Differential effects of oleic acid, sodium dodecyl sulfate, and protease inhibitors on the endopeptidase activities of the lobster multicatalytic proteinase.

1. Lobster muscles contain a latent multicatalytic proteinase; heating at 60 degrees C for 1-2 min converts the latent form to a heat-activated form with enhanced proteolytic activity. Both forms have three endopeptidase activities, which are classified as the trypsin-like, chymotrypsin-like, and peptidylglutamylpeptide bond hydrolyzing activities. 2. Sulfhydryl reagents (mersalyl acid, N-ethylmaleimide, hemin, iodoacetamide, and p-chloromercurisulfonic acid), benzamidine, and chloromethyl ketones inhibited all three activities of the heat-activated form. Leupeptin and antipain inhibited only the trypsin-like activity, while the chymotrypsin-like activity was the most sensitive to diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, aprotinin, and soybean trypsin inhibitor. Pepstatin and L-trans-epoxysuccinylpeptides had little effect on the peptidase activities. 3. Sodium dodecyl sulfate and oleic acid preferentially activated the peptidylglutamyl-peptide hydrolyzing activity of the latent form, whereas N-ethylmaleimide stimulated both the trypsin-like and peptidylglutamyl-peptide hydrolases. These results suggest that the lobster enzyme is an atypical serine proteinase.     

Purification and characterization of a latent form of multicatalytic proteinase from fish muscle.

1. A latent form of multicatalytic proteinase (MCP) was purified to apparent homogeneity from white croaker muscle by DEAE-Sephacel, Mono-Q, Sephacryl S-300 and second Mono-Q chromatographies. 2. The enzyme preparation was electrophoretically and immunologically similar to MCP purified from the same source by a different method (Folco et al., 1988b, Archs Biochem. Biophys. 267, 599-605) but showed much lower chymotrypsin- and trypsin-like activities. 3. These activities responded to sodium dodecyl sulphate (SDS), urea and heat treatments in different ways: SDS stimulated both activities, urea stimulated the former and inhibited the latter and heating stimulated the former and did not affect the latter. 4. The stimulation of chymotrypsin-like activity by the three treatments was irreversible. 5. Exposure of MCP to SDS or urea in the absence of substrate rapidly inactivated it, whereas heat activation took place irrespective of the presence of substrate. 6. The stimulating effect of SDS on chymotrypsin-like activity was lost in the presence of urea. 7. These results suggest that the enzyme may be activated by different mechanisms.     

Multicatalytic proteinase in fish muscle

A partially active and a latent form of multicatalytic protease (MCP) were isolated from fish skeletal muscle. Both forms were inactive against protein substrates, but their activity against peptide substrates differed in one order of magnitude. The chymotrypsin-like activity of the partially active form was moderately stimulated by fatty acids and SDS, whereas its trypsin-like activity was inhibited by the same reagents. In contrast, both activities of the latent form were strongly stimulated by SDS. The chymotrypsin-like activity of the latent form was also stimulated by heating or high urea concentrations, whereas its trypsin-like activity did not change or was inhibited respectively by these treatments. These activation effects were irreversible. Pre-treatment of the latent form with SDS or urea in the absence of substrate led to its irreversible inactivation, whereas activation by pre-heating occurred in the presence or absence of substrate. These results suggest that MCP can exist in several active states with distinct properties. Studies on the distribution of MCP in fish tissues showed a much higher level of the enzyme in gonads than in any other tissue, suggesting a role of MCP in development.Abbreviations MCP multicatalytic proteinase - Suc succinyl - Bz benzoyl - Z carbobenzoxy - NMec 4-methyl-7-coumarylamide - CTAB cetyl trimethylammonium bromide     

Multicatalytic proteinase in fish muscle

Proteinase II, a high-molecular-mass proteinase previously identified in white croaker skeletal muscle, was purified to apparent homogeneity by DEAE-Sephacel, phenyl-Sepharose CL 4B, and Sephacryl S-300 chromatographies. Under denaturing conditions, the enzyme dissociated into a cluster of subunits with Mr ranging from 18,000 to 26,000 and a large subunit with a Mr 60,000. The proteinase was able to hydrolyze N-terminal-blocked 4-methyl-7-coumarylamide substrates having either an aromatic amino acid (chymotrypsin-like activity) or an arginine residue (trypsin-like activity) adjacent to the fluorogenic group. The trypsin-like activity of the enzyme was inhibited by fatty acids and sodium dodecyl sulfate, whereas the chymotrypsin-like activity was stimulated by those compounds but inhibited by nonionic and cationic detergents. Several thiol reagents inhibited both proteinase II activities. However, leupeptin and Cu2+ strongly inhibited its trypsin-like activity but only slightly affected its chymotrypsin-like activity. Dithiothreitol stimulated both activities, but at different extents and in different concentration ranges. These results suggest that the enzyme is multicatalytic, having at least two different active sites.     

3,4-dichloroisocoumarin-induced activation of the degradation of beta-casein by the bovine pituitary multicatalytic proteinase complex.

The breakdown of beta-casein (caseinolytic activity) by the bovine pituitary multicatalytic proteinase complex (MPC) is initiated by a fourth active site different from the previously described chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide, where Cbz is benzyloxycarbonyl), trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide), and peptidylglutamyl peptide bond-hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) (Yu, B., Pereira, M. E., and Wilk, S. (1991) J. Biol. Chem. 266, 17396-17400). 3,4-Dichloroisocoumarin, a serine proteinase inhibitor, stimulated the caseinolytic activity of bovine pituitary or lens MPC, 3-18-fold under conditions under which the other three catalytic activities were inactivated. Addition of hydroxylamine to the modified enzyme did not reverse the effects of the inhibitor. A form of the proteinase exhibiting only 2-4% of control chymotrypsin-like, trypsin-like, and PGP activities degraded beta-casein with no accumulation of intermediate peptides. 3,4-Dichloroisocoumarin, by reacting with the chymotrypsin-like, trypsin-like, and/or PGP-active sites, may promote a conformational change of MPC, rendering the caseinolytic active site accessible to the substrate. Once bound to the active site, beta-casein is rapidly degraded either by the caseinolytic component itself or by a cooperative interaction with catalytic centers that are not affected by the serine proteinase inhibitor. These results imply that the caseinolytic component does not belong to the class of serine proteinases. Other proteins tested were not degraded by the 3,4-dichloroisocoumarin-treated enzyme, suggesting that the conformation of beta-casein may be more adequate for degradation by the caseinolytic component.     

High-molecular-weight serine proteinase from lobster muscle that degrades myofibrillar proteins

A latent alkaline serine proteinase (ASP) has been extracted from the soluble fraction of lobster claw and abdominal muscles. The enzyme, which was irreversibly activated 30- to 40-fold by brief (2-3 min) heating at 60 degrees C, had an optimal caseinolytic activity at pH 7.75. Its molecular weight was estimated to be 740,000 by gel filtration chromatography. Serine protease inhibitors (diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, aprotinin, benzamidine, and chloromethyl ketones) suppressed ASP activity 22 to 70%. In addition, sulfhydryl-blocking reagents and hemin inhibited activity 69 to 100%; leupeptin and E-64, however, did not. Pepstatin A, ethylenediaminetetraacetate, and adenosine triphosphate were without effect. These results suggest that the lobster ASP is a serine proteinase that contains one or more sulfhydryl groups essential for catalysis. ASP was stimulated by dithiothreitol and inhibited by CaCl2 and oleic and linoleic acids. The enzyme was partially activated by low concentrations of sodium dodecyl sulfate; 0.05% produced activities 13% of that of preparations heated at 60 degrees C. Neither poly-L-lysine, urea, dimethylsulfoxide, oleic acid, linoleic acid, nor N-ethylmaleimide activated the enzyme. The ASP degraded most myofibrillar proteins, but showed a preferential hydrolysis of paramyosin, troponin-I and -C, and myosin alpha light chain.     

Activation of the 20S Proteasome of Xenopus Oocytes by SDS: Evidence for the Substrate-Induced Conformational Change Characteristic of Trypsin-Like Peptidase

The 20S proteasome of eukaryotic cells has at least three distinct peptidase activities (trypsin-like, chymotrypsin-like and peptidylglutamylpeptide (PGP) hydrolase activities). These peptidases are latent and require appropriate activators. SDS has been widely used as an activator of these peptidases, but the mechanism of its activation remains unresolved. In this study, we investigated the kinetics of the SDS-activated hydrolysis of the above three types of peptidase of the 20S proteasome purified from Xenopus oocytes. When the reaction was started by simultaneous adding both SDS and substrate, maximal rates of hydrolysis were reached after appreciable lag phases with the trypsin-type substrate [t-butyloxycarbonylLeu-Arg-Arg-4-methylcoumaryl-7-amide (Boc-LRR-MCA)], but no such lag phases were observed with the chymotrypsin-type and PGP hydrolase-type substrates [succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (Suc-LLVY-MCA), and benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide (Cbz-LLE-2NA), respectively]. Similarly, changes in the hydrolysis rate to a reduced level upon dilution of SDS occurred after an appreciable lag phase again in the trypsin-like peptidase, but not in the other types. The lag phase characteristic of the trypsin-like peptidase was dependent on the substrate concentration. Thus, the lag phase was less discernible at very low concentrations of the substrate (e.g. at concentrations in the order of 1/100 of the Km value), but became more conspicuous with the increases in the substrate concentration. This lag phase also vanished upon preincubation of the activator (SDS) for a short period of 5 sec. These results suggest that the formation of the enzyme-substrate complex in the trypsin-like reaction induces a conformational change in the enzyme which makes the SDS activator site(s) in an occluded form, reducing the rates of SDS binding and dissociation.     

Chemical modification of the bovine pituitary multicatalytic proteinase complex by N-acetylimidazole. Reversible activation of casein hydrolysis

The effect of N-acetylimidazole, a mild acetylating reagent, on the catalytic activities and subunit structure of the bovine pituitary multicatalytic proteinase complex (MPC) was studied. The trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide) and the peptidylglutamyl-peptide bond hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) of MPC were rapidly inactivated by N-acetylimidazole, whereas the chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide) was inactivated slowly. However, the hydrolysis of casein was markedly stimulated. Hydrolysis of casein by the acetylated enzyme generated a stable intermediate (21 kDa) which could be further degraded by native MPC. Treatment of acetylated MPC with hydroxylamine reversed the changes in trypsin-like and caseinolytic activities but did not restore the PGP activity. N-Acetylimidazole did not dissociate MPC but altered its migration on nondissociating gels presumably by acetylation of epsilon-amino groups of lysine residues. Hydroxylamine did not alter the gel electrophoretic appearance of the acetylated enzyme. These results indicate that acetylation of thiol or tyrosyl groups changes the trypsin-like and caseinolytic activities, and that amino group acetylation inhibits the PGP activity. Degradation of casein by MPC appears to be a sequential process with initial cleavage catalyzed by a component distinct from the chymotrypsin-like, trypsin-like, and PGP activities. The latter three components likely participate in the secondary proteolysis of the generated intermediates.     

Proteasome inactivation upon aging and on oxidation-effect of HSP 90

Increases of oxidatively modified protein in the cell have been associated with the aging process. Such an accumulation of damaged protein may be the result of increase in the rate of protein oxidation and/or decrease in the rate of degradation of oxidized protein. The multicatalytic proteinase or proteasome is known to be the major proteolytic system involved in the removal of oxidized protein. We have reported that, after isolation of the 20S proteasome from the liver of young and old male Fischer 344 rat, out of the three peptidase activities (chymotrypsin-like, trypsin-like and peptidyl-glutamyl peptide hydrolase) we assayed with fluorogenic peptides, the peptidyl-glutamyl peptide hydrolase activity was declining with age to a value approximately 50% of that observed for protease purified from young rats. The proteasome was subjected to metal catalyzed oxidation to determine the susceptibility of the different peptidase activities to oxidative inactivation. Both trypsin-like and peptidyl-glutamyl peptide hydrolase activities were found sensitive to oxidation. Treatment of the proteasome with 4-hydroxy-2-nonenal, a major lipid peroxidation product, was also found to inactivate the trypsin-like activity. However, the trypsin-like activity was protected from inactivation by metal catalyzed oxidation in proteasome preparations contaminated with HSP 90, a protein that often copurifies with the proteasome. Upon addition of HSP 90 to pure 20S active proteasome, the trypsin-like activity was protected from inactivation by metal catalyzed oxidation and from inactivation by treatment with 4-hydroxy-2-nonenal. These results suggest a possible intervention of HSP 90 in response to oxidative stress in preventing the inactivation of the proteasome by oxidative damage. Abbreviations: AAF-amc – Ala-Ala-Phe-7-amido-4-methylcoumarin; LSTR-amc – N-t-Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin; LLE-na – Leu-Leu-Glu-b-naphthylamide; HSP 90: heat shock protein 90, MCP – multicatalytic proteinase or 20S proteasome.     

Pituitary multicatalytic proteinase complex. Specificity of components and aspects of proteolytic activity

The 700-kDa multicatalytic proteinase complex from bovine pituitaries separates in polyacrylamide gel electrophoresis under dissociating and reducing conditions into 11 components with molecular masses ranging from 21 to 32 kDa. No higher molecular mass components were detected. A rabbit polyclonal antibody raised against the complex recognizes five immunoreactive components. As reported previously, the complex exhibits three distinct proteolytic activities designated as chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing activities. All three activities are rather rapidly inactivated by 3,4-dichloroisocoumarin, a general serine protease inhibitor, however, the pseudo-first-order rate constants of inactivation of the three components differ within a wide range, with the chymotrypsin-like activity being most sensitive to inhibition. The peptidylglutamyl-peptide hydrolyzing activity is greatly activated by low concentrations of sodium dodecyl sulfate and fatty acids and seems to constitute the main component responsible for degradation of protein substrates. In addition to cleaving bonds on the carboxyl side of glutamyl residues, this activity also cleaves, albeit at a slower rate, bonds on the carboxyl side of hydrophobic residues; however, the secondary specificity of this component is clearly different from the chymotrypsin-like activity. Heparin selectively activates the chymotrypsin-like activity. The complex cleaves rapidly both native and dephosphorylated beta-casein in a reaction greatly accelerated by low concentrations of sodium dodecyl sulfate. The nature of proteolytic products, and also the rate of formation of acid-soluble, ninhydrin-reactive products, is different for the phosphorylated and dephosphorylated form of beta-casein, indicating that the degree of phosphorylation influences the rate and pattern of proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of membrane-associated proteasomes in WB rat liver epithelial cells

Although proteasomes are mainly located in the cytosol, it is known that significant amounts are also associated with endoplasmic reticulum (ER) membranes where they may play a role in the degradation of specific ER membrane proteins. The present studies were undertaken to compare ER and cytosolic proteasomal activities in WB rat liver cells. N-Heptyl-beta-thioglucopyranoside (HTG) extracts of membrane or cytosol fractions were chromatographed in glycerol/ATP buffers on size-exclusion and ion-exchange columns and the elution profiles of proteasomal peptidase activity and immunoreactive components of the 20S complex, 19S complex, and PA28 were compared. Cytosol fractions showed a single peak of chymotrypsin-like peptidase activity (Cht-L), which was inhibited completely by 5 microM lactacystin (LC) or SDS (0.03%) and corresponded to 26S proteasomes based upon the presence of both 20S and 19S components. By comparison, membrane fractions contained two major peaks of Cht-L activity. The first peak shared the same properties as the peak activity observed in cytosol fractions. However, the second peak was stimulated by SDS and was LC-insensitive (5 microM) and contained trypsin-like (T-L) and peptide-glutamyl peptidase (PGPH) but no cathepsin or calcium-activated protease activities. PA28 activator protein was present in both membrane and cytosol fractions. Thus, the principal difference between cytosolic and membrane activity was that the latter fractions contained a novel membrane-associated LC-insensitive protease(s) catalyzing three of the major peptidase activities of the proteasome.     

Kinetic studies of the differential effect of detergents on the peptidase activities of the multicatalytic proteinase from rat liver

We present here a detailed study of the effect of detergents on the three peptidase activities (hydrolysis of the LLVY, ARR, and LLE peptides) of the purified multicatalytic proteinase from rat liver. At Triton X-100 and sodium dodecyl sulfate (SDS) concentrations of 0.1%, all three peptidase activities are inhibited. Lower concentrations of the two detergents (0.01%) do not affect the hydrolysis of the ARR peptide, whereas they behave differently on the hydrolysis of the LLVY and LLE peptides. Triton X-100 inhibits and SDS strongly activates LLVY peptide hydrolysis by decreasing and increasing Vmax, respectively. In the absence of detergents, the saturation curve for the LLE peptide can be analyzed as the result of two components, one showing cooperative (nH = 1.6) with higher affinity (S0.5 = 60 microM) and lower Vmax than a second, noncooperative component (Km = 320 microM). SDS (0.01%) activates LLE peptide hydrolysis by suppressing cooperativity, slightly increasing Vmax, and decreasing the half-saturation concentration (Km = 30 microM) of the enzyme. Triton X-100 (0.01%) also suppresses the cooperativity and decreases the half-saturation concentration (Km = 25 microM) for the LLE peptide; in contrast, it reduces Vmax by inhibition of the low affinity, high Vmax component observed in the absence of detergents. Based on these observations, it can be concluded that both detergents behave like allosteric activators of peptidylglutamyl-peptide hydrolyzing activity and that the multicatalytic proteinase has at least three different classes of active sites: two independent noncooperative sites that catalyze the hydrolysis of trypsin and chymotrypsin-like substrates and one class for peptidylglutamyl-peptide hydrolysis having two components: one cooperative (two or more sites) and one noncooperative.     

Peptidase activities of the 20/26S proteasome and a novel protease in human brain

Many neurodegenerative diseases are characterized by ubiquitin-positive protein aggregates or inclusion bodies. Ubiquitin-conjugated proteins are degraded by the 20/26S proteasome, and reduced proteasome peptidase activities in brain homogenates have been reported in pathologic lesions of Parkinson's and Alzheimer's diseases. However, it is unknown whether crude extracts of human brain contain other proteases having peptidase activities. We found a novel protease of molecular weight of approximately 105 kDa in normal human brain, which exhibited trypsin-like (T-L) and chymotrypsin-like (ChT-L) activities (corresponding to 52% and 21% of the total activities in crude extracts) but not peptidyl glutamyl peptide hydrolase activity. Both T-L and ChT-L activities of this protease were partially inhibited by proteasome inhibitors (MG132, lactacystin) and, in contrast to those of the proteasome, also by sodium dodecyl sulfate. A simple method to obtain a brain fraction specific to the 20/26S proteasome was developed. Our human brain data suggest that T-L and ChT-L activity levels of the proteasome reported previously may include those of the 105 kDa protease, an enzyme of as yet unknown biological significance, and that it is necessary to separate the proteasome from this protease to evaluate the actual status of the ubiquitin-proteasome system in neurodegenerative disorders.     

Effect of N-3-methyladenine on peptidase and ribonuclease activity of proteasome

Using a culture of cardiomyocytes it has been shown, that a well-known inhibitor of autophagy, N-3-methyladenine causes a 1.4 fold increase (p = 0.023) of the chymotrypsin-like activity, a 1.5 fold increase (p = 0.09) of the peptidyl-glutamyl peptide-hydrolyzing activity and 1.5 fold decrease (p = 0.07) of the trypsin-like activity of the proteasome. N-3-methyladenine in a dose-dependent manner inhibits chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of the purified 20S proteasome, but activates it trypsin-like activity. Chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of the 26S proteasome from proteasome fraction II did change in the same way, as in the case of 20S proteasome, but trypsin-like activity decreased. Using the above method of determining ribonuclease activity, we have shown, that N-3-methyladenine and clasto-lactacystin b-lactone inhibit the RNase activity of the proteasome. Specific proteasome inhibitor exhibits more powerful action, almost completely preventing RNA of actin and myosin from degradation. These data show a multitarget action of N-3-methyladenine, resulting in changes of peptidase and ribonuclease activity of the proteasome.     

Purification and characterization of a multicatalytic proteinase from Atlantic salmon (Salmo salar) muscle

A high molecular mass alkaline proteinase was purified by DEAE-Sepharose and Mono Q chromatography. The mol. wt was estimated to be about 600,000. Under denaturing conditions, the enzyme dissociated into a cluster of subunits with mol. wt ranging from 25,000 to 30,000. The isoelectric point of the enzyme was about pH 7.3. The proteinase was able to hydrolyse N-terminal-blocked 4-methyl-7-coumarylamide substrates for either trypsin- or chymotrypsin-like activity. It was also able to hydrolyse haemoglobin and myosin at temperatures of about 60°C. The activities responded to pH and some chemicals in different ways. The trypsin-like activity was clearly inhibited by several serine protease inhibitors. These results suggest that the enzyme is multicatalytic, having at least two different active sites.     

Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays

Proteolytic activities in soluble protein extracts from Mamestra brassicae (cabbage moth) larval midgut were analysed using specific peptide substrates and proteinase inhibitors. Serine proteinases were the major activities detected, with chymotrypsin-like and trypsin-like activities being responsible for approximately 62% and 19% of the total proteolytic activity towards a non-specific protein substrate. Only small amounts of elastase-like activities could be detected. The serine proteinases were active across the pH range 7-12.5, with both trypsin-like and chymotrypsin-like activities maximal at pH 11.5. The digestive proteinases were stable to the alkaline environment of the lepidopteran gut over the timescale of passage of food through the gut, with 50% of trypsin and 40% of chymotrypsin activity remaining after 6h at pH 12, 37 degrees C. Soybean Kunitz trypsin inhibitor (SKTI) ingestion by the larvae had a growth-inhibitory effect, and induced inhibitor-insensitive trypsin-like activity. Qualitative and quantitative changes in proteinase activity bands after gel electrophoresis of gut extracts were evident in SKTI-fed larvae when compared with controls, with increases in levels of most bands, appearance of new bands, and a decrease in the major proteinase band present in extracts from control insects.     

Inhibition of the chymotrypsin-like activity of the pituitary multicatalytic proteinase complex.

The multicatalytic proteinase complex (MPC), also referred to as proteasome, is a large molecular mass intracellular particle (approximately 700 kDa), which exhibits three distinct proteolytic activities designated as chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing (PGPH), all sensitive to inhibition by 3,4-dichloroisocoumarin (DCI). The presence of a component resistant to inhibition by DCI with an apparent preference toward bonds on the carboxyl side of branched-chain amino acids has also been recently established. Peptide aldehydes and peptide alpha-keto esters containing a hydrophobic residue in the P1 position have been tested as potential inhibitors of the chymotrypsin-like activity. Three peptide aldehydes (benzyloxycarbonyl)-Leu-Leu-phenylalaninal (Z-LLF-CHO), N-acetyl-Leu-Leu-norleucinal (Ac-LLnL-CHO), and N-acetyl-Leu-Leu-methioninal (Ac-LLM-CHO) were found to be slow-binding reversible inhibitors with Ki values of 0.46, 5.7, and 33 microM, respectively. The simplest kinetic model for inhibition is consistent with a mechanism involving a slow and reversible association of the enzyme with the inhibitor to form a EI complex. The aldehyde inhibitors also inhibited the trypsin-like and PGPH activities of the complex albeit with much higher Ki values than those for chymotrypsin-like activity. Z-LLF-CHO, the most selective of the three aldehydes, did not inhibit the PGPH activity at concentrations of up to 200 microM and inhibited the trypsin-like activity with a Ki approximately 2 orders of magnitude higher than that for the chymotrypsin-like activity. The activity of the DCI-resistant component was not affected by Z-LLF-CHO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of midgut proteinases from Bacillus thuringiensis-susceptible and -resistant Heliothis virescens (Lepidoptera: Noctuidae)

Insects with altered proteinases can avoid intoxication by Bacillus thuringiensis (Bt) toxins. Therefore, proteinase activities from gut extracts of Bt-susceptible (YDK) and -resistant (YHD2-B, CXC and KCBhyb) Heliothis virescens strains were compared. The overall pH of gut extracts from YDK and CXC were statistically similar (9.56 and 9.62, respectively), while the pH of extracts from KCBhyb and YHD2-B were significantly more alkaline (9.81 and 10.0, respectively). Gut extracts from YHD2-B and CXC larvae processed Cry1Ac and Cry2Aa protoxin slower than extracts from YDK larvae, suggesting that differences in proteolysis contribute to resistance in these strains. Casein zymogram analysis of gut extracts revealed both qualitative and quantitative differences in caseinolytic activities among all strains, but the overall caseinolytic activity of YHD2-B gut extract was lower. Kinetic microplate assays with a trypsin substrate (l-BApNA) demonstrated that proteinases in YDK gut extract had increased alkaline pH optima compared to resistant strains YHD2-B, CXC and KCBhyb. Gut extracts from YHD2-B had reduced trypsin-like activity, and activity blots indicated that YHD2-B had lost a trypsin-like proteinase activity. In assays with a chymotrypsin substrate (SAAPFpNA), enzymes from all Bt-resistant strains had increased pH optima, especially those from KCBhyb. Activity blots indicated that CXC had lost a chymotrypsin-like proteinase activity. Because serine proteinases are a critical component of Bt toxin mode of action, these differences may contribute to decreased toxicity in the Bt-resistant strains.     

Use of serine-protease inhibitors as probes for the different proteolytic activities of the rat liver multicatalytic proteinase complex.

The multicatalytic proteinase (MCP) complex catalyses cleavage of bonds on the carboxy-group side of basic, hydrophobic or acidic amino acid residues. Originally, it was proposed that the complex contained three distinct types of catalytic component. MCP from rat liver has been assayed for so-called trypsin-like activity with Boc-Leu-Ser-Thr-Arg-NH-Mec (Mec, 4-methylcoumarin; Boc, t-butoxycarbonyl), for chymotrypsin-like activity with Ala-Ala-Phe-NH-Mec and Suc-Leu-Leu-Val-Tyr-NH-MEc (Suc, succinyl), and peptidyl-glutamylpeptide hydrolase activity with Cbz-Leu-Leu-Glu-Nap (Nap, naphthylamide; Cbz, benzyloxycarbonyl). Results of these studies suggest that as many as five distinct components can be distinguished, one for the trypsin-like activity and two for each of the others. The activities were tested with a variety of serine-protease inhibitors, and other novel effectors have also been identified. The two most effective inhibitors were 4-(2-amino-ethyl)benzenesulphonyl fluoride, which selectivity inactivates the trypsin-like activity, and 3,4-dichloroisocoumarin which inhibits chymotrypsin-like activity and the second, cooperative component [Djaballah, H. & Rivett, A. J. (1992) Biochemistry 31, 4133-4141] of peptidylglutamylpeptide hydrolase activity. The three activities inhibited by 3,4-dichloroisocoumarin can easily be distinguished by the effects of chymostatin analogues, diisopropylfluorophosphate, guanidine/HCl and casein. The results support the view that the enzyme is a novel type of serine protease and suggest that it may contain at least five distinct catalytic components. Marked differences in the reactivities of the different catalytic sites with different reagents can be used to distinguish between them.     

Sodium dodecyl sulfate-induced conformational and enzymatic changes of multicatalytic proteinase

Chymotrypsin-like activity of the multicatalytic proteinase (MCP) purified from eggs of the ascidian Halocynthia roretzi was activated by the addition of SDS. Complete activation was achieved simultaneously at the time of SDS addition, and this activity decreased as a function of time. Autonomous fluorescence of MCP also increased rapidly at the time of SDS addition and then decreased at a rate that depended on the SDS concentration. The decrease of autonomous fluorescence induced by SDS preceded that of the activity. These results suggest that a rapid conformational change of MCP induced by SDS results in the enhancement of chymotrypsin-like activity, followed by the decrease of this activity because of the lability of the activated conformation.