Nobuhiko Katunuma

Inhibitory Effect of di- and Tripeptidyl Aldehydes on Calpains and Cathepsins

Eight different di- and tripeptidyl aldehyde derivatives, each having at its C-terminus an aldehyde analog of L-norleucine, L-methionine, or L-phenylalanine with a preceding L-leucine residue, were synthesized and tested for their inhibitory effects on several serine and cysteine endopeptidases. These compounds showed almost no inhibition of trypsin, and only weak inhibition of alpha-chymotrypsin and cathepsin H, while they exhibited marked inhibition of cathepsin B less than calpain II congruent to calpain I less than cathepsin L, being stronger in this order. The mode of inhibition of these cysteine proteinases was competitive for the peptide substrate used and inhibitor constants (Ki) were calculated from the Dixon plot. The best inhibitors found were: 4-phenyl-butyryl-Leu-Met-H for calpain I (Ki, 36 nM) and calpain II (Ki, 50 nM); acetyl-Leu-Leu-nLeu-H for cathepsin L (Ki, 0.5 nM); acetyl-Leu-Leu-Met-H for cathepsin B (Ki, 100 nM).

Involvement of tryptase-related cellular protease(s) in human immunodeficiency virus type 1 infection.

Trypstatin, a new cellular Kunitz‐type protease inhibitor purified from rat mast cells, inhibited syncytium formation in human immunodeficiency virus type 1 (HIV‐1)‐infected CCRF‐CEM and uninfected Molt‐4 clone 8 at a concentration of 1 μM. Anti‐rat tongue mast cell tryptase antibodies reacted with Molt‐4 clone 8 cells, as determined by Western blot and by immunofluorescence. In addition, the antibody inhibited syncytium formation. These findings along with homologous sequences with trypstatin and a neutralizing epitope of gp120 of HIV‐1 suggest that a tryptase‐like cellular enzyme(s) is involved in HIV‐1 infection.

Structures and Functions of Lysosomal Thiol Proteinases and Their Endogenous Inhibitor

Publisher Summary This chapter discusses the structures and functions of lysosomal thiol proteinases and their endogenous inhibitor. There is a very high concentration of cathepsin L in liver, which is similar to that of cathepsin B. When lysosomes are broken during homogenization of tissues, the thiol proteinase inhibitor binds to proteinases immediately. Various proteinase inhibitors of microbial origin are useful in elucidating the importance of lysosomal thiol proteinases in protein degradation; however, these inhibitors do not distinguish between cathepsin B, cathepsin L, and other thiol proteinases. Thus, specific substrates for use in the assay of individual thiol proteinases and specific inhibitors of respective proteinases must be developed. Addition of proteinase inhibitors to cells or their injection in vivo inhibits lysosomal proteinase activities, causes formation of autophagic vacuoles, and induces synthesis of hemoglobin-hydrolyzing thiol proteinase. Various blood proteins—such as asialoproteins—are degraded in liver via processes involving receptor-mediated binding, pinocytosis, fusion with primary lysosomes, and subsequent hydrolysis in secondary lysosomes.

Studies on New Intracellular Proteases in Various Organs of Rat

1. Specific proteases which inactivate the apo-proteins of many pyridoxal enzymes were found in skeletal muscle, liver and small intestine of rats. The protease from these three organs were purified and their properties were compared. 2. The purified proteases from liver and skeletal muscle appeared homogeneous on acrylamide gel electrophoresis. Two different proteases were separated from small intestine. A homogeneous, crystalline enzyme was obtained from the muscle layer while enzyme from the mucosa was partially purified. 3. They showed substrate specificity for pyridoxal enzymes. Their pH optima were in an alkaline region. They showed activity with the substrate of chymotrypsin, N-acetyl-L-tyrosine ethyl ester, but not with that of trypsin, p-toluenesulfonyl-L-arginine ethyl ester. They were inhibited by pyridoxal phosphate or pyridoxamine phosphate and seryl residues were involved in their active center. 4. The four enzymes differed in the following characters: (a) molecular weights; (b) patterns of elution from a CM-Sephadex column; (c) rates of inactivation of substrate enzymes; (d) rates of cleavage of N-acetyl-L-tyrosine ethyl ester; (e) reactivities with antiserum against the enzyme from the muscle layer of small intestine; (f) specific activities. 5. The amino acid composition and effect of chemical modifications of the crystalline enzyme from the muscle layer of small intestine were examined to elucidate its active sites and mode of action. Serine and histidine residues were found to be essential for protease activity. A tyrosine residue was also necessary for activity. Modifications of its sulfhydryl group, amino residues and carboxyl group had no effect on its activity.

Regulation of the urea cycle and TCA cycle by ammonia

Abstract This paper presents the biochemical and enzymatic fundamentals of ammonia toxicity and automatic regulation of urea formation from ammonia. 1. 1.It is considered that the oxidation of isocitrate to 2-oxoglutarate is more sensitive to ammonia than the other dehydrogenases in the TCA cycle. 2. 2.Ammonia resulted in a decrease of intramitochondrial pyridine nucleotide levels, especially in their reduced forms. 3. 3.A new metabolic pathway of NADH2 and NADPH2 to nicotinamide including reduced pyridine nucleotide pyrophosphatase and nicotinamide mononucleotide oxidase was first proved to occur in rat liver mitochondria. 4. 4.As the result of specific ammonium activation of NMNH2 and NADH2 oxidase reaction, the new pathway described was accelerated by addition of ammonia. 5. 5.The following two coupling reactions of mitochondrial aspartate transaminase and each half of the TCA cycle were found to occur in rat liver mitochondria: Glu + 3 2 O 2 → Asp+CO 2 Asp+Acetyl CoA + 1 2 O 2 → Glu +CO 2 +H 2 O+CoA The former reaction supplies aspartate to urea cycle without liberation of ammonia and the latter reaction was found to be more sensitive to ammonium inhibition than the former one. 6. 6.Supernatant aspartate and alanine transaminase isozymes were markedly induced by the administration of high protein diet or hydrocortisone and/or under diabetic conditions, whereas no effect was observed with mitochondrial transaminase isozymes. 7. 7.Ornithine-keto acid transaminase activity was found to be an important regulator of liver ornithine contents and further urea cycle activities. The ornithine consumption was lowered when the TCA cycle was inhibited by ammonia or when ornithine transaminase activity was inhibited by branched chain amino acids in isolated mitochondria. 8. 8.Mice injected intraperitoneally with ammonia or branched chain amino acids resulted in 2 or 3 times increase of liver ornithine content and a marked increase of urea synthesis.

Chymotrypsin- and trypsin-type serine proteases in rat mast cells: Properties and functions

Two of the major enzymes present in and released from rat mast cells are chymotrypsin-type serine protease (chymase) and trypsin-type serine protease (tryptase), and these have been postulated to be important in the inflammatory reactions. There have been no clear data regarding the trypsin-type protease in rat mast cells. Tryptase was recently purified from rat peritoneal mast cells with an associated protein (trypstatin) that inhibited the protease activity above pH 7.5. Chymase was also purified from rat peritoneal cells by employing a one-step method involving hydrophobic chromatography on octyl-Sepharose 4B or arginine-Sepharose 4B. The properties of chymase and tryptase were described in relation to substrate specificity and their relative sensitivity to inhibitors. It was found that proteolytic activities of these enzymes were modulated by naturally occurring substances, such as phosphoglycerides, long-chain fatty acids, and trypstatin. There is as yet little evidence for the physiological roles of these enzymes in the inflammatory reaction. It has been found that the specific, low-molecular-weight inhibitor of chymase, chymostatin, and that of tryptase, leupeptin, inhibit histamine release induced by addition of anti-rat IgE to mast cells. However, the inhibitors with molecular weights of more than 6000 were found to have no effect in this process. The data suggest that chymase and tryptase in mast cell granules play a crucial or significant role in the process of degranulation.

Molecular cloning and sequencing of cDNA for rat cathepsin L

A near full‐length cDNA for rat cathepsin L was isolated. The deduced protein comprises 334 amino acid residues (M r 37 685) containing a typical signal sequence (N‐terminal 17 residues), pro‐peptide (96 residues), and the sequence for mature cathepsin L (221 residues). Rat cathepsin L shows 94% amino acid identity with mouse cysteine proteinase. Amino acid sequence homologies of rat cathepsin L with rat cathepsins H and B are 45 and 25%, respectively. These facts indicate that mouse cysteine proteinase is probably mouse cathepsin L and that cathepsin L is more closely related to cathepsin H than cathepsin B.

A new function of kininogens as thiol-proteinase inhibitors: inhibition of papain and cathepsins B, H and L by bovine, rat and human plasma kininogens.

Kininogen Thiol‐proteinase inhibitor Papain Cathepsin B Cathepsin H Cathepsin L

Structural basis of actin recognition and arginine ADP-ribosylation by Clostridium perfringens iota-toxin.

The ADP-ribosylating toxins (ADPRTs) produced by pathogenic bacteria modify intracellular protein and affect eukaryotic cell function. Actin-specific ADPRTs (including Clostridium perfringens ι-toxin and Clostridium botulinum C2 toxin) ADP-ribosylate G-actin at Arg-177, leading to disorganization of the cytoskeleton and cell death. Although the structures of many actin-specific ADPRTs are available, the mechanisms underlying actin recognition and selective ADP-ribosylation of Arg-177 remain unknown. Here we report the crystal structure of actin-Ia in complex with the nonhydrolyzable NAD analog βTAD at 2.8 Å resolution. The structure indicates that Ia recognizes actin via five loops around NAD: loop I (Tyr-60–Tyr-62 in the N domain), loop II (active-site loop), loop III, loop IV (PN loop), and loop V (ADP-ribosylating turn–turn loop). We used site-directed mutagenesis to confirm that loop I on the N domain and loop II are essential for the ADP-ribosyltransferase activity. Furthermore, we revealed that Glu-378 on the EXE loop is in close proximity to Arg-177 in actin, and we proposed that the ADP-ribosylation of Arg-177 proceeds by an SN1 reaction via first an oxocarbenium ion intermediate and second a cationic intermediate by alleviating the strained conformation of the first oxocarbenium ion. Our results suggest a common reaction mechanism for ADPRTs. Moreover, the structure might be of use in rational drug design to block toxin-substrate recognition.

Biosyntheses and processing of lysosomal cysteine proteinases in rat macrophages

The intracellular processing and release of three lysosomal cysteine proteinases, cathepsin B, H and L, by rat peritoneal macrophages were investigated by pulse‐chase experiments. Newly synthesized procathepsins B (39 kDa), H(41 kDa) and L (39 kDa) after 15 min labeling were processed to the mature, single‐chain enzymes within 1 h. The single‐chain forms of cathepsin B, H and L were further processed to two‐chain forms at different rates: conversion of cathepsin L to the two‐chain form was rapid, whereas the conversions cathepsin B and H took at least 6 h. Macrophages released 30% of the procathepsins B and L, and 10% of the procathepsin H.