The kinetics of hydrolysis of some synthetic substrates containing neutral hydrophilic groups by pig pepsin and chicken liver cathepsin D.

1. Several peptides containing either of the sequences -Phe(NO2)-Trp- and -Phe(NO2)-Phe- and an uncharged hydrophilic group were synthesized, and the steady-state kinetics of their hydrolysis by pig pepsin (EC 3.4.23.1) and chicken liver cathepsin D (EC 3.4.23.5) were determined. Despite the presence of a hydrophilic group to increase substrate solubility, it was not possible to achieve the condition [S]0 much greater than Km, and, in some cases, only values of kcat./Km could be determined by measuring the first-order rate constant when [S]0 much less than Km. 2. Occupancy of the P2 and P3 sites considerably enhanced the specificity constant, and alanine was more effective than glycine at site P2. 3. The specificity constants for the hydrolysis by pepsin of those substrates in the present series that contain an amino acid residue at site P3 are considerably lower than for comparable substrates containing a cationic group. This difference does not apply to cathepsin D. 4. Hydrolyses with cathepsin D commonly exhibited a lag phase, and a possible explanation for this is given.     

The effect of beef spleen cathepsin D on pig heart lactate dehydrogenase.

Pig heart lactate dehydrogenase (EC 1.1.1.27; H₄ isoenzyme) has been subjected to the action of beef spleen cathepsin D (EC 3.4.23.5) at pH 3.9. No proteolytic effect on the tetrameric form of the dehydrogenase could be detected. On the other hand, the acid proteinase appears to act on the dissociated monomers in such a manner that they are unable to reassociate into functional tetramers. This finding could be correlated with proteolytic modification of the monomers. The extent of proteolysis depends on the temperature and duration of the incubation.     

Interaction of human cathepsin D with the inhibitor pepstatin.

1. Because of the proposed role of cathepsin D in a variety of biological and pathological processes, the characteristics of inhibition by the potentially useful agent, pepstatin, were determined. 2. The beta and gamma forms of human cathepsin D, separated by isoelectric focusing, have identical specific extinction coefficients and specific activity in the degradation of haemoglobin. 3. Cathepsin D showed tight binding of 1 mol of pepstatin per 43000 g of protein, indicating that titration with the inhibitor represents a useful method for determination of absolute concentrations of the enzyme. 4. The titration curves were used to determine apparent dissociation constants (KD) for the binding of pepstatin and pepstatin methyl ester at pH3.5; values of approx. 5 X 10(-10)M were obtained. 5. Pepstatinyl-[3H]glycine was synthesized and shown to have a KD similar to that of pepstatin. Gel-chromatographic experiments showed that the binding of pepstatin and its derivatives is strongly pH-dependent. 6. The effect of pH on the KD for pepstatinyl-glycine was determined by equilibrium dialysis. As the pH was raised from 5.0 to 6.4, KD rose from 5 X 10(-10)M to 2 X 10(-6)M. 7. The catalytic activity of cathepsin D declines essentially to zero on going from pH5.0 to pH7.0, and we suggest that the binding site for substrate and pepstatin is abolished by a conformational change in the enzyme molecule. 8. The data indicate that, in biological experiments near neutral pH, large molar excesses of pepstatin over cathepsin D will be required for efficient inhibition.     

Effects of secondary binding by activator and inhibitor peptides on covalent intermediates of pig pepsin.

1. Cerebral-cortex synaptosomes were shown to synthesize (14C)acetylcholine after incubation with (14C)choline, and 25mM-KCl released (14C)acetylcholine (but not (14C)choline) into the medium by a Ca2+-dependent and Mg2+-sensitive process. 2. The K+-stimulated release of (14C)acetylcholine was inhibited by more than 80% after preincubation of the synaptosomes with 10(5) mouse lethal doses of botulinum toxin/ml. (14C)choline uptake, (14C)acetylcholine synthesis, intrasynaptosomal K+ and occluded lactate dehydrogenase were unaffected by the toxin. It also failed to prevent the K+-stimulated release of (3H)noradrenaline and (14C)glycine from synaptosomes. 3. Fractionation of hypo-osmotically shocked synaptosomes revealed that more than 75% of the radioactive acetylcholine was in the cytoplasmic compartment, although the vesicle pellet contained more total acetylcholine than the cytoplasmic pool. Consequently the specific radioactivity of acetylcholine in the cytoplasmic pool was almost 5 times that of the vesicles. This distribution was unaffected by preincubation with botulinum toxin. It is concluded that the toxin acts directly on the release of acetylcholine, rather than influencing its storage. 4. After K+-stimulation, toxin-inhibited synaptosomes contained increased amounts of total acetylcholine, which suggests that its rate of synthesis is controlled by depolarization rather than release.     

Improved pepsin inhibitor derived from activation peptide 1-16 of porcine pepsinogen.

The peptide Leu-Val-Lys-Val-Pro-Leu-Val-Arg-Lys-Lys-Ser-Leu-Arg-Gln-Asn-Leu, a known pepsin inhibitor, is derived from the first 16 amino acids of porcine pepsinogen. It was prepared from the activation mixture and was modified by guanidination of its three lysine residues to form homoarginine residues. The modified peptide is a better pepsin inhibitor than the native peptide; for 50% inhibition of the milk clotting action of pepsin at pH 5.3, the molar ratio of peptide to pepsin required is 9 for the native inhibitor and only 2 for the guanidinated inhibitor. The dissociation constants (k1) of the inhibitor-pepsin complexes are 7 X 10(-8) and 1.4 X 10(-8) M for the native and guanidinated peptides, respectively. The guanidinated peptide is more resistant to digestion by pepsin at pH 3.5. The native and modified peptides partially protect pepsin from inactivation at pH 7. Stepwise removal of the amino-terminal Leu-Val-Har residues from the guanidinated inhibitor by Edman degradation decreases the pepsin-inhibiting activity only slightly at the first step, but markedly at the second and third steps. Thus, all of the amino-terminal sequence except the leucine residue is necessary for full activity.     

Biotinylated fluorescent peptide substrates for the sensitive and specific determination of cathepsin D activity.

Cathepsin D (CatD) is a member of the mammalian aspartic protease family and is involved in cellular protein degradation and in several pathological processes. A sensitive and specific assay for the determination of CatD activity in biological samples was developed. The peptide amide substrates Amca-EDKPILF downward arrowFRLGK(biotin)-CONH2 (I), Amca-EEKPIC(Acm)F downward arrowFRLGK(biotin)-CONH2 (II) and Amca-EEKPISF downward arrowFRLGK(biotin)-CONH2 (III) contain a CatD cleavage site (F downward arrowF) flanked by a N-terminal Amca-fluorophore (7-amino-4-methylcoumarin-3-acetic acid) and a C-terminal biotin moiety. Substrates II and III proved to be specific substrates containing only one cleavage site for CatD. After cleavage of the Phe-Phe bond by CatD all biotin conjugated peptides were removed with streptavidin-coated magnetic beads. The remaining fluorescent peptides in solution represent the amount of digested substrate. The versatility of this CatD digest and pull down assay was demonstrated by measuring the activity of CatD in different subcellular fractions of human EBV-transformed B cells and human monocytes. The described method based on the designed CatD substrates represents a valuable tool for routine assays.     

A novel function for the cathepsin D inhibitor in tomato

Proteinaceous aspartic proteinase inhibitors are rare in nature and are described in only a few plant species. One of them corresponds to a family of cathepsin D inhibitors (CDIs) described in potato (Solanum tuberosum), involving up to 15 isoforms with a high sequence similarity. In this work, we describe a tomato (Solanum lycopersicum) wound-inducible protein called jasmonic-induced protein 21 (JIP21). Sequence analysis of its cDNA predicted a putative function as a CDI. The JIP21 gene, whose protein has been demonstrated to be glycosylated, is constitutively expressed in flowers, stem, and fruit, and is inducible to high levels by wounding and methyl jasmonate in leaves of tomato plants. The genomic sequence of JIP21 shows that the gene is intronless and reveals the presence of both a methyl jasmonate box (TGACT) and a G-box (CACGT) in the promoter. In contrast to the presumed role of JIP21 based on sequence analysis, a detailed biochemical characterization of the purified protein uncovers a different function as a strong chymotrypsin inhibitor, which questions the previously predicted inhibitory activity against aspartic proteinases. Moreover, Egyptian cotton worm (Spodoptera littoralis) larvae fed on transgenic tomato plants overexpressing JIP21 present an increase in mortality and a delay in growth when compared with larvae fed on wild-type plants. These larvae belong to the Lepidoptera family whose main digestive enzymes have been described as being Ser proteases. All these results support the notion that tomato JIP21 should be considered as a chymotrypsin inhibitor belonging to the Ser proteinase inhibitors rather than a CDI. Therefore, we propose to name this protein tomato chymotrypsin inhibitor 21 (TCI21).     

Recombinant prosegment peptide acts as a folding catalyst and inhibitor of native pepsin

Porcine pepsin A, a gastric aspartic peptidase, is initially produced as the zymogen pepsinogen that contains an N-terminal, 44 residue prosegment (PS) domain. In the absence of the PS, native pepsin (Np) is irreversibly denatured and when placed under refolding conditions, folds to a thermodynamically stable denatured state. This denatured, refolded pepsin (Rp) state can be converted to Np by the exogenous addition of the PS, which catalyzes the folding of Rp to Np. In order to thoroughly study the mechanism by which the PS catalyzes pepsin folding, a soluble protein expression system was developed to produce recombinant PS peptide in a highly pure form. Using this system, the wild-type and three-mutant PS forms, in which single residue substitutions were made (V4A, R8A and K36A), were expressed and purified. These PS peptides were characterized for their ability to inhibit Np enzymatic activity and to catalyze the folding of Rp to Np. The V4A, R8A and K36A mutant PS peptides were found to have nanomolar inhibition constants, K_i, of 82.4, 58.3 and 95.6 nM, respectively, approximately a two-fold increase from that of the wild-type PS (36.2 nM). All three-mutant PS peptides were found to catalyze Np folding with a rate constant of 0.06 min^? 1, five-fold lower than that of the wild-type. The observation that the mutant PS peptides retained their inhibition and folding-catalyst functionality suggests a high level of resilience to mutations of the pepsin PS.     

Two-step affinity-chromatographic purification of cathepsin D from pig myometrium with high yield.

Cathepsin D was purified by two-step affinity chromatography on concanavalin A-- and pepstatin--Sepharose. The main purification was achieved by washing the enzyme bound to the pepstatin--Sepharose column with buffered 6 M-urea. This step separated cathepsin D from all low- and high-molecular-weight impurities. Although the 1700-fold purified acid proteinase was homogeneous on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it still showed microheterogeneity.     

Inactivation studies of the leucocyte inhibitor of urokinase by cathepsin D

Leucocytes contain an urokinase inhibitor, that can be inactivated by cathepsin D. In this work biochemical and immunological studies on the inactivation of this inhibitor by cathepsin D are presented. Examinations by polyacrylamide gel electrophoresis and SDS electrophoresis indicate that cathepsin D inactivates urokinase inhibitor by hydrolysis of the inhibitor molecule and that the degradation needed for total inactivation is different from that for formation of the precipitin line with antibodies. The conversion of active inhibitor into inactive protein proceeds catalytically.     

Cloning and characterization of a cathepsin D inhibitor gene from Solanum tuberosum L.

A DNA clone encoding a cathepsin D inhibitor CathInh was isolated from a potato genomic library using a CathInh cDNA as hybridization probe. The amino acid sequence of the coding region is nearly identical with a CathInh cDNA and CathInh proteins previously isolated from a tuber-specific cDNA library and from tubers, respectively. Analysis of GUS activity resulting from expression of chimeric CathInh promoter-GUS genes in transgenic potato plants revealed expression exclusively confined to potato tubers. No GUS activity could be detected in any other organ of the transgenic plants either constitutively or after wounding or treatment with abscisic and jasmonic acid (JA). Interestingly, part of the promoter region of the CathInh gene, essential for GUS activity in tubers, shows striking similarity to promoter regions of tuber-specific class I patatin genes.     

Calcitonin gene-related peptide: an inhibitor of guinea pig gallbladder contraction.

Calcitonin gene-related peptide (CGRP) relaxes vascular and intestinal smooth muscle. This study localized CGRP in the guinea pig gallbladder, examined the effects of CGRP on KCl- and ACh-induced contraction, and determined CGRPs site of action in the gallbladder. The gallbladder of male Hartley guinea pigs was used in in vitro tension studies, radioimmunoassay, or immunocytochemical studies. Radioimmunoassay showed that 8.0 +/- 0.5 pmol/g of immunoreactive CGRP was present. Immunocytochemistry demonstrated that immunoreactive-CGRP nerve fibers occurred around blood vessels, in gallbladder smooth muscle layers, and were associated with ganglia. No immunoreactive cell bodies were observed, even after colchicine treatment. The in vitro tension studies showed that CGRP inhibits either KCl- or acetylcholine-stimulated contraction. CGRP may in part act directly on the gallbladder smooth muscle to inhibit contraction.     

The amino acid sequence of a novel inhibitor of cathepsin D from potato

The amino acid sequence of a cathepsin D inhibitor isolated from potato is described. It was determined by analysis of peptides generated by use of the glycine-specific proteinase PPIV. The order of the peptides was established by examination of tryptic peptides derived from the two cyanogen bromide peptides. The inhibitor comprises 187 amino acid residues, and has a calculated Mr of 20,450.