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)

1. The isolation of the ADP/ATP translocator from beef heart mitochondria as the bongkrekateprotein complex is described, using hydroxyapatite chromatography and gel filtration in Triton X-100 solution. 2. The inhibitor is bound to the protein prior to solubilization with detergent for protection against denaturation. Only the intact bongkrekate-protein passes easily through the hydroxyapatite column. Bongkrekate shileds the protein in contrast to carboxyatractylate only partially against proteinases present in the crude extract. 3. The isolated bongkrekate protein shows the same molecular weights in dodecylsulfate and Triton X-100, the same amino acid composition and the same isoelectric point as the earlier isolated carboxyatractylate-protein complex. It differs by its higher sensitivity against trypsin and thermolysin. 4. The identity of both proteins is demonstrated by interconversion of the bongkrekate-protein into the carboxyatractylate-protein. The process requires the catalysis by ADP or ATP, the natural substrates of the protein. 5. The formation of the extractable [3H]bongkrekate-protein complex in mitochondria requires the presence of ADP or ATP. 6. These data, the immunological studies presented earlier, and the differences in the reactivity of -SH groups of the isolated bongkrekate and carboxyatractylate complexes (to be published) indicate that both proteins represent different conformational states of the translocator protein (m-state and c-state).
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PMID:Isolation of the ADP/ATP translocator from beef heart mitochondria as the bongkrekate-protein complex. 64 34

Three-headed Tetrahymena 22S ciliary dynein was found to consist of three heavy chains (HCs) and decompose into two-headed and single-headed fragments upon chymotrypsin digestion. The three HCs (A alpha, A beta, and A gamma) were immunologically different, and presumed to be located on each of the head regions. The two-headed fragment contained A beta and A gamma HCs, while the A alpha HC originated in the single-headed fragment. Both fragments were associated with ATPase activity (Toyoshima, Y. (1987a) J. Cell Biol. 105, 887-895 and Toyoshima, Y. (1987b) J. Cell Biol. 105, 897-901). Using the two-headed dynein fragment, we attempted to determine the site of ATP hydrolysis in the fragment. After digestion of the fragment with 100 micrograms/ml thermolysin for 45 min, we noted eight thermolysin-digested polypeptides (TH 1, 2, 3, 4, 5 alpha, 5 beta, 6 alpha, and 6 beta). By precisely analyzing the degradation process and the products using peptide mapping, immunoblotting and high pressure liquid chromatography, it appeared that the two-headed fragment is dissociated as two separate fragments, each of which contained A beta or A gamma HC. Thermolysin digests, TH 1, 2, 5 alpha and 6 beta were found to be derived from A beta HC, while TH 3, 4, 5 beta and 6 alpha originated in the A gamma HC. Based on the measurements of ATPase activity of these polypeptides, we concluded that the ATPase site is located in the A beta and A gamma HCs, which may have their origins in each head of the two-headed fragment of Tetrahymena 22S ciliary dynein.
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PMID:ATPase sites in two-headed fragment of Tetrahymena 22S ciliary dynein. 153 22

The folding of the peptide chain of the beef heart ADP/ATP carrier in the inner mitochondrial membrane was investigated by enzymatic and immunochemical approaches, using specific proteases and polyclonal antibodies directed against the whole protein and specific regions of the carrier. The accessibility of the membrane-bound ADP/ATP carrier to proteases was followed by immunodetection of the cleavage products, using mitochondria devoid of outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors which are able to bind to the outer face or the inner face of the carrier, respectively. Four types of particles were investigated, namely, mitoplasts-CATR, mitoplasts-BA, SMP-CATR, and SMP-BA. Only the ADP/ATP carrier in SMP-BA was cleaved by two specific proteases, namely, trypsin and lysine C endoprotease, at low doses for short periods of time. Two initial cleavage sites were found between Lys-42 and Glu-43, and between Lys-244 and Gly-245. After a longer period of incubation, an additional cleavage site between Lys-146 and Gly-147 could be demonstrated. Despite cleavage of the membrane-embedded carrier, the binding capacity and affinity of SMP for BA were not altered. A number of other proteases tested, including V8 protease, proline C endoprotease, thrombin, alpha-chymotrypsin, and thermolysin had virtually no effect. These results are explained by a dynamic model of the arrangement of the peptide chain of the ADP/ATP carrier.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Topography of the membrane-bound ADP/ATP carrier assessed by enzymatic proteolysis. 156 52

We have used photoaffinity labelling to examine the chloroplast RNA polymerase components which come into contact with nascent transcripts during the in vitro transcription of plastid DNA. The transcripts were synthesized in the presence of a photoactive analogue (4-thio UTP) and alpha-32P-ATP, using enriched pea chloroplast RNA polymerase preparation and a recombinant plasmid containing the plastid 16S rRNA promoter. Brief irradiation of the transcriptional complex crosslinked the photoactive nascent RNA to proximal proteins. Labelling of the transcriptional complex was dependent on 4-thio UTP and template DNA. Two polypeptides of 51 and 54 kDa were consistently crosslinked to the nascent transcripts; about 60% of the total radioactivity of the crosslinked RNA was associated with these polypeptides. In some experiments, two additional polypeptides of 38 and 75 kDa were also found to be associated with about 13% and 17% of the total crosslinked RNA radioactivity, respectively. The UV-crosslinked transcriptional complexes were stable to either DNase or S1 nuclease hydrolysis but partially sensitive to RNase T1. Insensitivity of the complex to hydrolysis with RNase H suggested that the nascent transcripts were not crosslinked to the template. The complexes could also be hydrolysed by proteinase K and thermolysin. No crosslinkage was observed when labelled RNA molecules containing 4-thio UMP residues were added after synthesis to the polymerase preparation. This suggested that the method identified only those polypeptides which came into close contact with the transcript during its synthesis. Antibodies raised against the RNA-protein complex confirmed the presence of the polypeptides in the chloroplast RNA polymerase preparation on Western blots. Preincubation of these antibodies with the chloroplast RNA polymerase inhibited plastid DNA transcription. These data showed that the transcript-binding polypeptides were functional components of the chloroplast transcriptional complex.
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PMID:Photoaffinity labelling of the pea chloroplast transcriptional complex by nascent RNA in vitro. 171 36

The chloroplastic envelope is composed of two membranes, inner and outer, each with a distinct set of polypeptides. Like proteins in other chloroplastic compartments, most envelope proteins are synthesized in the cytosol and post-translationally imported into chloroplasts. Considerable knowledge has been obtained concerning protein import proteins. We isolated a cDNA clone from pea that encodes a 14-kilodalton outer envelope membrane protein. The precursor form of this protein does not possess a cleavable transit peptide and its import into isolated chloroplasts does not require either ATP or a thermolysin-sensitive component on the chloroplastic surface. These findings, together with similar observations made with a spinach chloroplastic outer membrane protein, led us to propose that proteins destined for the outer membrane of the chloroplastic envelope follow an import pathway distinct from that followed by proteins destined for other chloroplastic compartments.
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PMID:Targeting of proteins to the outer envelope membrane uses a different pathway than transport into chloroplasts. 184 25

The photosynthetic membranes of spinach (Spinacia oleracea L.) chloroplasts were incubated with [gamma-32P] ATP. When the thylakoid membrane kinase was activated with light, the 25- and 27-kDa forms of the light-harvesting chlorophyll a/b protein (LHC II) were phosphorylated on their amino termini. Treatment of the membranes with proteinase K or thermolysin released phosphopeptides which were purified by ferric ion affinity chromatography and reverse phase high performance liquid chromatography. Sequencing of the phosphopeptides was performed with tandem quadrupole mass spectrometry. Three different phosphopeptides Ac-RKTAGKPKT, Ac-RKTAGKPKN, and Ac-RKSAGKPKN originating from class I LHC II were examined after release by thermolysin. One phosphopeptide, Ac-RRTVKSAPQ, originating from class II LHC II was examined after release by proteinase K. Each of the four LHC II phosphopeptides was derived from the amino terminus of a distinct protein. Peptides were acetylated at their amino-terminal arginine and were phosphorylated on either threonine or serine in the third position. We conclude that proteolytic processing of pre-LHC II occurs at a conserved methionyl-arginyl bond and is followed by amino-terminal acetylation of the arginine and nearby phosphorylation of the mature LHC II. Eight different peptides were synthesized in acetylated and nonacetylated forms as substrates for the thylakoid membrane kinase. From a comparison of the kinetics of phosphate incorporation into the peptides, we conclude that basic residues on both sides of the phosphorylation site are important for enzyme recognition. Acetylation of the amino terminus is not required for phosphorylation.
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PMID:Tandem mass spectrometry identifies sites of three post-translational modifications of spinach light-harvesting chlorophyll protein II. Proteolytic cleavage, acetylation, and phosphorylation. 189 41

Myosin subfragment-1 from rabbit skeletal muscle was digested by thermolysin at 25 degrees, 12 degrees and 0 degree C. Thermolysin cleaves subfragment-1 heavy chain into two stable fragments, 28 kDa and 70 kDa, aligned in this order from the N-terminus [Applegate, D. & Reisler, E. (1983) Proc. Natl Acad. Sci. USA 80, 7109-7112]. The rate of digestion at 25 degrees C was significantly increased in the presence of MgATP and somewhat less in the presence of MgADP, or magnesium pyrophosphate. This activating effect of the nucleotides was decreased at 12 degrees C and completely eliminated at 0 degrees C. The results can be explained by assuming that there are two subfragment-1 conformers [Shriver, J. W. & Sykes, B. D. (1981) Biochemistry 20, 2004-2012], and that both the addition of ATP or its analogs, and lowering the temperature, shift the conformational equilibrium in the direction that is more susceptible to thermolysin. Actin inhibited thermolysin digestion of subfragment-1 at all three temperatures studied. Actin inhibition can be explained either by shifting the equilibrium of the conformers in the direction of the less susceptible form or by direct interference of actin with the binding of thermolysin to subfragment-1. Actin inhibition of thermolysin digestion also prevailed when subfragment-1 was in a ternary complex with nucleotide and actin, in both the strongly and weakly attached states. Similarly, actin inhibited the digestion of subfragment-1 modified by 4-phenylenedimaleimide [corrected], which also forms a weakly attached complex with actin. No difference could be found in the accessibility of the thermolysin-susceptible site of subfragment-1 at the 28-70 kDa junction in either rigor, strongly or weakly attached states, which indicates the similarity of the structure proximal to this specific site in the three attached states.
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PMID:Effect of nucleotides, actin and temperature on thermolysin digestion of myosin subfragment-1. 220 15

The photochemical reaction of MgADP-vanadate with the active site of myosin has been used to place a serine at the binding site for the gamma-phosphate of ATP. Irradiation of the MgADP-vanadate myosin subfragment 1 transition state-like complex with UV light specifically photooxidizes the hydroxyl group of a serine residue to an aldehyde (Cremo, C. R., Grammer, J. C., and Yount, R. G. (1988) Biochemistry 27, 8415-8420). Reduction of photooxidized myosin with Na-B3H4 gave only 3H-labeled serine. Here, subsequent extensive proteolytic digestion of 3H-labeled myosin subfragment 1 with trypsin and thermolysin yielded two 3H-labeled peptides, both of which contained the sequence Gly-Glu-Ser-Gly-Ala-Gly-Lys-Thr, in which all the 3H was associated with the serine. This sequence is conserved in all myosin heavy chains sequenced to date and corresponds to residues 178-185 in the rabbit myosin heavy chain (Tong, S. W., and Elzinga, M. (1983) J. Biol. Chem. 21, 13100-13110). These results place Ser-180 at the gamma-phosphate-binding site for ATP and indicate that the glycine-rich loop around the serine provides essential elements of the phosphate-binding site for ATP in all myosin molecules. Such a role was previously suggested based on the common sequence Gly-X-X-X-X-Gly-Lys-Thr/Ser, found in myosin and many other nucleotide-binding enzymes (Walker, J. E., Saraste, M., Runswick, M. H., and Gay, N. J. (1982) EMBO J. 1, 945-951).
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PMID:Direct chemical evidence that serine 180 in the glycine-rich loop of myosin binds to ATP. 252 83

Adenosinetriphosphopyridoxal (AP3PL) specifically modifies Lys684 of Ca2(+)-ATPase of sarcoplasmic reticulum (SR-ATPase) in the presence of Ca2+, leading to its inactivation (Yamamoto, H. et al. (1988) J. Biochem. 103, 452-457). We have now investigated the effects of AP3PL on SR-ATPase in the absence of Ca2+. Similarly to its action in the presence of Ca2+, AP3PL inhibited the Ca2(+)-transporting activity in a dose-dependent manner in the absence of Ca2+ as well. ATP and ADP protected SR-ATPase against inactivation by this reagent. One mole of AP3PL was bound per mol of SR-ATPase with concomitant loss of the Ca2(+)-transporting activity. Binding of AP3PL to SR-ATPase was prevented by ATP. AP3PL-labeled SR membranes were digested with thermolysin and labeled thermolytic peptides were purified through C18 reversed-phase HPLC. Two major AP3PL-labeled peptides were obtained in approximately 1:1 ratio; one was an octapeptide corresponding to 679-ValGluProSerHisLys*SerLys-686, and the other, a nonapeptide corresponding to 487-PheSerArgAspSerLys*ArgMetSer-495 (Lys* indicates a labeled Lys residue) of SR-ATPase. Lys684 in the former turned out to be the same as the highly specific target of AP3PL in the presence of Ca2+ which was identified previously. The target site specificity of AP3PL thus changed significantly but not entirely on binding of Ca2+ to SR-ATPase. This indicates that the spatial arrangement around the gamma-phosphoryl group of the bound ATP is affected by Ca2+ ions bound at the transport site. It is also likely that Lys492 and Lys684 are situated close together in the ATP binding site of SR-ATPase.
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PMID:Ca2(+)-dependent conformational change of the ATP-binding site of Ca2(+)-transporting ATPase of sarcoplasmic reticulum as revealed by an alteration of the target-site specificity of adenosine triphosphopyridoxal. 253 25

To understand the nature of the ATP-induced structural change in myosin subfragment-1, rabbit and chicken skeletal subfragments-1s were cleaved by various proteolytic enzymes in the absence, and in the presence, of ATP and the exact locations of the cleavage sites that were affected by ATP were determined from the amino end analysis of fragments by the use of a protein sequencer. It was found that subtilisin cleaved a site between Gln27 and Asn28 of rabbit subfragment-1 and between Gln28 and Asn29 of chicken subfragment-1 only in the presence of ATP. Thermolysin cleaved a site between Pro31 and Phe32 of chicken subfragment-1 in the presence of ATP, but the same site of rabbit subfragment-1 was not cleaved. The location of these sites is quite similar to the ATP-induced chymotryptic cleavage site of chicken gizzard heavy meromyosin, between Trp29 and Ser30 as reported by others. It is suggested, therefore, that the structure and the ATP-induced structural change in the regions are similar in these subfragment-1s. ATP also changes the cleavage rate of the 26K-50K junction by many proteases. Exact cleavage sites were determined and the relationship between their location and the suppression or the enhancement by ATP of the cleavage was studied. It was found that the cleavage sites were restricted to a quite narrow region and only the cleavage by thermolysin that attacked the middle of the region was enhanced by ATP. The distribution of the cleavage sites and the effect of ATP suggest that ATP induces drastic structural change at the middle of the 26K-50K junction region. The region attacked easily by many proteases coincided very well with a hydrophilic region indicated by the hydropathy index. The region probably protrudes outside and is, therefore, easily attacked by many proteases.
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PMID:ATP-induced structural change in myosin subfragment-1 revealed by the location of protease cleavage sites on the primary structure. 258 5


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