Takashi Murachi

Proteases of macrophages in rat peritoneal exudate, with special reference to the effects of actinomycete protease inhibitors

Abstract The glycogen-induced macrophages in rat peritoneal exudate were separated from most other nucleated cells by means of a glass bead column. The lysate of the glass-adhering cells was subjected to characterization of its proteolytic activities using several specific protease inhibitors, recently discovered from the culture media of actinomycetes. The hemoglobin-hydrolyzing activity at pH 4.0 was strongly inhibited by pepstatin, a pepsin (EC 3.4.4.1) inhibitor. The caseinolytic activity at pH 7.0 was sensitive to chymostatin, a chymotrypsin (EC 3.4.4.45) inhibitor. In addition to these two major proteases, i.e. , cathepsin D (EC 3.4.4.23) and chymotrypsin-like enzyme, there are two other minor proteolytic activities. One is active at acidic pH and sensitive to leupeptins, a trypsin (EC 3.4.4.4) inhibitor, and the other is active at neutral pH and sensitive to Hg 2+ but not to leupeptins.

Structural studies on stem bromelain isolation, characterization and alignment of the cyanogen bromide fragments

Stem bromelain [EC 3.4.22.4.], a proteolytic enzyme of pineapple stem, has a mol. wt. of about 28 000 [1 ]. In this paper, we describe the isolation and the alignment of three peptides obtained by means of cyanogen bromide cleavage. The tryptic peptides from the parent protein and its CNBr fragments were also isolated by a combination of column and paper separation techniques. The peptides have been characterized by amino acid analyses and end group determinations. Isolation of overlapping peptides to determine the alignment of all of the tryptic peptides is still in progress. However, the partial sequence we report here covers unique portions of the molecule, such as the amino and carboxyl-termini, the branching point for the carbohydrate moiety, and the catalytic center residues, so that it deserves comparison with the sequence of the corresponding portions of papain [2].

The appearance of a 34,000-dalton inhibitor of calpain (Ca2+-dependent cysteine proteinase) in rat liver after the administration of phenylhydrazine

A 34,000-dalton inhibitor of calpain (Ca2+-dependent cysteine proteinase) was found in the cytosol of anemic rat liver. When phenylhydrazine hydrochloride was continuously administered to rats, a 280,000-dalton calpain inhibitor that existed originally in the liver gradually disappeared within two weeks and, concomitantly, a 34,000-dalton inhibitor appeared. The purified 34,000-dalton inhibitor resembles 280,000-dalton inhibitor in that both are heat-stable proteins and do not inhibit papain and trypsin. Unlike the protomers of a 280,000-dalton inhibitor, 34,000-dalton inhibitor does not show any sign of self-association.

Normal apparent pKa value for the ionization of the histidine residue of papain and stem bromelain as determined by photooxidation reaction

During the course of investigating photosensitized inactivation of stem bromelain, we found that the photooxidation of the enzyme in the presence of methylene blue yielded a modified enzyme which, upon reduction in 5 mM dithiothreitol, fully retained its essential sulfhydryl group, showed a markedly decreased catalytic activity toward casein, and had decreased hisitidine and methionine contents [l] . The reaction showed a pronounced pH dependence. It is well known that the unprotonated species of an imidazole is readily photooxidized while the protonated species is hardly oxidized under the conditions employed in our studies [2] . Thus the photooxidation method has permitted us to determine directly the apparent pK, value of the single histidine residue in the stem bromelain molecule. The same technique was also applied to papain. We report here that the apparent pK, values for the ionization of the histidine imidazole as determined by the pH dependence of the rate of the photooxidation in papain and stem bromelain are 6.7 and 6.4, respectively, which are in the normal range for an imidazole group.

Structure and Function of Stem Bromelain

Publisher Summary This chapter discusses individual differences among papain, ficin, and stem Bromelain. Bromelain enzymes are proteolytic enzymes found in tissues of pineapple plant and other species of family Bromeliaceae . Stem bromelain is a sulfhydryl protease of plant origin and it resembles papain and ficin. Unlike papain, ficin, or chymopapain, stem bromelain is a glycoprotein. Bromelain has four methionine residues, while papain is known to be devoid of methionine. In connection with some differences in protein structure and in catalytic function of stem bromelain from papain and ficin, it must be pertinent to point out that the pineapple plant belongs to Monocotyledonae, whereas the papaya and fig belong to Dicotyledonae. Stem bromelain favors a basic amino acid ester like BAEE, but it can also hydrolyze at an appreciable rate benzoylglycine ester. In the case of papain, benzoylglycine amide is hydrolyzed only at one-hundredth of the rate for benzoylarginine amide. One may say that stem bromelain has broad substrate specificity. Papain and ficin are also said to have broad specificities. However, bromelain does not so closely resemble papain as papain resembles ficin.

A rapid and quantitative determination of amino acid phenylthiohydantoins by direct densitometry on thin-layer chromatogram

Abstract Thin-layer chromatograms on silica gel plate of amino acid phenylthiohydantoins were simultaneously used for identification and quantitation. With a scanning, two-wavelength densitometer, linear calibration curves have been obtained for 15 different derivatives, which can be employed for a rapid and simple quantitation of sample spots. A satisfactory application of the method to the determination of amino-terminal residues of bovine α-chymotrypsin is described.