Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The occasional cleavage of the Pseudomonas cytochrome-c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) molecule into two well-defined fragments during the preparation of the enzyme is shown to be identical to that caused by elastase isolated from the culture solution of Pseudomonas aeruginosa. A cyanogen bromide fragmentation of proteolytically cleaved and of intact enzyme shows the cleaved peptide bond to be situated in cyanogen bromide fragment II. The amino-acid sequence of this fragment was established by sequencing peptides obtained with trypsin, thermolysin, chymotrypsin and o-iodosobenzoate. It is concluded from the sequence homology that the polypeptide chain of Pseudomonas peroxidase is wrapped around the high-potential heme in a similar manner as in high-potential cytochromes c in general. The specific proteolytic cleavage occurs at a Ser-Val (Leu-Pro) region which is assumed to be the site of attachment between enzyme and membrane. The cleavage of the Ser-Val bond renders the peroxidase molecule enzymatically inactive by impeding the conformational changes essential for the function of the native enzyme.
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PMID:Specific cleavage of Pseudomonas cytochrome-c peroxidase by elastase from Pseudomonas aeruginosa. 282 23

An active form of phosphorylase phosphatase of Mr = 33,000, referred to as the catalytic subunit for over a decade, was purified to near-homogeneity from rabbit skeletal muscle. Repeated immunization of a sheep produced immunoglobulins that blocked the activity of the phosphatase. These immunoglobulins were affinity-purified on columns of immobilized phosphorylase phosphatase and used as macromolecular probes in a "Western" immunoblotting procedure with peroxidase-conjugated rabbit anti-sheep immunoglobulins. Only one protein, of Mr = 33,000, was stained in samples of the immunogen, attesting to the specificity of the probes. However, the Mr = 33,000 phosphatase protein was not detected in muscle extracts or in partially purified preparations. Instead, a single protein of Mr = 70,000 was detected. Limited proteolysis, in particular by Staphylococcus aureus V8 protease and thermolysin, converted the immunoreactive protein from Mr = 70,000 to Mr = 33,000. Coagulation of the phosphatase preparation with 80% ethanol at room temperature rendered the Mr = 70,000 protein insoluble, but allowed extraction of the Mr = 33,000 protein from the precipitate. Thus, we conclude that the immunoreactive protein of Mr = 70,000 is the "catalytic subunit" of phosphorylase phosphatase with a catalytic domain of Mr = 33,000. Previous purification schemes have yielded only the fragment of Mr = 33,000 due to its relative resistance to proteolysis and coagulation. Gel filtration chromatography of the "native" form of phosphorylase phosphatase showed Mr approximately 230,000. Both the Mr = 70,000 catalytic subunit and a Mr = 60,000 protein related to inhibitor-2 were detected by immunoblotting in the same fractions that exhibited activity after treatment with Co2+ and trypsin. Only the Mr = 60,000 protein was degraded during this activation process. We propose that the native phosphorylase phosphatase is an elongated structure with two-fold symmetry, containing one catalytic subunit of Mr = 70,000 and one regulatory subunit of Mr = 60,000.
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PMID:Phosphorylase phosphatase catalytic subunit. Evidence that the Mr = 33,000 enzyme fragment is derived from a native protein of Mr = 70,000. 298

Porcine alveolar macrophages (AM), as we reported in 1981, inactivate slow-reacting substance of anaphylaxis (SRS-A) in a time- and cell-number-dependent fashion. In the present study, metabolism of synthetic leukotriene D4 (LTD4) to leukotriene E4 (LTE4) by AM was demonstrated (mean conversion, 216 pmoles LTD/10(7) AM/60 min). The metabolism was inhibited by cooling or by removal of calcium ions plus addition of EDTA but not by dinitrophenol or puromycin, lipoxygenase inhibitors, the peroxidase inhibitor aminotriazole, or the hydroxyl ion scavenger benzoic acid. Similarly, although the addition of catalase plus superoxide dismutase significantly augmented (169 +/- 25% control) SRS release from dispersed porcine lung cells, the presence of these enzymes did not prevent LTD4 metabolism by AM. Inhibitors of gamma-glutamyl transpeptidase were also without effect but the dipeptidase inhibitors L-cysteine and dithiothreitol significantly reduced the conversion, as did pretreatment of AM with thermolysin (100 micrograms/ml). These data indicate that an AM dipeptidase, which may at least partly be located on the cell surface, converts LTD4 to the less bioactive LTE4.
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PMID:Metabolism of leukotriene D4 by porcine alveolar macrophages. 632 Jun 96

Incubation of purified prostaglandin endoperoxide synthetase from sheep vesicular glands with aspirin results in a covalent binding of the acetyl group of acetylsalicylic acid to the protein. During this acetylation, the cyclooxygenase activity is lost, but not the peroxidase activity. The reaction is completed when almost one acetyl group is bound per polypeptide chain (Mr = 68 000). After proteolysis of [3H]acetyl-protein with pronase, radioactive N-acetylserine was obtained. Originally, however, the hydroxyl group of an internal serine residue in the chain is acetylated. The formation of N-acetylserine can be explained by a rapid O leads to N acetyl shift as soon as the NH2 group of serine is liberated. A radioactive dipeptide was isolated from a thermolysin digest of the [3H]acetyl-enzyme containing phenylalanine and serine, phenylalanine being its N-terminal amino acid. Automatic Edman degradation of native and acetylated enzyme showed that only one polypeptide sequence was present: Ala-Asp-Pro-Gly-Ala-Pro-Ala-Pro-Val-Asn-Pro-X-X-Tyr-. The N-terminal sequence has an apolar character.
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PMID:Acetylation of prostaglandin endoperoxide synthetase with acetylsalicylic acid. 677 69

The unfolding and refolding rates of the heme-and Ca2+ -containing Coprinus cinereus peroxidase (CIP) have been measured at pH 12.1, in 4 M urea, and at 61.2 degrees C. The change in peroxidase activity paralleled the change in the Soret band absorbance of the heme group. The unfolding rate constant (ku), was determined independently in thermolysin digestion and EDTA experiments at 59.4 degrees C. Both gave ku values of 1.5 ms-1, and also showed that the presence of 4 mM EDTA made CIP unfolding practically irreversible. Unfolding and refolding rates could therefore be determined under identical conditions of denaturation having either EDTA or Ca2+ in excess. The refolding rates at high pH and in 4 M urea were measured by adding Ca2+ to the unfolded CIP, whereas refolding at 61.2 degrees C was evaluated by comparing the unfolding carried out under reversible (excess of Ca2+) and irreversible conditions (excess EDTA). The activation energies for the unfolding at 61.2 degrees C are approximately delta G++(u) 100, T delta S++(u) 200, and delta H++(u) 300 kJ/mol. Five different additives, glycerol, EtOH, Na2SO4, guanidinium chloride (GdmCl), and NaCl, all at 100 mM, were used as probes to evaluate the mechanism of base, urea, and heat on unfolding and refolding. Salts destabilized CIP at high pH, primarily by enhancing the unfolding rate but also by decreasing the refolding rate. Glycerol had the reverse effects and thus stabilized CIP at high pH. The unfolding rate in urea was only slightly affected by the additives, with the exception of GdmCl which enhanced the unfolding rate. The refolding rate was decreased in urea by EtOH and GdmCl, in contrast to glycerol and Na2SO4 which increased the refolding rate. At high temperature the unfolding was affected only slightly by the additives, except for GdmCl, and to a lesser extent NaCl, which enhanced the unfolding rate. The refolding rates were greatly decreased by Na2SO4, EtOH, and GdmCl, whereas glycerol and Nacl enhanced the process. It appears that 100 mM NaCl functions as a catalyst for the temperature-induced process, enhancing both the unfolding and refolding rates. The results indicate that the mechanisms of CIP unfolding and refolding are similar in urea and at high temperature but different at high pH.
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PMID:Unfolding and refolding of Coprinus cinereus peroxidase at high pH, in urea, and at high temperature. Effect of organic and ionic additives on these processes. 865 38

The present study was initiated to identify the region(s) of ovotransferrin involved in binding to the bacterial transferrin receptors from Haemophilus paragallinarum and Haemophilus avium. Ovotransferrin was digested with either trypsin or thermolysin to obtain its N-lobe and C-lobe fragments. The individual fragments were then purified by a combination of gel exclusion and ion-exchange chromatography. Solid phase binding experiments with the individual fragments demonstrated that the C-lobe fragments blocked the binding of horse radish peroxidase-conjugated ovotransferrin to the transferrin receptors and that much higher concentrations of the N-lobe fragment were required for any detectable blocking. Affinity isolation of the bacterial transferrin receptor from the two Haemophilus species revealed that both native ovotransferrin and its C-lobe fragment were capable of isolating two iron repressible outer membrane proteins. These 95 and 60 kDa outer membrane proteins correspond to Tbp1 and Tpb2, respectively. In contrast, the N-lobe fragment was capable of isolating Tbp2 of H. paragallinarum but not that of H. avium. The inability of the N-lobe and C-lobe fragments from ovotransferrin and human transferrin to support the growth of iron-limited cultures of H. paragallinarum and Neisseria meningitidis, respectively, suggested that interaction with both lobes is necessary for efficient iron acquisition.
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PMID:Transferrin binding protein two interacts with both the N-lobe and C-lobe of ovotransferrin. 872 96