Common epitopes of bovine lens multicatalytic-proteinase-complex subunits.
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ABSTRACT:
SUBMITTER: Wagner BJ
PROVIDER: S-EPMC1135565 | biostudies-other | 1989 Jan
REPOSITORIES: biostudies-other
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Project description:The multicatalytic proteinase (MCP) is a high-molecular-mass non-lysosomal proteinase that gives rise to a characteristic pattern of bands of molecular mass 22-34 kDa on SDS/PAGE gels. Isoelectric-focusing gels of the enzyme purified from rat liver show 16 bands with isoelectric points in the range of pH 5-8.5. Two-dimensional PAGE gels reveal that there are more than the previously reported 13 polypeptides associated with the MCP from rat liver and show a pattern of 15-20 major spots and several minor ones, similar to that of MCP isolated from some other sources. Possible relationships between the different polypeptides were investigated by immunoblot analysis of electrophoretically purified proteinase subunits with affinity-purified subunit-specific antibodies as well as antibodies raised against individual denatured subunits of the complex. The results demonstrate that many of the major polypeptide components of the MCP complex are antigenically distinct. Moreover comparison of immunoreactive material in crude cell extracts with that in purified MCP preparations has shown that the polypeptides are not derived from a smaller number of higher-molecular-mass subunits. Also, individual subunits have the same apparent molecular mass in a variety of rat tissues, suggesting close similarity between MCPs of different tissues. The highest concentrations of MCP subunits occur in liver and kidney. Gel-filtration analysis of crude extracts has demonstrated that MCP polypeptides are also associated with a higher-molecular-mass complex, which may be the 26 S proteinase that has been implicated in the degradation of ubiquitin-protein conjugates.
| S-EPMC1151464 | biostudies-other
Project description:The multicatalytic proteinase (MCP) complex is a major nonlysosomal proteinase which plays an important role in non-lysosomal pathways of protein degradation and which has recently been implicated in antigen processing. The mammalian MCP complex is composed of more than 20 different types of polypeptide, but it is not yet clear which of these components are responsible for its proteolytic activities. The complex has at least three distinct types of proteolytic activity. One of these, the so-called 'trypsin-like' activity, which involves cleavage on the carboxy side of basic amino acid residues, can be selectively and completely inhibited by peptidyl arginine aldehydes (such as leupeptin and antipain), and is also the most sensitive to inhibition by thiol-reactive reagents. In the present study N-[ethyl-1-14C]ethylmaleimide has been used to specifically label thiol groups protected by leupeptin binding. The results suggest that one or two polypeptide components within the complex can be protected against modification by N-ethylmaleimide. These components may be responsible for the 'trypsin-like' activity of the complex or may be adjacent to the catalytic component(s) and play an important role in substrate binding.
| S-EPMC1132128 | biostudies-other
Project description:Hydrolysis of carbobenzoxy-Leu-Leu-Glu 2-naphthylamide by bovine lens neutral-proteinase preparations is not affected by the esterase inhibitor di-isopropyl fluorophosphate, whereas hydrolysis of carbobenzoxy-Gly-Gly-Leu p-nitroanilide is completely inhibited. Hydrolysis of alpha-crystallin, a lens structural protein, can be inhibited by only 50% after prolonged treatment with di-isopropyl fluorophosphate. These data suggest that the lens neutral-proteinase preparation contains at least two enzymes, one of which may be a serine proteinase. This may account, in part, for the previously observed complex response of the preparation to inhibitors.
| S-EPMC1145011 | biostudies-other
Project description:The multicatalytic proteinase complex or proteasome is a high-molecular-mass multisubunit proteinase which is found in the nucleus and cytoplasm of eukaryotic cells. Electron microscopy of negatively stained rat liver proteinase preparations suggests that the particle has a hollow cylindrical shape (approximate width 11 nm and height 17 nm using methylamine tungstate as the negative stain) with a pseudo-helical arrangement of subunits rather than the directly stacked arrangement suggested previously. The side-on view has a 2-fold rotational symmetry, while end-on there appears to be six or seven subunits around the ring. This model is very different from that proposed by others for the proteinase from rat liver but resembles the structure of the simpler archaebacterial proteasome. The possibility of conformational changes associated with the addition of effectors of proteolytic activity has been investigated by sedimentation velocity analysis and dynamic light-scattering measurements. The results provide the first direct evidence for conformational changes associated with the observed positive co-operativity in one component of the peptidylglutamylpeptide hydrolase activity as well as with the stimulation of peptidylglutamylpeptide hydrolase activities by MnCl2. In the latter case, there appears to be a correlation between changes in the shape of the molecule and the effect on activity. KCl and low concentrations of SDS may also act by inducing conformational changes within the complex. Sedimentation-velocity measurements also provide evidence for the formation of intermediates during dissociation of the complex by urea, guanidinium chloride or sodium thiocyanate. Dissociation of the complex either by these agents or by treatment at low pH leads to inactivation of its proteolytic components. The results suggest that activation and inhibition of the various proteolytic activities may be mediated by measurable changes in size and shape of the molecules.
| S-EPMC1134193 | biostudies-other
Project description:The activity of multicatalytic proteinase against synthetic substrates and the kinetics of its inhibition by a range of class-specific inhibitors have been investigated. The enzyme was found to have a broader pH activity profile than previously noted, being active against succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin optimally at pH 4.5 and against benzyloxycarbonyl-Gly-Gly-Arg-7-amino-4-methylcoumarin optimally at pH 10.5. Neither activity was inhibited by the class-specific inhibitors 1,10-phenanthroline, EDTA, pepstatin, di-isopropyl fluorophosphate, peptidyl chloromethanes, peptidyl diazomethanes or L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido-(4-guanidin o)butane (E-64), indicating that the enzyme is not a typical metallo-, aspartic, serine or cysteine proteinase. Inhibition by HgCl2, iodoacetamide and N-ethylmaleimide suggests that free thiols are necessary for the enzyme to maintain activity, but that these thiols are not particularly reactive as is the case for cysteine proteinases of the papain superfamily. The peptidyl aldehydes chymostatin and leupeptin were found to be reversible inhibitors of multicatalytic proteinase. Chymostatin inhibited activity against succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin at pH 4.5 (Ki 160 +/- 22 microM) whereas leupeptin (200 microM) was not inhibitory. Inhibition of activity against benzyloxycarbonyl-Gly-Gly-Arg-7-amino-4-methylcoumarin by these compounds was more complex, in that they behaved as slow tight-binding inhibitors. kon values were determined to be 12 +/- 2 M-1.s-1 and 1290 +/- 125 M-1.s-1 for chymostatin and leupeptin, respectively. The upper limit for Ki values for these two inhibitors was estimated as 5 +/- 1.5 microM and 25 +/- 5 nM, respectively. The different inhibition characteristics for each substrate were also apparent at an intermediate pH of 8.5, showing that the two activities are distinct. Dichloroisocoumarin, a mechanism-based inhibitor of serine proteinases, did inhibit activity against succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin with a rate constant of 250 M-1.s-1, suggesting that multicatalytic proteinase is an atypical serine proteinase.
| S-EPMC1136909 | biostudies-other
Project description:1. Two new assay methods were developed for the lens proteinase. In both, the substrate was alpha2-crystallin (a major lens protein); in the first method, the products were detected by reaction with trinitrobenzenesulphonate in the presence of SO32-, whereas in the second method, 3H-labelled substrate was used, and the products were detected as radioactivity soluble in trichloroacetic acid. 2. The neutral proteinase from bovine lens was partially purified by extraction of the lens at pH5.0 and column chromatography on hydroxyapatite and Sepharose 6B gel. 3. The purified enzyme had no detectable activity against haemoglobin, azo-casein or gamma-crystallin under optimum conditions for alpha2-crystallin. 4. The enzyme showed greatest activity and stability at pH7.5. It was reversibly inhibited by EDTA and 1,10-phenanthroline, and activated by Ca2+ and Mg2+. 5. Molecular weights obtained for the enzyme by chromatography on Sepharose 6B were approx. 500,000 in buffer of I = 0.02, and 250,000 at I = 1.02. 6. The properties of the purified lens proteinase are such as to suggest that this enzyme could account for the entire endopeptidase activity of the lens.
| S-EPMC1165261 | biostudies-other
Project description:On the basis of recent reports that suggested that proteasomes, via an ATP-dependent process, become integral components of a '26 S' complex possessing 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity, we have investigated the molecular interaction of proteasomes in ATP-stabilized fraction II (proteins absorbed on DEAE-matrix and eluted with 0.5 M-KCl) of rabbit reticulocytes and mouse liver. Analysis of the various extracts by (NH4)2SO4 fractionation, velocity-gradient centrifugation, non-denaturing PAGE and SDS/PAGE and immunoblotting with proteasome-specific antisera failed to identify the proteasome as part of a higher-molecular-mass '26 S' multienzyme complex. In all instances proteasomes are identified in their 'free' 650 kDa '20 S' form. In addition to the proteasome and independent of the presence of MgATP, we isolated a high-molecular-mass proteinase whose electrophoretic migration behaviour and sedimentation rate correspond to that of the previously described '26 S' proteinase. This '26 S' proteinase possesses a strong 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity and is composed of several non-identical polypeptides in the molecular-mass range 20-150 kDa. Despite its similarity to proteasomal enzyme activity, protein analysis and immunoblotting experiments demonstrate that neither the intact proteasome nor subunits thereof are components of the '26 S' proteinase complex.
| S-EPMC1130624 | biostudies-other
Project description:An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes in vitro as revealed by two types of native electrophoresis. Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I+III2+IV (S1) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover.
| S-EPMC4331043 | biostudies-literature
Project description:A key innovation for high resolution eyes is a sophisticated lens that precisely focuses light onto photoreceptors. The eyes of holometabolous larvae range from very simple eyes that merely detect light to eyes that are capable of high spatial resolution. Particularly interesting are the bifocal lenses of Thermonectus marmoratus larvae, which differentially focus light on spectrally-distinct retinas. While functional aspects of insect lenses have been relatively well studied, little work has explored their molecular makeup, especially in regard to more complex eye types. To investigate this question, we took a transcriptomic and proteomic approach to identify the major proteins contributing to the principal bifocal lenses of T. marmoratus larvae. Mass spectrometry revealed 10 major lens proteins. Six of these share sequence homology with cuticular proteins, a large class of proteins that are also major components of corneal lenses from adult compound eyes of Drosophila melanogaster and Anopheles gambiae. Two proteins were identified as house-keeping genes and the final two lack any sequence homologies to known genes. Overall the composition seems to follow a pattern of co-opting transparent and optically dense proteins, similar to what has been described for other animal lenses. To identify cells responsible for the secretion of specific lens proteins, we performed in situ hybridization studies and found some expression differences between distal and proximal corneagenous cells. Since the distal cells likely give rise to the periphery and the proximal cells to the center of the lens, our findings highlight a possible mechanism for establishing structural differences that are in line with the bifocal nature of these lenses. A better understanding of lens composition provides insights into the evolution of proper focusing, which is an important step in the transition between low-resolution and high-resolution eyes.
| S-EPMC5886344 | biostudies-literature
Project description:A proteolytic enzyme was purified from the post-myofibrillar fraction of rat skeletal muscle. The purification procedure consisted of fractionation of the muscle extract by (NH4)2SO4, chromatography on DEAE-Sephacel, fast protein liquid chromatography on Mono Q and gel filtration on Sepharose 6B. The enzyme preparation appeared to be homogeneous as judged by disc electrophoresis in polyacrylamide gels and by immunoelectrophoresis. The isoelectric point of the proteinase is at 5.1-5.2. The enzyme has an Mr of about 650 000 and dissociates into eight subunits of Mr 25 000-32 000 when subjected to electrophoresis in sodium dodecyl sulphate/polyacrylamide gels. The proteinase contains hydrolytic activity against N-blocked tripeptide 4-methyl-7-coumarylamide substrates with an arginine or phenylalanine residue adjacent to the leaving group. Maximum activity with the first group of substrates was at pH 10.5, and this activity was inhibited by leupeptin, chymostatin and Ca2+. Maximum activity with the latter group of substrates was at pH 7.5, and was also inhibited by the two microbial inhibitors, but was activated by Ca2+ ions. By using [14C]methylcasein as a substrate, maximum activity was observed at pH9.0, and this proteolytic activity was not affected by leupeptin, was enhanced by chymostatin and inhibited by Ca2+. Similar effects were observed when benzyloxycarbonyl-Leu-Leu-Glu 2-naphthylamide was used as a substrate. These enzymic activities were abolished by p-hydroxymercuribenzenesulphonic acid or mersalyl acid, whereas a small activation was observed with cysteine or dithiothreitol.
| S-EPMC1144965 | biostudies-other