnatal stage. cDNA clones encoding all of these pepsinogens were obtained, with the exception of pepsinogens I and M, and the nucleotide sequences were determined. Each cDNA contained a leader region (signal peptide), a pro-region (activation segment), and a pepsin region, of 15, 44, and 328 residues, respectively, with the exception of the cDNA for pepsinogen F in which the pro- and pepsin regions were composed of 43 and 330 residues, respectively. Pepsinogens in groups II and III exhibited a high degree of similarity with one another, whereas many substitutions were found in pepsinogen F. A unique substitution in the activation segment of pepsinogen F, namely, Gly----Asp at position 21, was found, which made the structural features of this segment more specific. A phylogenic tree was constructed from the differences in nucleotide sequences and showed clearly that each pepsinogen in groups II and III could be classified as pepsinogen A, a major pepsinogen in mammals. Pepsinogen F diverged significantly from these groups and may be a new type of pepsinogen. Northern analysis revealed that the expression of the gene for pepsinogen F was restricted to the early postnatal stage, and the expression of genes for pepsinogens in groups II and III was detected predominantly at later stages, a result that shows the switching of gene expression from fetal pepsinogen to adult pepsinogens during development.     

Solubilization and localization of weakly polar lipids in unsonicated egg phosphatidylcholine: A 13C MAS NMR study

The weakly polar lipids cholesteryl ester, triacylglycerol, and diacylglycerol incorporate to a limited extent into the lamellar structure of small unilamellar vesicles. The localization of the carbonyl group(s) at the aqueous interface was detected by [13C]carbonyl chemical shift changes relative to the neat unhydrated lipid [Hamilton, J.A., & Small, D.M. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6878-6882; Hamilton, J.A., & Small, D.M. (1982) J. Biol. Chem. 257, 7318-7321; Hamilton, J.A., Bhamidipati, S.B., Kodali, D.R., & Small, D.M. (1991) J. Biol. Chem. 266, 1177-1186]. This study uses 13C NMR to investigate the interactions of these lipids with unsonicated (multilamellar) phosphatidylcholine, a model system for cellular membranes and surfaces of emulsion particles with low curvature. Magic angle spinning reduced the broad lines of the unsonicated dispersions to narrow lines comparable to those from sonicated dispersions. [13C]Carbonyl chemical shifts revealed incorporation of the three lipids into the lamellar structure of the unsonicated phospholipids and a partial hydration of the carbonyl groups similar to that observed in small vesicles. Other properties of interfacial weakly polar lipids in multilayers were similar to those in small unilamellar bilayers. There is thus a general tendency of weakly polar lipids to incorporate at least to a small extent into the lamellar structure of phospholipids and take on interfacial properties that are distinct from their bulk-phase properties. This pool of surface-located lipid is likely to be directly involved in enzymatic transformations and protein-mediated transport. The 13C magic angle spinning NMR method may be generally useful for determining the orientation of molecules in model membranes.     

Renin-dependency of glycyrrhizin-induced pseudoaldosteronism

A prospective study was carried out on 12 patients with chronic hepatitis who were taking 546 mg/day of glycyrrhizin for 4 weeks in order to identify the factors responsible for the development of hypertension and hypokalemia. In 5 patients, blood pressure increased and serum potassium decreased after the treatment (responders). In the remaining 7 patients, these values were unchanged (nonresponders). There were no significant differences in age, plasma aldosterone, the catecholamine concentrations or serum transaminases. The basal plasma renin activity (PRA) in the responders was more than 1.5 ng/m/h (2.5 +/- 0.3 ng/m/h), while that in the non-responders was less than 1.5 ng/m/h (0.7 +/- 0.1 ng/ml/h). Furthermore, a positive correlation between the basal RPA and the changes in blood pressure, and a negative correlation between the basal PRA and the changes in potassium were found. These results suggest that patients with higher PRA levels are more likely to develop hypertension and hypokalemia when treated with glycrrhizin.     

Development-dependent expression of isozymogens of monkey pepsinogens and structural differences between them

The developmental changes in the expression of monkey pepsinogens and structural differences between the polypeptides were investigated. Monkey pepsinogens included five different components, namely, pepsinogens A-(1-4) and progastricsin. Their respective relative levels and specific activities changed significantly during development. The sequential expression of genes for type-A pepsinogens was particularly noteworthy. Pepsinogen A-3 was the major zymogen at the newborn stage, accounting for nearly half of the total pepsinogens at this stage. Pepsinogen A-2 became predominant at the 4-month stage, and pepsinogen A-1 predominated at the juvenile and adult stages. Enzymatic properties of pepsinogens A-1, A-2 and A-3 were similar but not identical to those of pepsinogen A-4 and progastricsin, in particular with respect to the activation processes. Each pepsin digested various protein substrates but some differences in specificity were evident. cDNA clones for five pepsinogens were isolated, and the nucleotide sequences were determined. Each cDNA contained leader, pro, and pepsin regions that encoded 15, 47, and 326 amino acid residues, respectively, with the exception of the cDNA for progastricsin in which the pro and pepsin regions encoded 43 and 329 amino acid residues, respectively. Type-A pepsinogens exhibited a high degree of similarity, with over 96% of bases in the nucleotide sequences of the protein-coding regions being identical. Northern analysis revealed that the level of expression of genes for type-A pepsinogens and for progastricsin was significant at the fetal stage and increased with development.     

Influence of zoysiagrass rhizosphere fungal isolates on growth and yield of soybean plants

Among 21 rhizosphere fungi tested, eight sterile fungi and oneTrichoderma isolate (GT2-1) from zoysiagrass rhizosphere promoted the overall growth of soybean varieties when grown in the greenhouse. Out of nine effective isolates, GS7-4, GS8-2, GS8-3, GU23-3 (all sterile fungi) and GT2-1 (Trichoderma sp.) promoted plant growth and increased yield of Toyosuzu (variety 1) significantly, while GS8-3, GS10-1, GS10-2 (sterile fungi), and GT2-1 significantly caused plant growth promotion and yield increase of Kitamusume (variety 2). Among these efficient isolates, GS8-3 and GS10-2 induced considerable and consistent increases in length, biomass and yield of plants of varieties 1 and 2, respectively. In the field, however, only GS8-3 and GU23-3 among seven selected isolates, induced consistent and significant increases in plant growth and yield of varieties 1 and 2, while the ability of other isolates decreased. The plant growth promotion by these isolates in the field followed a similar trend to that in the greenhouse, but the effect was less marked. Some isolates which were effective in the greenhouse were less effective in the field. The degree of growth promotion by different isolates depended on the variety of soybean. The nutrient condition of soils used in experiments also seemed to play a vital role, since notable growth promotion by these isolates was observed in nutrient-depleted soil.The author is grateful to Ministry of Education, Science and Culture, Japan, for financial assistance.     

Proliferation, differentiation and morphogenesis of fetal rat glandular stomach transplanted under the kidney capsule of syngeneic hosts

Undifferentiated glandular stomach tissue fragments from 16.5-day fetal rats were transplanted under the kidney capsule of syngeneic adult rats, and the proliferation, differentiation and morphogenesis of the transplanted tissues were investigated. Gastric epithelial cells began to invaginate 3–4 days after the transplantation and immature glands were formed after 1 week. During the period, there was a gradual increase in the expression of pepsinogen and cathepsin E, markers of cytodifferentiation of the stomach epithelia, both at protein and mRNA levels. Cathepsin E was weakly expressed in undifferentiated gastric epithelial cells at 16.5 days of gestation, and a higher level of the expression was observed in differentiated epithelia of the transplants. In contrast, the pepsinogen-producing cells first appeared around days 3–4 after transplantation and gradually increased in number to about 30% of the epithelial cells and became localized at the bottom of the gland. During the period of the experiment up to 1 month, the pepsinogen-producing cells were all positive for class III mucin and cathepsin E, indicating the immature character of these cells. In addition, no parietal cells were observed. When the tissue fragments were transplanted into adrenalectomized animals, the epithelial differentiation and morphogenesis was suppressed, but its proliferation was enhanced. The observed changes were reversed by hydrocortisone replacement. These results suggest that the development of the 16.5-day fetal stomach is regulated intrinsically to a certain extent by the genetic program of the cells involved and various gastric functions develop in the absence of luminal stimulation, stage-specific systemic hormonal change, neuronal regulation or other systemic influences, and that glucocorticoids modulate the developmental program of the fetal stomach tissues.