High performance affinity chromatography of Bacillus neutral proteases.

Bacillus neutral proteases were purified using bacitracin-silica as an affinity medium. Several chromatographic procedures were investigated, including high speed runs on columns with 40- to 60-microns silica particles. The high speed procedure enabled the purification of 4.9 mg of B. subtilis neutral protease directly from 165-ml culture supernatant within 1.5 h. The neutral proteases of B. polymyxa and B. stearothermophilus were also purified. The latter enzyme was further concentrated by a second affinity chromatography step, using Sepharose with glycyl-D-phenylalanine as a ligand. During the purification procedures isopropanol was used to prevent autodigestion of the enzymes.     

Esterase activity of liver alcohol dehydrogenase.

Liver alcohol dehydrogenase is found to possess, in addition to its dehydrogenase and dismutase activities, the ability to hydrolyze octanoate esters at a rate approximately $\text{1}\text{500}\text{–}\text{1}\text{1000}$ of that of the dehydrogenase reaction. The esterase and dehydrogenase activities exhibit an identical isozyme pattern indicating that the same protein catalyzes both reactions. Inhibition studies suggest that the esterase activity presumably shares the catalytic domain with the dehydrogenase activity.     

Human liver aldehyde dehydrogenase. Esterase activity.

Human liver aldehyde dehydrogenase has been found to be capable of hydrolyzing p-nitrophenyl esters. Esterase and dehydrogenase activities exhibited identical ion exchange and affinity properties, indicating that the same protein catalyzes both reactions. Competitive inhibition of esterase activity by glyceraldehyde and chloral hydrate furnished evidence that p-nitrophenyl acetate was hydrolyzed at the aldehyde binding site for dehydrogenase activity. Pyridine nucleotides modified esterase activity; NAD+ accelerated the rate of p-nitrophenyl acetate hydrolysis more that 5-fold, whereas NADH increased activity by a factor of 2. Activation constants of 117 muM for NAD+ and 3.5 muM for NADH were obtained from double reciprocal plots of initial rates as a function of modifier concentration at pH 7. The kinetics of activation of ester hydrolysis were consistent with random addition of pyridine nucleotide modifier and ester substrate to this enzyme.     

Affinity chromatography of thermolysin and of neutral proteases from B. subtilis

Affinity chromatographic systems are described for the purification of neutral metalloendopeptidases on columns of acetyl-$\text{D}$-phenylalanine or succinyl-$\text{D}$-leucine covalently linked to Sepharose by spacers of various lengths. The neutral proteases of $\text{B. subtilis}$ are separated in a single chromatographic procedure from all other proteins of the culture filtrates and subfractionated into two active species. An analogous chromatographic system is effective in the purification of thermolysin of $\text{B. thermoproteolyticus}$.     

Esterase activity of hydrocarbon-oxidizing bacteria

The activity of esterase was studied in bacteria oxidizing hydrocarbons and belonging to the genera Rhodococcus, Arthrobacter and Pseudomonas. Indophenyl acetate was used as a substrate of the reaction catalysed by the enzyme. Exocellular esterases were not found. Endocellular esterases differed in their activity and thermostability both among the genera and among species of one and the same genus.     

Human leucocyte neutral proteases, with special reference to collagen metabolism.

Three different types of neutral proteases related to collagen metabolism have been found in the granule fraction of human leucocytes from normal adults, using collagen, gelatin, and synthetic peptides as substrates. These are collagenase, an enzyme showing a potent hydrolytic activity against gelatin but little against native collagen, and one splitting the cross-links region of collagen. Their molecular weights were estimated to be about 75,000 150,000, and 25,000, respectively, by gel chromatography. The former two enzymes were inhibited by a alpha2-macroglobulin and ethylenediaminetetraacetate, but not by alpha1-proteinase inhibitor (alpha1-antitrypsin) or phenylmethylsulfonylfluoride, while the latter enzyme, associated in behavior with an enzyme hydrolyzing succinyl-(l-alanyl)3-p-nitroanilide, was inhibited by alpha1-proteinase inhibitor, alpha2-macroglobulin, and phenylmethylsulfonylfluoride, but not by ethylenediaminetetraacetate. A possible cooperative function of these enzymes in collagen catabolism is discussed.     

Esterase activity of synthetic fragments of human adrenocorticotrophic hormone.

The anterior pituitary hormone adrenocorticotrophin (ACTH) has been extensively studied in terms of structure-function relationships and in vivo and in vitro activities of different synthetic fragments of ACTH have been characterized. Here we describe the ability of synthetic fragments of ACTH to hydrolyze a fluorogenic esterase substrate 4-methylumbelliferyloleate (MUBO). The measured esterase activities (in mumol 4-MU mol-1 s-1) were 79.7 for ACTH1-13, 385.9 for ACTH3-18, 503.0 for ACTH1-19, 1249.9 for ACTH1-24 D-ser3, and 1350 for ACTH1-24. Although the significance of the observed esterase activities in the actual molecular mechanisms of action of ACTH remains to be established it is worth noticing that the esterase activities of the different ACTH fragments closely parallel their reported ability to activate the brain lipase as well as their in vivo ability to induce steroidogenesis in adrenal cortex.     

Identification of the secreted neutral proteases from Anisakis simplex

Ingestion of larval nematodes (family: Anisakidae) can cause the human disease known as anisakiasis. After ingestion, Anisakis larvae can be invasive, penetrating host stomach or intestinal wall. Observation of larvae penetrating the tissue layers of human stomach in vitro by SEM showed tunnels and burrows were formed in the mucosa and submucosa. Based on these observations, we hypothesized that secreted proteases may be involved in the degradation of host tissue macromolecules to allow tunnel formation. Using a model of connective tissue extracellular matrix (ECM), we found that as few as 5 Anisakis simplex larvae could degrade approximately 25% of the ECM in a 16-mm culture well in 24 hr. Further characterization of the secreted proteases using synthetic peptide substrates and inhibitors revealed that there were 2 classes of proteases present: a metallo aminopeptidase and a trypsinlike serine protease. Extracts of Anisakis larvae contained a 25-kDa protease that was recognized by rabbit anti-rat trypsin antibody on western blots. This suggests that there is structural as well as functional similarity between the Anisakis trypsin and vertebrate trypsins.     

One-step affinity purification of Bacillus neutral proteases using bacitracin-silica

A purification procedure for neutral proteases from bacilli is described, in which bacitracin-silica was used as affinity medium. This enabled a one-step purification of the proteases directly from culture supernatant. Since neutral proteases are extremely sensitive towards autodigestion, conditions were chosen such, that autodigestion was largely prevented. Isopropanol appeared to be useful in both eluting the enzymes from the affinity medium, and inhibiting enzymatic activity during this step. The bacitracin-silica medium allowed high flow rates: with columns prepared for use in an FPLC system flow rates up to one column volume per minute were feasible, and still gave satisfactory results. The neutral proteases purified by this method were found to be homogeneous both by SDS-PAGE and analytical gel filtration.     

Esterase activity during the life cycle of Blastocladiella emersonii.

Total esterase activity was measured in extracts on Blastocladiella throughout its life cycle by the degradation of alpha-naphthyl acetate. A fivefold incease in activity, apparently due to the synthesis of new enzymes, was found during sporulation.     

Effect of neutral proteases from human granulocytes on colony forming cells in vitro.

Marked inhibition of colony formation is observed after incubation of mouse and human bone marrow cells with the human granulocytic neutral proteases elastase and chymotrypsin as well as with pancreatic chymotrypsin. The corresponding enzymes inactivated with diisopropylfluorophosphate were almost inactive. Incubation of different colony inducing agents either resulted in no change or in an increase of their colony stimulating activity. The data suggest a direct proteolytic action of the proteases on colony forming cells which may alter receptor sites for colony stimulating activities.