Addition of ATP increases the apparent molecular mass of the multicatalytic proteinase, ingensin

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.     

RNA degrading activity is tightly associated with the multicatalytic proteinase, ingensin

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.     

Putative N-terminal splitting enzyme of amyloid A4 peptides is the multicatalytic proteinase, ingensin, which is widely distributed in mammalian cells

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.     

Ingensin, a high-molecular-mass alkaline protease from rabbit reticulocyte

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.     

Purification of the two forms of the high-molecular-weight neutral proteinase ingensin from rat liver

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.     

Ingensin, a fatty acid-activated serine proteinase from rat liver cytosol

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.     

Crosslinking and modification of Na,K-ATPase by ethyl acetimidate.

Monofunctional imidoesters such as ethyl acetimidate can induce crosslinking of subunits of the (Na⁺ + K⁺) ion-stimulated ATPase. The cross-linked product is shown to be composed of equal parts of two subunits: one phosphorylated by γ-[³²P]ATP, the other a glycoprotein. Because crosslinking of proteins by imidoesters normally requires reaction at both ends of a bifunctional reagent, the reaction is unexpected. A model for the reaction is proposed, in which a favorably positioned amino group on one subunit displaces the amidino group on the other, forming a covalent diamidino crosslink between the two subunits.Reaction with imidoesters also partially inhibits the Na,K-ATPase and reduces the sensitivity of the phosphorylated form of the enzyme to potassium ion. This modification resembles the effect of ouabain, a specific inhibitor of Na,K-ATPase, and is independent of crosslinking.     

Purification and characterization of a high-molecular-weight protease, ingensin, from human placenta

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.     

Evidence that the intracellular domain of FGF receptor 2IIIb affects contact of the ectodomain with two FGF7 ligands

Models of the oligomeric FGF signaling complex, including those derived from crystal structures, vary in stoichiometry and arrangement of the three subunits comprised of heparin/heparan sulfate chains, FGFR tyrosine kinase and activating FGF. Here, using covalent affinity crosslinking of radiolabeled FGF7 to binary complexes of FGFR2IIIb and heparin, we show that two molecules of FGF7 contact each FGFR2IIIb. This supports models that propose a dimeric complex of two units with stoichiometry 1 FGF:1 FGFR in which each FGF contacts both FGFR. The bivalent FGF7 contact was dependent on the full-length amino terminus of FGF7alpha and the intracellular domain of FGFR2IIIb extending through the juxtamembrane domain and the beta1 and beta2 strands of the kinase which is required for ATP binding. We propose that the differences in crosslinking report differences in relationships among subunits in the ectodomain of the complex that are affected by the amino terminus of FGF and the FGFR intracellular domain. From this, we suggest the corollary that conformational relationships among subunits in the ectodomain are transmitted to the intracellular and ATP binding domains during activation of the complex.     

Glutaraldehyde crosslinking analysis of the C12E8 solubilized H,K-ATPase

A soluble porcine H,K-ATPase preparation was obtained with the nonionic detergent, C12E8. ATP hydrolysis by the soluble H,K-ATPase was stimulated with respect to the native preparation at pH 6.1, while the K(+)-phosphatase activity was comparable to the native enzyme. The soluble enzyme demonstrated characteristic ligand-dependent effects on ATP hydrolysis, including ATP activation of K(+)-stimulated hydrolysis with a K0.5 of 28 +/- 4 microM ATP, and inhibition with an IC50 of 2.1 mM ATP. The activation and inhibition of ATP hydrolysis by K+ was also observed with a K0.5 for activation of 2.8 +/- 0.4 mM KCl at 2.0 mM ATP (pH 6.1) and inhibition with an IC50 of 135 mM KCl at 0.05 mM ATP. 2-Methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine-3-acetonitrile (SCH 28080), a specific inhibitor of the native H,K-ATPase, competitively inhibited the K(+)-stimulated activity with a Ki of 0.035 microM. The soluble enzyme was stable with a t0.5 for ATPase activity of 6 h between 4 and 11 degrees C. The demonstration of these related ligand responses in the catalytic reactions of the soluble preparation indicates that it is an appropriate medium for investigation of the subunit associations of the functional H,K-ATPase. Subunit associations of the active soluble enzyme were assessed following treatment with the crosslinking reagent, glutaraldehyde. The distribution of crosslinked particles was independent of the soluble protein concentration in the crosslinking buffer within the protein range 0.3 to 2.0 mg/ml or the detergent to protein ratio varied from 1 to 15 (w/w). The crosslinked pattern was unaffected by the presence or absence of K during crosslinking or nucleotide concentration. These observations suggest that crosslinking occurs in associated subunits that do not undergo rapid associations dependent upon enzyme turnover. Phosphorylation of the soluble enzyme with 0.1 mM MgATP produced a phosphoprotein at 94 kDa. A phosphoprotein obtained after glutaraldehyde treatment exhibited identical electrophoretic mobility to the crosslinked particle identified by silver stain. Glutaraldehyde treatment of soluble protein fractions resolved on a linear 10-35% glycerol gradient revealed several smaller peptides partially resolved from the crosslinked pump particle, but no active fraction enriched in the monomeric H,K-ATPase. This data indicates that the functional porcine gastric H,K-ATPase is organized as a structural dimer.     

The sites of phosphorylation of rabbit brain phosphofructo-1-kinase by cyclic AMP-dependent protein kinase

The three isozymic subunits of phosphofructo-1-kinase present in rabbit brain and designated A, B and C were phosphorylated in vitro by cyclic AMP-dependent protein kinase with 32P-labeled ATP. Limited digestion of the labeled enzymes with trypsin or with Staphylococcus aureus V8 proteinase led to the solubilization of radiolabeled peptides derived from the three isozymic subunits. Limited digestion by V8 proteinase was accompanied by a slight reduction in the apparent sizes of the subunits, indicating that the phosphorylated sites are located near either the amino or carboxyl termini of the protein. V8 proteinase digestion led to no change in the maximal activity of the enzyme but did abolish sensitivity to ATP inhibition. The phosphopeptides of the tryptic and the V8 digests were purified by chromatography and their amino acid sequences were determined and compared to the previously established sequence from rabbit muscle isozyme A. PFK-A E H I S R K R S G E A T V PFK-B H V T R R S L S M A K G F PFK-C V S A S P R G S Y R K F L In each instance, the phosphorylated serine, underlined in the above sequences, was found to be one or two residues toward the C-terminus of one or more basic residues. No other similarities in structure were noted.     

Reticulocyte lipoxygenase, ingensin, and ATP-dependent proteolysis

Lipoxygenase purified from rabbit reticulocyte lysate has a molecular mass of 68 kDa on SDS gel and a pI of 5.97. Lipoxygenase is inhibited by nordihydroguaiaretic acid (NDGA), 3-amino-1-(m-(trifluoromethyl)phenyl)-2-pyrazoline (BW755C), 5,8,11,14-eicosatetraynoic acid (ETYA), salicylhydroxamate (SHAM) or hemin. Metal ions or nucleotides do not affect its activity. The addition of certain of these inhibitors to the reticulocyte extract also inhibited the ATP-dependent proteolysis of casein, one of the distinct characteristics of reticulocytes. No clear correlation between lipoxygenase activity and ATP-dependent proteolysis could be detected. Hemin and NDGA inhibited both processes, but the concentrations necessary for inhibition were quite different. SHAM completely inhibited lipoxygenase, but not proteolysis. o-Phenanthroline inhibited ATP-dependent proteolysis, but had no effect on lipoxygenase activity. We have also purified a high-molecular-mass protease, ingensin, from reticulocyte extract. This protease accounted for more than 90% of the casein-degrading activity in reticulocyte extract. NDGA inhibited ingensin at the same concentrations required for inhibition of ATP-dependent proteolysis. These results suggest that lipoxygenase is not indispensable for the ATP-dependent proteolysis and the novel high-molecular-mass protease, ingensin, may be involved in the process.     

The presence of ATP + ubiquitin-dependent proteinase and multicatalytic proteinase complex in bovine brain

The presence of two distinct high-molecular-weight proteases with similar pH optima in the weakly alkaline region was shown in cytosol of the bovine brain cortex. They were separated by ammonium sulfate fractionation and each was further purified by DEAE-Sephacel Sephacryl S-300, DEAE-Cibacron Blue 3GA-agarose, heparin-agarose, and Sepharose 6B chromatography. The larger enzyme (Mr 1,400 kDa), which precipitates at 0–38% ammonium sulfate saturation, seems to be active in ATP+ubiquitin (Ub)-dependent proteolysis; it has low basal caseinolytic activity that is stimulated 3-fold by ATP, and when Ub is present ATP causes a 4.5-fold stimulation. A second proteinase was also found to be present (Mr 700 kDa) that precipitates at 38–80% ammonium sulfate saturation, is composed of multiple subunits ranging in Mr from 18 to 30 kDa, and degrades both protein and peptide substrates, demonstrating trypsin-, chymotrypsin- and cucumisin-like activities. Catalytic, biochemical, and immunological characteristics of this proteinase indicate that it is a multicatalytic proteinase complex (MPC), whose enzyme activity, in contrast to that of MPC from bovine pituitaries (1–3), is stimulated 1.7-fold by addition of ATP in the absence of ubiquitin at the early steps of purification; this property is lost during the course of further purification. Both proteinases are present in the nerve cells, since the primary chicken embryonic telencephalon neuronal cell culture extracts contain both ATP+Ub-dependent proteinase and MPC activities.Special issue dedicated to Dr. Paola S Timiras     

Structure of the actin-myosin complex produced by crosslinking in the presence of ATP

The structure of the actin-myosin complex during ATP hydrolysis was studied by covalently crosslinking myosin subfragment 1 (S1) to F-actin in the presence of nucleotides (especially ATP) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The fluorescence energy transfer was measured between N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine and 6-(iodoacetamide)fluorescein bound to the SH1 thiol of S1 and the Cys374 thiol of actin. The covalent acto-S1, produced by crosslinking in the absence of nucleotide or in the presence of ADP, showed transfer efficiency of 0.50 to 0.52 and intersite distance of 4.5 to 4.7 nm, which were equal to those obtained with non-crosslinked acto-S1 in the absence of nucleotide. However, the covalent acto-S1, produced by crosslinking in the presence of either 5'-adenylyl imidodiphosphate (AMPPNP) at high ionic strength or ATP, showed a significant decrease in the efficiency to 0.26 to 0.34 and hence an increase in the distance to 5.2 to 5.5 nm. These results suggest that AM-ATP and/or AM-ADP-P (formed during ATP hydrolysis) and AM-AMPPNP have a very different conformation from AM and AM-ADP (in which A is actin and M is myosin).     

Modification of phenylalanyl-tRNA-synthetase from Escherichia coli MRE600 by adenosine-5'-trimetaphosphate

Modification of phenylalanyl-tRNA synthetase from E. coli MRE600 by adenosine-5'-trimetaphosphate, phosphorylating analog of ATP was shown to bring about the enzyme inactivation in the reactions of tRNA aminoacylation and ATP-[32P]pyrophosphate exchange. ATP when added in the reaction mixture protects the enzyme against inactivation in both reactions and decreases the level of covalent attachment of the analog. Phenylalanine has no protective effect. tRNA exhibits slight protective effect. Adenosine-5'-trimetaphosphate modifies both types (alpha and beta) of subunits of phenylalanyl-tRNA synthetase which is of alpha 2 beta 2 structure. ATP protects both types of the enzyme subunits against the covalent attachment of the analog. Disposition of the ATP-binding centers in the contact region of the nonequivalent subunits of the enzyme was proposed. The level of covalent attachment of the analog to the enzyme exceeds the number of the enzyme active sites that may be a consequence of the other nucleotide-binding center labeling.     

Interaction of various nucleotide-dependent enzymes with bifunctional analogs of ATP, derivatives of polymethylene diamines

The interaction of bifunctional ATP derivatives, Appp5'[NH-(CH2) n-NH]ppp5'A (n = 0 or 2-8) with tyrosyl-, valyl-, lysyl-, tryptophanyl-tRNA synthetases and creatine kinase was investigated. ATP derivatives don't inhibit the tRNA aminoacylation catalyzed by tyrosyl-tRNA synthetase. These derivatives behave as mixed-type inhibitors with respect to ATP in the case of valyl- and lysyl-tRNA-synthetases. In the case of the other enzymes all analogs of ATP manifest competitive inhibition towards ATP. The affinity of all ATP derivatives to tryptophanyl-tRNA synthetase does not differ significantly (Ki = 0.2 divided by 0.6 mM). The Ki values for these derivatives in the case of creatine kinase are also very similar with the exception of A5'ppp-NH-(CH2)3-NH-ppp5'A. The Ki value for this derivative is one order of magnitude lower than for other ones. The affinity reagents received by periodate oxidation of bifunctional ATP analogs derivatives of di-, tetra- and heptamethylenediamine modify non-identical subunits of creatine kinase with different velocities, but modification of M- and M'-subunits proceeds independently. An analogues derivative of trimethylenediamine interacts simultaneously with two centers of the dimeric form of kinase forming non-equivalent complexes. The covalent attachment of the reagent to one subunit of creatine kinase does not except the complex formation and covalent binding of bifunctional ATP analogs with the other subunit of the dimer, but results in a one order of magnitude decrease in affinity of the ATP derivative to the nonmodified centre of the enzyme. These data permit to evaluate the distance between ATP binding sites of creatine kinase in its dimeric form as 5-6 A approximately. Such a distance between active sites may be the reason for the higher activity of the M- and M'-creatine kinase subunits taken separately as compared to the enzyme dimeric form.     

Effects of linoleic acid and cations on the activity of a novel high-molecular weight protease, ingensin, from human placenta

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.     

Inhibitory mechanism of a cross-class serpin,the squamous cell carcinoma antigen 1

The squamous cell carcinoma antigen (SCCA) 1 and its homologous molecule, SCCA2, belong to the ovalbumin-serpin family. Although SCCA2 inhibits serine proteinases such as cathepsin G and mast cell chymase, SCCA1 targets cysteine proteinases such as cathepsin S, K, L, and papain. SCCA1 is therefore called a cross-class serpin. The inhibitory mechanism of the standard serpins is well characterized; those use a suicide substrate-like inhibitory mechanism during which an acyl-enzyme intermediate by a covalent bond is formed, and this complex is stable against hydrolysis. However, the inhibitory mechanism of cross-class serpins remains unresolved. In this article, we analyzed the inhibitory mechanism of SCCA1 on a cysteine proteinase, papain. SCCA1 interacted with papain at its reactive site loop, which was then cleaved, as the standard serpins. However, gel-filtration analyses showed that SCCA1 did not form a covalent complex with papain, in contrast to other serpins. Interaction with SCCA1 severely impaired the proteinase activity of papain, probably by inducing conformational change. The decreased, but still existing, proteinase activity of papain was completely inhibited by SCCA1 according to the suicide substrate-like inhibitory mechanism; however, papain recovered its proteinase activity with the compromised level, when all of intact SCCA1 was cleaved. These results suggest that the inhibitory mechanism of SCCA1 is unique among the serpin superfamily in that SCCA1 performs its inhibitory activity in two ways, contributing the suicide substrate-like mechanism without formation of a covalent complex and causing irreversible impairment of the catalytic activity of a proteinase.     

Human placental calcium activated neutral proteinase: Separation and functional characterization of subunits

The subunits of human placental milli calcium activated neutral proteinase and micro calcium activated neutral proteinase have been separated by partial denaturation with urea followed by molecular sieving, with a recovery of 82–91% of activity. The separated subunits were homogeneous, as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Their molecular sizes, catalytic activities and sulphydryl contents suggest that both the subunits of these two calcium activated neutral proteinases are distinct. The subunits were highly specific and could not be interchanged. Both the subunits of micro calcium activated neutral proteinase were catalytically active whereas only the 80 k subunit of milli calcium activated neutral proteinase was active. 30 k subunit of milli calcium activated neutral proteinase has a regulatory role since maximum activity of the 80 k subunit was elicited only in its presence. Activity of the reassociated subunits indicated that interaction is essential for the expression of optimum activity. Interaction of subunits rendered the enzymes less susceptible to inhibition by endogenous calcium activated neutral proteinase inhibitor.