Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

35S-labeled Drosophila melanogaster apocytochrome c was made by in vitro transcription/translation of the gene and purified to the monomeric, fully reduced form. It was found that in the presence of a wheat germ extract factor there was a high-affinity phase of the uptake into mouse liver mitochondria at 10-300 pM apocytochrome c, and a lower-affinity phase through 4000 pM. Without the factor, the high-affinity phase was absent. The stimulatory effect of the factor could not be elicited with various reductants, such as NADH, FMN, and ferrous protoheme IX. Conversely, when mitochondria loaded with apocytochrome c were resuspended in fresh medium, the protein readily reequilibrated. Successive washings depleted greater than 95% of the associated apoprotein but removed no holoprotein. Proteases (proteinase K or trypsin) added to a suspension of mitochondria loaded with apoprotein digested an amount of apoprotein similar to that which would have been dissociated during the same time, as measured by successive washings in the absence of protease. Mitochondria loaded with apoprotein and similarly treated with protease continued exporting the apoprotein, even after the protease was inhibited and removed, suggesting that most of the apoprotein associated with the organelle was in a protease-resistant compartment. Apocytochrome c mutants in which serines or alanines replaced cysteines 14 and 17, which bind the prosthetic group, behaved like the cysteine-containing protein, indicating that the covalent attachment of the heme is unrelated to the translocation of the apoprotein.
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PMID:Reversible import of apocytochrome c into mitochondria. 216 15

The proton motive force (delta mu H+) plays an important role, although it is not absolutely essential, in the in vitro translocation of secretory proteins, such as OmpA, across the cytoplasmic membrane of Escherichia coli (Yamada, H., Tokuda, H., and Mizushima, S. (1989) J. Biol. Chem. 264, 1723-1728). The transient accumulation in membrane vesicles of a possible translocation intermediate of OmpA was observed in the absence of delta mu H+. The intermediate was detected on a polyacrylamide gel as a proteinase K-resistant band corresponding to a molecular weight of 26,000. The intermediate did not possess the signal peptide. The appearance of this band was inhibited in the absence of ATP or the presence of adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP) and enhanced upon the addition of SecA. Upon the addition of NADH that energizes the membrane, the intermediate was converted to the translocated form of OmpA, even in the presence of AMP-PNP. These results suggest different requirements of ATP and delta mu H+ for the early and late stages of the translocation reaction. The SecA requirement for the early stage of the translocation has also been suggested. In addition to this band, two other bands were observed at higher positions on the gel, when the translocation reaction was performed in the absence of delta mu H+. Although these two bands also represented the mature form of OmpA, which was partly protected from the proteinase K treatment by the membrane vesicles, the accumulation was not transient. These bands did not appear when the translocation reaction was performed in the presence of dithiothreitol. Together with other evidence, the above observations suggest that OmpA, which has an intramolecular disulfide bridge, cannot undergo the translocation unless delta mu H+ is imposed.
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PMID:In vitro analysis of the process of translocation of OmpA across the Escherichia coli cytoplasmic membrane. A translocation intermediate accumulates transiently in the absence of the proton motive force. 255 15

The translocation into Escherichia coli cytoplasmic membrane vesicles of a protein containing an uncleavable signal peptide was studied. The signal peptide cleavage site of the ompF-lpp chimeric protein, a model secretory protein, was changed from Ala-Ala to Phe-Pro through oligonucleotide-directed site-specific mutagenesis of the ompF-lpp gene on a plasmid. The mutant protein was no longer processed by the signal peptidase. When proteinase K treatment was adopted as a probe for protein translocation into inverted membrane vesicles, the mutant protein exhibited rapid and almost complete translocation, most likely due to the lack of premature cleavage of the signal peptide before the translocation. This result also indicates that cleavage of the signal peptide is not required for translocation of the mature domain of the protein. The establishment of an efficient system made it possible to perform precise and quantitative analysis of the translocation process. The translocation was time-dependent, vesicle-dependent, and required ATP and NADH. Translocation into membrane vesicles was also observed with the uncleavable precursor protein purified by means of immunoaffinity chromatography, although the efficiency was appreciably low. The translocation required only ATP and NADH. Addition of the cytosolic fraction did not enhance the translocation.
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PMID:Efficient in vitro translocation into Escherichia coli membrane vesicles of a protein carrying an uncleavable signal peptide. Characterization of the translocation process. 328 38

The subcellular localization of l-lactate dehydrogenase (LDH) in rat hepatocytes has been studied by analytical subcellular fractionation combined with the immunodetection of LDH in isolated subcellular fractions and liver sections by immunoblotting and immunoelectron microscopy. The results clearly demonstrate the presence of LDH in the matrix of peroxisomes in addition to the cytosol. Both cytosolic and peroxisomal LDH subunits have the same molecular mass (35.0 kDa) and show comparable cross-reactivity with an anti-cytosolic LDH antibody. As revealed by activity staining or immunoblotting after isoelectric focussing, both intracellular compartments contain the same liver-specific LDH-isoforms (LDH-A4 > LDH-A3B) with the peroxisomes comprising relatively more LDH-A3B than the cytosol. Selective KCl extraction as well as resistance to proteinase K and immunoelectron microscopy revealed that at least 80% of the LDH activity measured in highly purified peroxisomal fractions is due to LDH as a bona fide peroxisomal matrix enzyme. In combination with the data of cell fractionation, this implies that at least 0.5% of the total LDH activity in hepatocytes is present in peroxisomes. Since no other enzymes of the glycolytic pathway (such as phosphoglucomutase, phosphoglucoisomerase, and glyceraldehyde-3-phosphate dehydrogenase) were found in highly purified peroxisomal fractions, it does not seem that LDH in peroxisomes participates in glycolysis. Instead, the marked elevation of LDH in peroxisomes of rats treated with the hypolipidemic drug bezafibrate, concomitantly to the induction of the peroxisomal beta-oxidation enzymes, strongly suggests that intraperoxisomal LDH may be involved in the reoxidation of NADH generated by the beta-oxidation pathway. The interaction of LDH and the peroxisomal palmitoyl-CoA beta-oxidation system could be verified in a modified beta-oxidation assay by adding increasing amounts of pyruvate to the standard assay mixture and recording the change of NADH production rates. A dose-dependent decrease of NADH produced was simulated with the lowest NADH value found at maximal LDH activity. The addition of oxamic acid, a specific inhibitor of LDH, to the system or inhibition of LDH by high pyruvate levels (up to 20 mm) restored the NADH values to control levels. A direct effect of pyruvate on palmitoyl-CoA oxidase and enoyl-CoA hydratase was excluded by measuring those enzymes individually in separate assays. An LDH-based shuttle across the peroxisomal membrane should provide an efficient system to regulate intraperoxisomal NAD+/NADH levels and maintain the flux of fatty acids through the peroxisomal beta-oxidation spiral.
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PMID:L-lactate dehydrogenase A4- and A3B isoforms are bona fide peroxisomal enzymes in rat liver. Evidence for involvement in intraperoxisomal NADH reoxidation. 863 3

Sera from patients with a variety of cancers, including solid carcinomas, leukemias, and lymphomas, contain a ca. 33.5-kDa protein absent from sera of healthy volunteers or patients not diagnosed as having cancer. The protein exhibits an NADH oxidase activity inhibited by 8-methyl-N-vanillyl-6-noneamide (capsaicin). The activity and the protein are resistant to digestion by proteases (trypsin, chymotrypsin, proteinase K, subtilisin) and to heat. Following protease digestion to reduce the content of major serum proteins, the 33.5-kDa protein could be detected on Western blots of SDS-PAGE transferred to nitrocellulose membranes using polyclonal antisera to a corresponding partially purified 33.5-kDa protein shed into culture media conditioned by growth of HeLa cells. No corresponding protein was seen with control sera. The findings confirm the capsaicin-inhibited NADH oxidase activity of cancer sera as a circulating marker potentially specific to sera of cancer patients and identify a ca. 33.5-kDa protein resistant to proteases and heat as the source of the circulating capsaicin-inhibited NADH oxidase activity.
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PMID:A 33.5-kDa heat- and protease-resistant NADH oxidase inhibited by capsaicin from sera of cancer patients. 918 12

Peroxisomes were isolated from pea (Pisum sativum L.) leaves and the peroxisomal membranes were purified by treatment with Na2CO3. The production of superoxide radicals (O2) induced by NADH was investigated in peroxisomal membranes from intact organelles incubated with proteases (pronase E and proteinase K). Under isoosmotic conditions, in the presence of pronase E, the production of O2-. radicals was inhibited by 80%. SDS-PAGE of peroxisomal membranes after protease treatment demonstrated a decrease in the 18-kDa PMP. This suggests that this polypeptide has a small fragment exposed to the cytosolic side of the peroxisomal membrane which is essential for O2-. production. The 18-kDa PMP was purified by preparative SDS-PAGE and in the reconstituted protein the NADH-driven production of O2-. radicals was investigated. The isolated polypeptide showed a high generation rate of superoxide (about 300 nmol O2-. x mg-1 protein x min-1) which was completely inhibited by 50 mM pyridine. The 18-kDa PMP was recognized by a polyclonal antibody against Cyt b5 from human erythrocytes. The presence of b-type cytochrome in peroxisomal membranes was demonstrated by difference spectroscopy. Results obtained show that in the NADH-dependent O2-. radical generating system of peroxisomal membranes, the 18-kDa integral membrane polypeptide, which appears to be Cyt b5, is clearly involved in superoxide radical production.
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PMID:Superoxide radical generation in peroxisomal membranes: evidence for the participation of the 18 kDa integral membrane polypeptide. 921 43

The partitioning behaviour of a drug (capsaicin)-responsive NADH oxidase (tNOX) activity released from HeLa cells by low pH treatment followed by heat and proteinase K was determined. When partitioned in a standard 6.4% PEG 3350/6.4% dextran T-500 two-phase system, the bulk of the tNOX activity was in the dextran-rich lower phase. The activity was inhibited by and bound to the triazine dye, Cibacron blue. Affinity partition, where the Cibacron blue was coupled to amino PEG 5000 and added to the first two-phase separation step, resulted in the partitioning of activity to the upper PEG phase. A second partition with PEG-salts resulted in the release of the tNOX from the Cibacron blue amino PEG enriched phase into the salt-enriched lower phase. The phase-purified protein exhibited anomalous behavior and tended to multimerize in sodium dodecyl sulphate (SDS) prior to SDS-polyacrylamide gel electrophoresis (PAGE). Multimerization appeared to be enhanced by PEG. The multimerization was enhanced with the reduced protein in the presence of detergent prior to SDS-PAGE. In addition, the activity was precipitated by PEG 8000 at concentrations between 6 and 30% by weight. In the presence of or after exposure to PEG 3350 or PEG 8000, the protein could not be detected by Western blot analysis after SDS-PAGE suggesting that the protein failed to enter the gel even though other HeLa cell surface proteins were unaffected. The anomalous multimerization behavior has thus far precluded the use of phase partition as a practical purification step for the oxidase.
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PMID:Aqueous two-phase partition and detergent precipitation of a drug-responsive NADH oxidase from the HeLa cell surface. 969 86

Our laboratory described a ca. 34-kDa protein of the HeLa S cell surface that bound an antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl) urea (LY181984) with high affinity and that exhibited NADH oxidase and protein disulfide-thiol interchange activities also inhibited by LY181984. The quinone site inhibitor 8-methyl-N-vanillyl-6-noneamide (capsaicin) also blocked these same enzymatic activities. Using capsaicin inhibition as the criterion, the drug-responsive oxidase was released from the surface of HeLa S cells and purified. The activity of the released capsaicin-inhibited oxidase was resistant to heating at 50 degrees C and to protease digestion. After heating and proteinase K digestion, the activity was isolated in >90% yield by FPLC as an apparent 50- to 60-kDa multimer. Final purification by preparative SDS-PAGE yielded a capsaicin-inhibited NADH oxidase activity of a specific activity indicative of >500-fold purification relative to the plasma membrane. The final activity correlated with a ca. 34-kDa band on SDS-PAGE. Matrix-assisted laser desorption mass spectroscopy as well as reelectrophoresis of the 34-kDa band indicated that the ca. 34-kDa material was a stable mixture of 22-, 17-, and 9.5-kDa components which occasionally migrated as a ca. 52-kDa complex. The purified complex tended to multimerize and formed insoluble 10- to 20-nm-diameter amyloid rods. The components of the purified 34-kDa complex were blocked to N-terminal amino acid sequencing and were resistant to further protease digestion. After multimerization into amyloid rods, the protein remained resistant to proteases even under denaturing conditions and to cyanogen bromide either with or without prior alkylation.
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PMID:A drug-responsive and protease-resistant peripheral NADH oxidase complex from the surface of HeLa S cells. 975 Jan 73

NADH oxidases of low specific activities from urine of cancer patients were found to be inhibited or stimulated by the vanilloid capsaicin (8-methyl-N-vanillyl-6-noneamide). Similar activities, inhibited or stimulated by capsaicin, were reported previously for sera of cancer patients but not for sera of normal volunteers or for patients with disorders other than cancer. Like those from sera, the activities from urine were resistant to heat and to digestion with proteinase K. Two different fractions with capsaicin-responsive NADH oxidase activities were obtained by FPLC. One fraction in which the 33-kDa band was the major component exhibited NADH oxidase activity stimulated by capsaicin. Another fraction in which 66-kDa and 45-kDa bands were major components exhibited NADH oxidase activities inhibited by capsaicin. A monoclonal antibody generated to a ca 34-kDa form of the NADH oxidase from sera reacted with a urine protein of a ca 33-kDa band in the capsaicin-stimulated fraction. The 33-kDa protein was of low abundance and was estimated to be present in amounts between 5 and 100 microgram/L, depending on the particular patient.
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PMID:Capsaicin-responsive NADH oxidase activities from urine of cancer patients. 978 48

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ammonium sulfate fractionation were employed in series to purify and concentrate a 12.5-kDa protein fragment with a periodic (24-min period) proteinase K-resistant and drug-unresponsive NADH oxidase (CNOX) activity from pooled sera from healthy volunteers. The activity was unresponsive to capsaicin to distinguish it from the previously isolated cancer-associated NOX form (tNOX). Polyclonal antisera generated to the CNOX fragment cross-reacted with 20.5- to 24-kDa proteins of human sera, human lymphocytes, and plasma membranes from Escherichia coli with the molecular weight depending on source and conditions of treatment with proteinase K.
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PMID:A drug-unresponsive and protease-resistant CNOX protein from human sera. 1136 Sep 93


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