Purification, characterization, and N-terminal amino acid sequence of the adenylyl cyclase-activating protease from bovine sperm.

We previously reported the extraction of a factor from bovine sperm that activated adenylyl cyclases of rat brain and human platelets, and identified it as a trypsin-like protease that was referred to as "ninhibin." This proteolytic activity was purified to near homogeneity from an alkaline extract of washed sperm particles by sequential chromatography on p-aminobenzamidine agarose and CM-Sephadex. Purification was greater than 100-fold with nearly 30% recovery of protease activity exhibiting a major band of approximately 40 kDa. An approximately 45-kDa form of the protease was also evident in crude extracts and was preferentially isolated when the enzyme was prepared in the presence of a mixture of protease inhibitors. The larger form of the protease was substantially less effective in stimulating adenylyl cyclase than was the smaller form; it is likely to be a zymogen form from which the smaller, more active form is derived. Purified forms of acrosin and ninhibin exhibited similar mobilities on PAGE, similar capacities for activating adenylyl cyclase, similar patterns of proteolytic fragmentation, and similar immunoblot patterns obtained with an antibody against purified bovine acrosin. More importantly, the N-terminal amino acid sequence of bovine ninhibin was found to be identical with that of bovine acrosin and caprine acrosin and more than 75% identical with porcine acrosin. The data support the conclusion that the adenylyl cyclase-activating protease previously referred to as ninhibin is, in fact, acrosin.

Chemical modification of chalcone isomerase by diethyl pyrocarbonate: histidine residues are not essential for catalysis.

Chalcone isomerase form soybean is inactivated by treatment with diethyl pyrocarbonate (DEP). The competitive inhibitor 4',4-dihydroxychalcone provides kinetic protection against inactivation by DEP with a binding constant at the site of protection in agreement with its binding constant at the active site. Very high concentrations of the competitive inhibitors 4',4-dihydroxychalcone or morin hydrate offer a 10- to 40-fold maximal protection, suggesting a second slower mechanism for inactivation which cannot be prevented by blockage of the active site. Blockage of the only cysteine residue in chalcone isomerase with p-mercuribenzoate does not affect the rate constant for DEP-dependent inactivation and indicates that the modification of the cysteine residue is not responsible for the activity loss observed in the presence of DEP. Treatment of inactivated enzyme with hydroxylamine does not restore catalytic activity, indicating that the modification of histidine or tyrosine residues is not responsible for the activity loss. All five histidines of chalcone isomerase are modified by DEP at pH 5.7 and ionic strength 1.0 M. The rate constant for the modification of the histidine residues of chalcone isomerase is close to that for the reaction of N-acetyl histidine with DEP, indicating that the histidine residues are quite accessible to the modifying reagent. The rate of histidine modification is the same in native enzyme, in urea-denatured enzyme, and in the presence of a competitive inhibitor. In the presence of the competitive inhibitor morin hydrate, all of the histidine residues of chalcone isomerase can be modified without significant loss in catalytic activity. These results demonstrate that the histidine residues of chalcone isomerase are not essential for catalysis and therefore cannot function as nucleophilic catalysts as previously proposed.

Functional properties of a new crosslinked hemoglobin designed for use as a red cell substitute.

A new crosslinking agent, bis-pyridoxal tetraphosphate, (bis-PL)P4, was used to prevent dissociation of the hemoglobin (Hb) tetramer. Yields in excess of 75 percent of intramolecularly crosslinked (bis-PL)P4Hb have been obtained using stoichiometric amounts of the crosslinking reagent. Some functional properties of (bis-PL)P4Hb have been evaluated in vitro and in vivo. Oxygen affinity was substantially reduced (p50 = 31 torr at 37 degrees C, pH 7.4, pCO2 = 40 torr), while the Bohr coefficient was -0.27 of H+ per mol of O2. Owing to its right-shifted dissociation curve, (bis-PL)P4Hb still yielded a p50 of 15 torr at a low temperature (16 degrees C), as compared with only 3 torr for normal adult Hb (HbA). Clearance of (bis-PL)P4Hb from plasma was significantly delayed (t1/2 = 171 min, at a dose of 0.2 g/kg of body weight compared with that of HbA (t1/2 = 54 min). Heart rate, mean arterial blood pressure, and respiration remained stable or returned to normal values within hours after bolus injection of the hemoglobin. The (bis-PL)P4Hb was not excreted in the urine, in contrast to HbA (21% of the total dose of HbA appeared in the urine within the first 2 hrs). These results show that the covalent beta-beta crosslink prevents the renal excretion of (bis-PL)P4Hb, thereby significantly prolonging vascular retention. These properties, together with an increased ability to unload O2, make (bis-PL)P4Hb a promising new candidate as a red cell substitute.

Hammett rho sigma correlation for reactions of horseradish peroxidase compound II with phenols.

The rates of reduction of horseradish peroxidase compound II by p-methoxyphenol (4-hydroxyanisole) have been studied from pH 6.0 to 10.5. The kinetics are influenced by an acid group of pKa 8.7 on compound II. The acidic form of compound II is reactive; the basic form is not. Only the electrically neutral, unionized form of p-methoxyphenol is reactive. Fifteen different phenols were reacted with compound II at either pH 7.6 or pH 7.0 (three of them at both pH's). Rate constants varied from zero for p-nitrophenol to 3.2 X 10(7) M-1 for p-aminophenol. The reactive m- and p-substituted phenols yield a rho value of -4.6 +/- 0.5 when plotted according to the Hammett relation. This compares to the rho value of -6.9 obtained for horseradish peroxidase compound I reactions with phenols (1976, D. Job and H. B. Dunford, Eur. J. Biochem. 66, 607). The difference in sensitivity of compounds I and II to electron donating substituents on the phenols can be explained in terms of the relative simplicity of the reactions. Electron donation occurs to the electron-deficient porphyrin pi-cation radical of compound I accompanied by single proton addition to the protein. For compound II the electron is fed to the ferryl group at the center of the porphyrin in a reaction accompanied by two proton additions to the ferryl oxygen atom, one from the protein and the other from the substrate or solvent. This is followed by loss of water from the inner coordination sphere of the ferric ion. The relative reactivities of three o-substituted phenols can be explained in terms of steric hindrance which is minimal for a single o-substituent.