Connell L. Marsh

Trypsin and chymotrypsin inhibitors from Ascaris suum.

Abstract Body wall and perienteric fluid of Ascaris suum were extracted with buffer, fractionated, and purified on carboxymethylcellulose. A trypsin inhibitor was eluted between pH 4.5 and 4.8 from perienteric fluid and body wall extract. The latter inhibited 3380 and 5170 μg of trypsin per milligram of protein, respectively. Two distinct chymotrypsin inhibitors were found. One inhibitor was eluted from carboxymethylcellulose between pH 4.3 and 4.6, while the second was eluted between pH 6.3 and 9.0. These inhibited 1930 and 2740 μg of chymotrypsin per milligram of protein, respectively.

Thermal enzymes. V. Properties of a malic dehydrogenase

Abstract Malic dehydrogenase from a thermophilic bacterium oxidizes malate to oxalacetate. Its optimum pH is 7.5–8.2, its Michaelis constant 0.012. The optimum temperature for the enzyme is about 60 °C. An activation energy of 3700 cal./mole has been found for the temperature range 32 to 55 °C. Operation of the Warburg apparatus at temperatures above 50 °C. has been effected by housing the apparatus in a constant temperature air bath.

Thermal enzymes. VIII. Properties of a heat-stable inorganic pyrophosphatase☆

A highly active inorganic pyrophosphatase from a thermophilic bacterium has been partially purified and studied in some detail. The enzyme was dependent upon magnesium ions for activation and was activated to a lesser extent by cobalt. Neither calcium nor manganese was effective as an activating ion. The pH-activity curve was quite broad and exhibited a distinct plateau in the range from pH 5.5 to pH 9.5. The usual Lineweaver-Burk plot of (S)v versus (S) did not give a straight line. Plotting (S)2v versus (S)2 gave a straight line from which Ks was calculated to be 5 × 10−3. Evidence is presented that the formation of a complex metallosubstrate is necessary for the hydrolysis of pyrophosphate by the enzyme. The energy of activation was found to be 21,000 cal./mole in the range from 45 to 70 °C. and 34,400 cal./mole in the range 30–45 °C. Apparent thermodynamic values were calculated from heat-inactivation data. The most unusual of these were the entropies of activation, ΔS++. For a 20-min. period of inactivation the mean ΔS++ value was 13.0 cal./mole/deg. After a 40-min. period of inactivation, the ΔS++ value was 45 cal./mole/deg. The low ΔS++ values suggest that there may be inherent structural differences in the thermal enzymes which render them less vulnerable to the denaturing effects of heat.

STUDIES IN HELMINTH ENZYMOLOGY. IV. IMMUNE RESPONSES TO MALIC DEHYDROGENASE FROM ASCARIS SUUM.

Abstract Purified malic dehydrogenase from Ascaris suum when injected into guinea pigs and swine stimulated the production of specific antibodies, which were demonstrated by enzyme inhibition and immunodiffusion analysis. Fractionation of swine serum on a cellulose ion exchange column resulted in the separation of the inhibiting and precipitating antibodies. Malic dehydrogenases from closely related parasites were inhibited by the antiserum and gave crossreactions in immunodiffusion analyses. However, malic dehydrogenases from swine heart did not react with the antiserum. Guinea pigs receiving injections of malic dehydrogenase from A. suum did appear to receive some protection against migrating A. suum larvae.

The effect of varying liquid-to-powder ratio to zinc oxide and eugenol of rat pulpal respiration

A study was conducted to determine the effect of varying the liquid to-powder ratio of zinc oxide and eugenol placed in contact with rat pulp tissue. The pulp tissue was suspended in phosphate buffer saline solution (PBS) to which 14 C-succinic acid had been added. The 14 CO 2 produced was used as an index of the effect on pulpal respiration. Findings indicated that the thicker mix of zinc oxide and eugenol produced the least effect in depression of pulpal tissue respiration.

Thermal enzymes. VII. Further data on an adenosinetriphosphatase

Abstract A heat-stable ATPase, previously characterized by Militzer and Tuttle (1), has been the subject of further investigation. The enzyme has been established as a true ATPase, catalyzing the removal of the terminal phosphorus from ATP. Experiments with varying magnesium-ion concentrations suggest that the role of the activating ion may be the formation of a chelate complex with the pyrophosphate group of ATP to make available to the enzyme a substrate upon which it is active. The Michaelis behavior also emphasizes the importance of substrate-magnesium-ion stoichiometry in promoting optimum catalytic activity. The Michaelis constant at pH 8.5 was found to be 3.2 × 10−3M, identical with the previously determined value at this pH using an excess of magnesium ions.

Haemonchus contortus: Enzymes: I. Isoelectric focusing of malate dehydrogenase in sucrose and in polyacrylamide gels

Abstract Soluble extracts were prepared from adult male and female Haemonchus contortus and fractionated by DEAE-cellulose chromatography. The initial unadsorbed fraction contained 73% of the total malate dehydrogenase activity present in the crude soluble extract. This was fractionated by isoelectric focusing using LKB Ampholine carrier ampholytes in a sucrose-density gradient column. Seven peaks of malate dehydrogenase were obtained with isoelectric points ranging from 5.9 to 9.0. In addition, six hemoglobin peaks were separated. Composites of fractions from each peak of malate dehydrogenase were then fractionated by gel filtration on Sephadex G-200. Aldolase, malate dehydrogenase, and hemoglobin (when present) were separated from one another. Variations in the pH optimum and the pH optimum curves were noted for the various malate dehydrogenase fractions. In addition, twofold differences in the Michaelis constants were found. The maximum purification of malate dehydrogenase was 22 times compared with that of the crude soluble extract. Maximum purification of the other enzymes investigated was 11 times for aldolase, 3 times for inorganic pyrophosphatase, and 123 times for asparate aminotransferase. Isoelectric focusing experiments performed in polyacrylamide gels resolved the malate dehydrogenase into multiple bands. Polyacrylamide or starch gel electrophoretic analyses failed to give discrete bands of MDH activity.

Studies in helminth enzymology. II. Properties of an inorganic pyrophosphatase from Ascaridia galli a nematode parasite of chickens.

A partially purified inorganic pyrophosphatase from Ascaridia galli, a nematode parasite of chickens has been investigated. The enzyme was activated by Mg++ and Zn++ and to a lesser extent by Co++, Mn++, Fe++, Cu++, and Ca++. In combination with Mg++ the other divalent ions were inhibitory. Cadmium and fluoride, both effective anthelmintics, inhibited markedly at low concentrations. Piperazine adipate, another anthelmintic, did not inhibit the enzyme. Tetracycline, oxytetracycline, chloromycetin, EDTA and ATP all inhibited the PPase. Optimum pH with Mg++ as activator was 6.8 to 7.2 but with Zn++ 6.2 to 6.5 was the optimum range. A Michaelis constant (Km) was estimated by the conventional Lineweaver-Burk treatment. Km was 6.4 × 10−4M, in fair agreement with Km values of PPases from other sources.

Ascaris suum: Purification and characterization of an intestinal aminopeptidase

Abstract Semipurified preparations of an aminopeptidase from intestinal extracts of Ascaris suum had low specific activity (.03 units per milligram of protein), a large molecular size, and a limited solubility. Treatment of these preparations with a mixture of chymotrypsin and trypsin resulted in the release of ninhydrin-staining components during the incubation period (6 hours at 37 °C) but the aminopeptidase retained over 90% of its enzymatic activity. The enzyme was now soluble at pH 5 and migrated as a single band by starch-gel electrophoresis after it had been further purified by sucrose-density gradient centrifugation. The purified aminopeptidase was maximally activated by Co 2+ compared to other divalent metallic ions. The enzyme had a pH optimum of 6.8, a specific activity of 2.7 units per milligram of protein, and a calculated Michaelis constant of 2.0 × 10 −4 M . Progressive autolysis of crude intestinal homogenates also resulted in increasing amounts of a component which appeared similar to that of the purified aminopeptidase.

Studies in helminth enzymology. V. An aminopeptidase of Ascaris suum which hydrolyzes l-leucyl-β-naphthylamide☆

Abstract Aminopeptidases which hydrolyze l -leucyl-β-naphthylamide were found in extracts of intestines, ovaries, uteri, and body walls, and in perienteric fluid of adult Ascaris suum. A 27-fold purification of one peptidase from the intestinal extract was achieved by using centrifugation, butanol extraction, pH 5 precipitation, and density-gradient centrifugation. The enzyme was maximally activated by cobaltous ion but other divalent metallic ions activated to a lesser extent. Other characteristics of the purified aminopeptidase are given.