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The ATP-ubiquitin-dependent proteolytic pathway (ubiquitin pathway) is believed to be involved in the formation of various neuronal inclusion bodies including Lewy bodies (LBs), a pathological hallmark of Parkinson disease and diffuse Lewy body disease (DLBD). Since multicatalytic proteinase (MCP) is involved in the ubiquitin pathway, an investigation of whether MCP is involved in neuronal inclusion bodies would provide a clue to the mechanism underlying the formation of neuronal inclusion bodies as well as to the pathogenesis of degenerative neurological disorders. In this study, we investigated detailed immunolocalization of MCP in LBs in DLBD brains using light and electron microscopy. We raised three different monoclonal antibodies against purified human MCP. Each of them recognized different sets of MCP subunits on Western blotting. Immunohistochemically, anti-MCP antibodies recognized all ubiquitin-positive cortical LBs in situ as well as those isolated from frozen DLBD cortices, suggesting that MCP is present in LBs as a whole molecule exhibiting protease activity. In electron microscopy, MCP immunoreactivity (MCP-IR) was exclusively localized on a characteristic oval structure with an approximate diameter of 100 nm. This structure was distributed throughout the LBs and was devoid of ubiquitin immunoreactivity. Treatment of isolated LBs with 2% SDS, but not with 0.5% Triton X-100, removed this structure from LBs in which fibrous materials predominated. Ubiquitin immunoreactivity was also decreased in isolated LBs treated with 2% SDS, suggesting that the fibrous structures in LBs were not ubiquitinated in situ. Thus, it is suggested that LBs are subjected to a proteolytic process in which MCP plays a role via processing of specific components of LBs.

Changes in multicatalytic proteinase activity during differentiation were investigated using Me2SO-induced differentiation of murine erythroleukemia cells as a model. The apparent ATP-dependent multicatalytic proteinase activity decreased in the Me2SO-treated cells with ATP-dependent incorporation of [3H]diisopropyl fluorophosphate decreasing notably after Me2SO-treatment. This decrease in activity does not seem to arise from a cessation of cell-proliferation, because no significant changes in proteinase activity were observed under different culture conditions. Hydroxyapatite column chromatography was employed to analyze the form of multicatalytic proteinase. It was clearly demonstrated that the 26S form of the proteinase decrease in the differentiated cells relative to normal cells. Multicatalytic proteinase-associated proteins that bind to the proteinase in an ATP-dependent manner were purified on an anti-multicatalytic proteinase IgG conjugated column. Only a small amount of protein was recovered from the differentiated cells. These results suggest that the decrease in multicatalytic proteinase-associated proteins that occurs upon cell-differentiation abolishes the ATP-dependent activity of the proteinase.

A non-lysosomal multicatalytic proteinase (MCP) is immunohistochemically visualized in Lewy bodies and loosely arranged globose tangles but not in the majority of compact neurofibrillary tangles in brains with cortical diffuse Lewy body disease. Virtually all Lewy bodies are immunoreactive to MCP, exhibiting various staining patterns where they are moderately diffuse, intense in the central core or intense in the peripheral rim. MCP may be involved in the formation of these inclusions.

The genes encoding three subunits of Saccharomyces cerevisiae proteasome were cloned and sequenced. The deduced amino acid sequences were homologous not only to each other (30 to 40% identity) but also to those of rat and Drosophila proteasomes (25 to 65% identity). However, none of these sequences showed any similarity to any other known sequences, including various proteases, suggesting that these proteasome subunits may constitute a unique gene family. Gene disruption analyses revealed that two of the three subunits (subunits Y7 and Y8) are essential for growth, indicating that the proteasome and its individual subunits play an indispensable role in fundamental biological processes. On the other hand, subunit Y13 is not essential; haploid cells with a disrupted Y13 gene can proliferate, although the doubling time is longer than that of cells with nondisrupted genes. In addition, biochemical analysis revealed that proteasome prepared from the Y13 disrupted cells contains tryptic and chymotryptic activities equivalent to those of nondisrupted cells, indicating that the Y13 subunit is not essential for tryptic or chymotryptic activity. However, the chymotryptic activity of the Y13 disrupted cells is not dependent on sodium dodecyl sulfate (SDS), an activator of proteasome, since nearly full activity was observed in the absence of SDS. Thus, the activity in proteasome of the Y13 disrupted cells might result in unregulated intracellular proteolysis, thus leading to the prolonged cell cycle. These results indicate that cloned proteasome subunits having similar sequences to the yeast Y13 subunit are structural, but not catalytic, components of proteasome. It is also suggested that two subunits (Y7 and Y8) might occupy positions essential to proteasome structure or activity, whereas subunit Y13 is in a nonessential but important position.

The cDNAs for a number of multicatalytic proteinase (MCP) subunits have been cloned, characterized, and their primary structures have been determined. The mechanism for how MCP demonstrates its multicatalytic nature, especially protease activities, however, is still obscure, since no sequences similar to known protease sequences can be found in the sequences of MCP subunits thus far determined. To explain this fact, we propose a structural model for MCP: MCP consists of two classes of subunits, structural and catalytic, and the structural subunits constitute a "test-tube"-like container in which the other catalytic subunits sit and react with substrate. Most of the observations thus far obtained can be explained easily by this hypothesis, although various other possibilities are not excluded.

cDNA clones for two subunits (designated subunits K and L) of rat liver multicatalytic proteinase (MCP) were isolated using oligonucleotide probes synthesized according to their partial amino acid sequences. The encoded polypeptides of subunits K and L consisted of 255 and 261 amino acid residues with calculated molecular mass of 28.3 kDa and 29.5 kDa, respectively. Northern blot analysis revealed that subunits K and L were expressed in all tissues examined and their expression patterns were almost identical. The deduced amino acid sequences showed no similarities to known protein sequences other than the recently reported sequences of rat and Drosophila MCP subunits. Sequence comparison of MCP subunits of rat and Drosophila revealed that the N-terminal two-thirds of the sequence (especially the N-terminal approximately 20 residues) is conserved, but the C-terminal third of the sequence shows no similarity, suggesting functional and structural roles for both regions. Implications for the structural and functional aspects of MCP subunits are discussed based on the sequence similarity.

1. A high-molecular-mass multicatalytic proteinase, ingensin, has been purified from rat liver and biochemically characterized. Trypsinization in the presence of ATP prevented the degradation of ingensin subunits. 2. Glutaraldehyde, which copolymerizes proteins, increased the apparent molecular mass of the subunits on SDS-PAGE, indicating the occurrence of covalent crosslinking of subunits. ATP, in this case, lowered the extent of covalent crosslinking. These results suggest that ATP altered the conformation of ingensin subunits. 3. Urea-induced autodigestion experiments demonstrated that some low-molecular-weight subunits selectively disappeared without changes in the contents of other subunits. The chymotryptic activity of the proteinase was more resistant to autodigestion than its tryptic activity. Therefore, we conclude that separate subunits of the enzyme are responsible for the different peptide-hydrolyzing activities.

The multicatalytic proteinase, ingensin, was purified to homogeneity from chicken liver. rRNA-degrading activity was co-eluted with the purified multicatalytic proteinase from a TSK-3000SW column. This RNA-degrading activity was inactivated by heat treatment and the addition of a low concentration of SDS. Therefore, the RNA-degrading activity co-eluted with the multicatalytic proteinase was not due to contamination by low-molecular-mass RNases. These results strongly suggest that this RNA-degrading activity was tightly associated with the multicatalytic proteinase, ingensin.

Two forms of a neutral protease that catalyzed the hydrolysis of succinyl-Leu-Leu-Val-Tyr-MCA were isolated from porcine skeletal muscle cytosol by fractionation on DEAE-cellulose, hydroxyapatite and Sephadex G-100. The native enzyme had a molecular mass of above 1000 kDa. Peak A, which was eluted from hydroxyapatite at 50 mM phosphate, was activated 37-fold by the detergent, SDS, while peak B which was eluted at 150 mM phosphate, was activated only 2-fold. After dialysis against water, the B form showed restored ability to be activated by SDS (9.6-fold with 0.04% SDS). The activated peak B was extremely sensitive to divalent and monovalent cations such as Ca2+, Mg2+, Na+, K+ and NH+4 as well as protease inhibitors such as leupeptin, chymostatin and DFP. These results suggest that these proteases are generally latent in the cells and may be regulated by changes in the concentrations of cations in the cytosol. We call this new type of protease, ingensin.

A high-molecular-mass protease, ingensin, was purified to homogeneity from rabbit reticulocytes by DEAE-cellulose, HPLC gel filtration, and hydroxyapatite chromatographies. By these procedures, ingensin activity was separated from the activities of two other unique aminopeptidases, one of which is activated by ATP. Ingensin had the following properties: the optimum activity was seen around pH 9.0 and at 50 degrees C; addition of 0.04% SDS and 1 mg/ml linoleic acid resulted in 8- and 4-fold increases in peptide-hydrolyzing activity, respectively. The molecular mass was found to be 700,000 +/- 100,000 daltons on gel filtration, but SDS electrophoresis revealed that the enzyme is composed of several subunits with molecular weights of less than 35,000. The N-terminal-blocked tyrosine- and arginine-MCA derivatives, but not Arg-MCA, were hydrolyzed rapidly by ingensin. The approximate Km values for the reaction of ingensin with Suc-Leu-Leu-Val-Tyr-MCA and Z-Ala-Arg-Arg-MCA were 0.32 and 0.12 mM, respectively. The degradation of several proteins in the reticulocyte extract was stimulated by the addition of SDS and linoleic acid. The activator concentrations necessary for stimulation of the protein hydrolysis are similar to those of the purified reticulocyte ingensin for synthetic substrates. Ingensin did not associate with either right-side-out or inside-out red cell membranes. These results suggest that ingensin is a cytosolic fatty acid-stimulated protease, which is involved in the protein turnover in reticulocyte extracts.

The enzyme responsible for the succinylleucylleucylvalyltyrosine methylcoumarylamide- (SLLVT-) degrading activity was purified from the postmitochondrial supernatant of rat liver (Yamamoto, T., Nojima, M., Ishiura, S. and Sugita, H. (1986) Biochim. Biophys. Acta 882, 297-304). The enzyme, named ingensin, was activated by saturated fatty acids, especially myristic acid, as well as by unsaturated linoleic acid and arachidonic acid. Although 2-mercaptoethanol activated ingensin 2-fold and p-chloromercuribenzoate and HgCl2 completely inhibited its peptide-hydrolyzing activity, the enzyme is activated by the addition of a thiol-blocking reagent, monoiodoacetic acid. Ingensin was also inhibited by a specific serine proteinase inhibitor, diisopropyl fluorophosphate, but not by a specific cysteine proteinase inhibitor, E-64-c. These results suggest that the enzyme is a serine proteinase with an active thiol group(s) near the active site. We have found that the addition of glycerol and nordihydroguaiaretic acid lowered the extent of its activation by fatty acids as well as its intrinsic peptide-hydrolyzing activity.

A high-molecular mass ATP-dependent proteinase was shown to be identical to a multicatalytic proteinase, ingensin [(1988) Eur. J. Biochem. 177, 261-266]. The molecular mass of this proteinase increased in crude extracts of the rat liver and porcine brain, but not in the purified sample, only when the proteinase was extracted with ATP. The higher-molecular form of ingensin may be the intact one, because the concentration of ATP in vivo never decreases below 1 mM. This form of the proteinase is latent and it requires a high concentration of detergent for activation. On chromatography, it was found that the high-molecular form corresponds to the previously reported minor isoenzyme of ingensin [(1986) Biochim. Biophys. Acta 882, 297-304], ingensin A, or possibly to the ATP/ubiquitin-dependent 26S protease [(1987) J. Biol. Chem. 262, 8303-8313], and the low-molecular form to major ingensin B or the ATP/ubiquitin-independent 20 S protease.

We investigated and characterized the ATP-dependent protease in human erythroleukemia, K562 cells. The succinyl-leucyl-leucyl-valyl-tyrosine-methylcoumarinamide hydrolytic activity in a K562 lysate at pH 9 rose more than 10-fold with the addition of 1 mM ATP. The effect of ATP on the protease activity was dose-dependent and inhibited by the addition of ADP. This activity was not inhibited by EDTA, L-3-carboxy-trans-2,3-epoxypropionyl-leucylamide-(4-guanidin o)butane or leupeptin, but was strongly inhibited by chymostatin and diisopropylfluorophosphate. The protease activity was eluted just after the void volume from a G3000SW HPLC column. The above results suggest that this protease is identical to the high-molecular-mass protease, ingensin, previously reported by us. The ATP-dependent increase in the protease activity was due to prevention of the inactivation of the protease by ATP, and not to activation of the protease itself in the reaction mixture at 37 degrees C. The depressed succinyl-leucyl-leucyl-valyl-tyrosine-methylcoumarinamide hydrolytic activity in the ATP-depleted lysate was restored to the same level by the detergent, SDS. Therefore, we conclude that the inactivation of ingensin occurring on preincubation is not irreversible.

The main characteristic changes observed in Alzheimer's disease (AD) are the presence of neurofibrillary tangles and the deposition of amyloid A4 peptides. The most abundant amyloid A4 peptide species in AD (which we tentatively named A4') is composed of 39 amino acids, which is devoid of the 3 N-terminal amino acids, Asp-Ala-Glu, of the originally reported A4 peptide. We synthesized a model peptide substrate, Suc-Ala-Glu-methylcoumarinamide (MCA), to identify the proteinase that splits the A4' peptide. DEAE-cellulose column chromatography of rat liver and porcine brain extracts showed that only one peak material digested the synthetic substrate at pH 8. The results for the final preparation indicate that the Suc-Ala-Glu-MCA-degrading enzyme is a high-molecular-mass proteinase, with a molecular mass of above 500,000, and is composed of several low-molecular-mass subunits. These results suggest that a non-lysosomal multicatalytic proteinase (we named this enzyme ingensin (ingens = large in Latin). Ishiura, S. et al. (1985) FEBS Lett. 189, 119-123) catalyzes the above reaction. Antiserum against the purified multicatalytic proteinase, ingensin, crossreacted with the purified Suc-Ala-Glu-MCA-degrading proteinase. It is likely that ingensin shows a similar action toward amyloid precursor protein (APP) in vivo.

A high molecular weight, fatty acid- and SDS-sensitive protease named ingensin was purified from rat brain in this study. The enzyme purified from rat brain has the same biochemical properties as those purified from other tissues, e.g., porcine skeletal muscle, human placenta, and rat liver in our laboratory, and rat skeletal muscle and bovine pituitary gland in other laboratories, independently. Immunoblot bands were detected in the same positions as those in the case of ingensin from rat liver. In addition, its topographical distribution was studied in rat brain and muscle by means of the immunohistochemical method. The cytoplasm of motor neurons of the spinal cord, pyramidal cells, and granular cells of the hippocampus, Purkinje cells, and glial cells were stained. Axons were stained. The cytoplasm of muscle was also stained, especially that of type 2 fibers.

Two forms of a high-molecular-weight proteinase were isolated from rat liver. The purification procedure involved homogenization of the tissue, chromatography on DEAE-cellulose, high-performance liquid chromatography (HPLC: TSK 3000 SWG) and hydroxyapatite chromatography. The breakthrough fraction from the hydroxyapatite column contained the sodium dodecyl sulphate (SDS)- and linoleic acid-activated proteinase, ingensin A, but the other form, ingensin B, which was also activated by SDS and linoleic acid, was bound to the hydroxyapatite and eluted at 200 mM phosphate. A distinct feature of ingensin A was its activation by a brief sonication procedure. The optimum pH of the two forms was 7.5-9.5, and both of them were activated by monovalent cations. Although both enzymes show similar molecular weights of 700,000 on gel filtration, ingensins A and B were separated into a major subunit of 120,000 and subunits of 25,000-35,000, respectively, under the denaturing conditions.

A linoleic acid-sensitive protease, ingensin, was purified to homogeneity from human placenta. The physical properties of the placental ingensin were found to be very similar to those of skeletal muscle ingensin [Ishiura et al. (1985) FEBS Lett. 189, 119-123]. The purified ingensin was activated by linoleic acid and SDS. The linoleic acid-activated form was inhibited preferentially by divalent cations, whereas the SDS-activated form was inhibited by monovalent cations instead.

We purified a high-molecular-weight protease, ingensin, from extract of human placenta by successive DEAE-cellulose, hydroxyapatite, and high performance liquid chromatographies. The activity of ingensin was determined by using a synthetic substrate, succinyl-leucyl-leucyl-valyl-tyrosine-methylcoumarinamide (MCA). The purified ingensin, which gave a single band in 6.5% nondenaturing polyacrylamide gel electrophoresis, was activated by linoleic acid and sodium dodecyl sulfate (SDS). Maximum activity was observed at pH 9.5 in the presence of 0.06% SDS, but at pH 8.0 in the presence of linoleic acid. A subcellular fractionation study showed that a large amount of ingensin activity was present in the cytosol or microsome fraction rather than in the precipitate of low-speed centrifugation. The effect of protease inhibitors on the activated ingensin was also investigated.