Arnold A. White

Separation of cyclic 3′,5′-nucleoside monophosphates from other nucleotides on aluminum oxide columns application to the assay of adenyl cyclase and guanyl cyclase

A method is described for separating 3′,5′-nucleoside monophosphates from other nucleotides on columns of aluminum oxide. It was found that 3′,5′-cyclic nucleotides, nucleosides, purines, and pyrimidines will all pass through columns of alumina prepared with Tris-HCl buffer in the neutral range. However, all other nucleotides tested will be adsorbed. Since the only phosphorylated compounds passed are the 3′,5′-cyclic nucleotides, an α-32P-labeled nucleoside triphosphate provides a specific precursor for use in assaying cyclase enzymes. Methods are described for determining the activity of adenyl cyclase and guanyl cyclase.

Improved two-step method for the assay of adenylate and guanylate cyclase

Abstract A two-step assay for adenylate and guanylate cyclase is described utilizing α- 32 P-labeled ATP or GTP as substrate and involving purification of the resulting 32 P-labeled cAMP or cGMP by sequential chromatography on Dowex 50 and alumina. The Dowex 50 chromatography is performed in acid, 50 m m HCl for cGMP and 10 m m HClO 4 for cAMP, and achieves complete separation from the radiochemical impurities in the substrate which are responsible for blank. The cAMP or cGMP peaks are collected directly onto alumina columns and, under acid conditions, are completely retained by the alumina. After washing the alumina with water, the 32 P-labeled cAMP or cGMP is eluted with 0.2 m imidazole buffer and counted. The method delivers blanks amounting to .0005% of the substrate radioactivity, high recoveries, and excellent reproducibility.

Opossum kidney contains a functional receptor for the Escherichia coli heat-stable enterotoxin

The Escherichia coli heat-stable enterotoxin (ST1 or STa) binds to specific receptors on mammalian intestinal brush border membranes, and stimulates guanylate cyclase in those membranes. We have found a similar signal transduction system in brush border membranes prepared from kidney cortex of the American opossum (Didelphis virginiana, and in a cell line (OK cell) derived from that tissue. Activation of guanylate cyclase by ST1 is therefore not limited to intestinal cells. Furthermore, since it is unlikely that ST1 which is produced in the intestinal lumen, would have access to kidney receptors, this suggests the existence of an endogenous peptide resembling ST1, at least in marsupials.

ALKALINE PHOSPHATASE AND OSTEOGENESIS IN VITRO.

Summary A study has been made of the role of alkaline phosphatase in the osteogene-sis of 7-day embryonic chick femora during 21 days in organ culture. The femora were analyzed for their content of inorganic P, acid-soluble ester P, lipid P, total nucleic acid P and residual P, after increasing culture periods. The results indicate that there is a close parallel between inorganic P content and alkaline phosphatase activity. Acid soluble ester P content shows a roughly reciprocal relationship to phosphatase activity, while the other P fractions do not appear to be related.

Effect of alamethicin, gramicidin S and melittin uppn the particulate guanylate cyclase from rat lung

The channel-forming antibiotic alamethicin activated rat lung particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing) EC 4.6.1.2), and the activated enzyme was further stimulated by sodium nitroprusside when a thiol such as 2-mercaptoethanol was present. Similar effects were seen with the antibiotic gramicidin S and with melittin, a polypeptide purified from bee venom. All of these agents are amphiphilic polypeptides. Nitroprusside was not able to stimulate both particulate and soluble enzyme treated with the nonionic amphiphile, Lubrol PX, suggesting that the membrane-active polypeptides had a different mechanism of action. These polypeptides are known to alter the membrane matrix by binding to phospholipid, and we suggest that this alteration allowed greater access of substrate and of nitroprusside to the enzyme. Lubrol PX, however, may interact preferentially with the enzyme, and thus block nitroprusside activation. The most potent of these agents was melittin, which stimulated nitroprusside activation at a concentration which had little effect by itself (7 microns), and at which others have demonstrated lytic effects on cells.

Activation of rat lung soluble guanylate cyclase by sodium nitroprusside: Effects of hemoglobin and reducing agents

Abstract A potent vasodilator, sodium nitroprusside, activated rat lung soluble guanylate cyclase about 2.0-fold; this activation was potentiated by reducing agents such as ascorbic acid and thiols, 4.5 to 9-fold. In the presence of 2-mercaptoethanol and sodium nitroprusside maximal enzymatic activity in crude enzyme preparation was evident after a lag of several minutes, after which the activity declined. Hemoglobin blocked sodium nitroprusside activation of a partially purified enzyme by causing a lag in the activation, and this inhibition was reversed by 2-mercaptoethanol. Therefore, the extent of sodium nitroprusside activation measured is affected by the concentration of hemoglobin and reducing agent present, and the activation time.

Differential effects of detergents upon the soluble and particulate guanylate cyclases from rat lung

Abstract The enzyme guanylate cyclase is present in both particulate and soluble form in rat lung homogenates. As previously reported, the soluble enzyme can be activated by preincubation in the presence of O 2 . The inactive (nonactivated) soluble enzyme is also stimulated by nonionic detergents, in the order Tween 20 > Lubrol PX > Triton X-67 > Triton X-100. The activated enzyme, however, was inhibited by these detergents in the reverse order. Sodium deoxycholate and lysolecithin were potent inhibitors of both inactive and activated enzyme. The activity of the particulate enzyme was stimulated by Lubrol PX > Triton X-100 > Triton X-67 > Tween 20. At a low concentration of lysolecithin or deoxycholate the particulate activity was increased; however, when detergent/protein > 1, inhibition was seen. In the case of deoxycholate, the inhibition could be reversed if excess deoxycholate was removed either by chromatography or by forming mixed micelles with Lubrol PX; however, deoxycholate inhibition of the soluble enzyme was irreversible. The stimulation by detergents of the particulate enzyme was apparently the result of solubilization. The effects upon the activity of the soluble enzyme were interpreted in terms of a model which assumes two hydrophobic regions on the enzyme surface. The two regions differ in hydrophobicity with the more hydrophobic region only being exposed as a result of activation. Interaction of a nonionic detergent with the less hydrophobic region stimulates activity, while interaction with the more hydrophobic region results in inhibition.

Regulation of intestinal mucosa guanylate cyclase by hemin, heme and protoporphyrin IX

Mg2+-dependent activity of intestinal brush border guanylate cyclase was stimulated 4-5-fold by 50-100 microM hemin. Higher concentrations were inhibitory. In the presence of 25% dimethyl sulfoxide, which stimulated activity 9-times, 50 microM hemin further increased activity 1.7-fold. However, when activity was stimulated 32-fold by the Escherichia coli heat-stable enterotoxin, or 26-fold by Lubrol PX, hemin produced only concentration-dependent inhibition. The first type of activation was more sensitive to hemin than the second. Reduction of hemin by dithiothreitol eliminated stimulation of basal activity, while inhibition of Lubrol PX-stimulated activity remained. Protoporphyrin IX also had no effect on basal activity, however, it inhibited enterotoxin- and Lubrol PX-stimulated activities similarly, but only to half the extent of hemin. Substitution of Mn2+ for Mg2+ elevated basal activity 15-fold, and this Mn2+-dependent activity was inhibited by hemin. Mn2+-dependent activity was stimulated (43%) by enterotoxin, however, the stimulated activity was more sensitive to hemin inhibition than the basal Mn2+-dependent activity and both inhibition curves were congruent above 50 microM hemin. Hemin inhibition of Lubrol PX-stimulated activity was much less with Mn2+ than with Mg2+. These results were interpreted as suggesting two sites of hemin inhibition; on an inhibitory regulator and on the enzyme. We also found that the secretory effect of enterotoxin in the suckling mouse bioassay was reduced 56% by the oral administration of hemin.

Stimulation of the Growth of Organ Cultures by Methyl and Propyl Parabens.

Summary When 10-day-old chick embryo femora were cultured for 2 days in medium containing methyl or propyl paraben, there resulted an increase in dry weight as compared with femora cultured in the control medium. The methyl paraben concentrations found to stimulate growth were 10-5 and 10-6 M. Propyl paraben stimulated at 10-5 to 10-7 M. The effects of the two were not additive. It is suggested that these compounds may stabilize lysosomes.

Culture Medium for Suitable Growth and Development of Embryonic Chick Femora in vitro

Summary We have found that Way-mouths medium MB 752/1, when modified (MB 752/1a has a higher level of calcium and a lower level of magnesium), plus embryo extract will support the growth and differentiation of cultured 7-day embryonic chick femora. The optimum level of embryo extract was 15 to 25% of the medium, and 25% was routinely used. In this medium the femora attain an 8-fold increase in dry weight and a 3-fold increase in calcium content during an 8-day culture period. Addition of chicken serum at 1, 5 or 10% of the medium did not improve these results.