Lipolytic and Esterolytic Activities in Posterior Lingual Glands of Rat: A Histochemical Study

Catalytic activities of lingual lipase were investigated by enzyme histochemistry in post-mortem tongues from male rats. Sections of fresh-frozen or formalin-calcium fixed tissue were incubated with naphthol-AS-nonanoate and α-naphthyl acetate substrate mixtures. The effects of pH level, sodium taurocholate activator and E600 inhibitor were also examined. The use of cryostat sections of tissues fixed in formalin-calcium and of nonanoate substrate within the range of pH 4.4–6.4, were optimal for localizing maximum reaction product, captured by Fast Blue BB, in acini and demilunes of the posterior deep and superficial lingual glands respectively. The reaction product corresponded with the distribution of secretory granules and failed to develop when taurocholate was omitted from the incubation medium. Similarly localized E600-resistant reaction product occurred with the acetate substrate and hexazotized New Fuchsin at pH 7.4, in the absence of taurocholate. Lipase and conventional esterase activities appear to be superimposed in posterior lingual glands of rat. The ability of their acini and demilunes to hydrolyse nonanoate substrate at an acidic pH optimum, when activated by sodium taurocholate, seems attributable to lipase destined for secretion into saliva – hence convenient for routine histochemical identification of the enzyme.     

Author index for volume 219

Proteinase inhibitors I and II were purified to electrophoretic homogeneity from leaves of tomato plants induced by either wounding intact plants or by supplying excised plants with the proteinase inhibitor inducing factor. Affinity chromatography with chymotrypsin-Sepharose was employed as a final purification step for each inhibitor. The tomato leaf inhibitors are very similar to potato tuber inhibitors I and II in subunit molecular weight, composition, and inhibitory activities against chymotrypsin, trypsin, and subtilisin. However, unlike the potato tuber which contains multiple isoinhibitors by isoelectric focusing, the tomato leaf exhibits only two isoinhibitor forms of inhibitor I and a single form of inhibitor II. The molecular weight of native potato inhibitor I was reevaluated by rigorous ultracentrifugal analysis and compared with data from previous analyses. The data confirm that native inhibitor I has a native M_r of about 41,000 and is a pentamer. Inhibitor II has a molecular weight of near 23,000 and is a dimer.     

The biosynthesis of rat serum albumin. Metabolism of the NH2-terminal hexapeptide of proalbumin

Proalbumin differs from serum albumin in containing a leading hexapeptide segment, Arg-Gly-Val-Phe-Arg-Arg. This propeptide is removed in the Golgi complex immediately prior to secretion of the albumin, but its fate and possible functions are unknown. We have tested for the presence of the propeptide and its immediate catabolic products in rat liver and plasma and have studied both the disappearance of 3H-propeptide after intravenous injection and the breakdown of synthetic propeptide by rat liver cell components and plasma in vitro. We found no detectable propeptide or its two pentapeptide derivatives in rat liver or plasma at a sensitivity of less than 1 microM. Injected 3H-propeptide was completely cleared from blood within 2 min. No binding of free propeptide to serum albumin was observed. Liver cell fractions as well as blood plasma degraded added propeptide, with the highest activity being observed in smooth microsomes, the Golgi-enriched fraction, and plasma membrane. These preparations chiefly removed the terminal arginine residues, whereas enzymes in the cytosol degraded the peptide completely to amino acids. The activity in plasma resided largely in an alpha-globulin with molecular mass of about 280,000 Da which appears to be carboxypeptidase N. We conclude that the liberated propeptide is quickly broken down within the liver cell and does not accumulate in an amount sufficient to exert feedback or other effects on albumin synthesis.     

Further evidence for a cell surface proteinase essential to the growth of cultured fibroblasts

Specific antibodies and protein proteinase inhibitors will inhibit cell-surface proteinase activity on human fibroblasts and cause a concomitant inhibition of DNA synthesis and of cell multiplication. An insolubilized proteinase inhibitor also inhibits cell multiplication. The same reagents partially inhibit the multiplication of mouse L cells, both in monolayer and suspension culture, and inhibit the mitogenic effect of epidermal growth factor (EGF) on both types of cell.     

Optical single-channel analysis of the aerolysin pore in erythrocyte membranes.

Scanning microphotolysis (Scamp), a recently developed photobleaching technique, was used to analyze the transport of two small organic anions and one inorganic cation through single pores formed in human erythrocyte membranes by the channel-forming toxin aerolysin secreted by Aeromonas species. The transport rate constants of erythrocyte ghosts carrying a single aerolysin pore were determined to be (1.83 +/- 0.43) x 10(-3) s-1 for Lucifer yellow, (0.33 +/- 0.10) x 10(-3) s-1 for carboxyfluorescein, and (8.20 +/- 2.30) x 10(-3) s-1 for Ca2+. The radius of the aerolysin pore was derived from the rate constants to be 19-23 A, taking steric hindrance and viscous drag into account. The size of the Ca2+ rate constant implies that at physiological extracellular Ca2+ concentrations (> 1 mM) the intracellular Ca2+ concentration would be elevated to the critical level of > 1 microM in much less than a second after formation of a single aerolysin pore in the plasma membrane. Thus changes in the levels of Ca2+ or other critical intracellular components may be more likely to cause cell death than osmotic imbalance.     

Sperm cell surface characteristics of Plumbago zeylanica L. in relation to transport in the embryo sac

Myosin associated with the male germ cells of angiosperms interacts with actin, promoting transport of the non-motile generative and later sperm cells in the pollen tube. Myosin localizing on the sperm cell plasma membrane seems negligible in Plumbago, as reflected by the absence of: (i) anti-myosin labeling using immunoelectron microscopy, (ii) sperm motility on actin matrices, and (iii) electrophoretic movement changes after addition of antibody. Sperm cells injected directly into actively streaming Nitella internodal cells, however, follow actin bundles and their movement is sensitive to ATP and Mg²⁺. This may be based on simple charge binding since negatively charged latex beads also migrate on actin, whereas neutral or positively-charged latex beads do not. Sperm cells are negatively charged according to capillary microelectrophoresis, whereas killed sperm cells, which are positively charged do not migrate. The sperm cell that normally fertilizes the egg has a higher calculated charge (8.277 × 10³ esu/cm²) compared with the sperm cell that fuses with the central cell (6.120 × 10³ esu/cm²). Received: 15 December 1998 / Accepted: 21 January 1999