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Derivatives of the thiirancarboxylic acid building-block containing a peptide bond were synthesised and screened against the model cysteine protease papain. The most active of the series showed a second-order rate constant of inactivation comparable to that of the parent compound. The insertion of a peptide moiety seems to compensate the lack of a free carboxylate interacting with the histidinium ion at the enzyme's active site.
The capability of the cysteine proteases ficin, papain and clostripain to form peptide bonds in frozen aqueous solutions was investigated. Freezing the reaction mixture resulted in increased peptide yields in kinetically controlled coupling of Bz-Arg-OEt with various amino acid amides and dipeptides. Under these conditions, peptide yields increased up to 70% depending on the enzyme and the amino component used. Enzyme-catalysed peptide syntheses were carried out under optimized reaction conditions (temperature, amino component concentration and pH before freezing) using the condensation of Bz-Arg-OEt and H-Leu-NH2 as a model reaction.
Electrostatic forces are involved in a wide variety of molecular interactions that are of biological interest, including, among others, DNA-Protein interactions, protein folding, and the interactions between enzymes and their substrates and inhibitors. In this work, the interaction between papain and an inhibitor, leupeptin, is analyzed from the point of view of their electrostatic interaction. The computations enable one to suggest that negatively charged amino acids located in the region of the active site are responsible for creating an environment that enables efficient binding of the inhibitor. This binding occurs despite the fact that the net global charge of both molecules is positive; an explanation for this apparent contradiction is proposed.
The influence of caldesmon on papain digestion of chicken gizzard monomeric myosin in folded (10S) conformation depends on its phosphorylation. Caldesmon exposes the head/rod junction of myosin in phosphorylated form to proteolytic attack (particularly in the presence of Ca2+) and slightly screens it in unphosphorylated form. In both folded forms RLCs are protected by caldesmon, more in unphosphorylated than in phosphorylated myosin. The results indicate that the conformations of folded unphosphorylated and phosphorylated myosin are distinct and suggest that caldesmon destabilizes the regulatory domain in folded conformation of phosphorylated myosin.
Papain has been shown, for the first time, to exhibit hydantoinase activity. It hydrolyzes the 5-mono and 5,5'-disubstituted hydantoins with linear and cyclic substituents, with a higher activity for the latter, to the corresponding N-carbamoyl amino acids, which on chemical hydrolysis yield the corresponding amino acids. The up-scaling of this simple procedure could be a major break-through for amino acid synthesis in chemical and pharmaceutical industries.
Nitric oxide (NO) is a pluripotent regulatory molecule showing, among others, an antiparasitic activity. Moreover, NO inhibits cysteine proteinase action by nitrosylating the Cys catalytic residue. In the present study, the inhibitory effect of the substrate N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methyl coumarin) and of NO on the catalytic activity of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi (the hemoflagellate protozoan parasite which causes the American trypanosomiasis), is reported. In particular, NO-donors S-nitroso-glutathione (GSNO), (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), 3-morpholinosydnonimine (SIN-1), S-nitroso-acetyl-penicillamine (SNAP), and sodium nitroprusside (SNP) dose-dependently inhibited cruzipain, this effect being likely attributable to the S-nitrosylation of the Cys25 catalytic residue. These results were analyzed in parallel with those concerning the inhibitory effect of the substrate and of NO on the catalytic activity of falcipain, the cruzipain-homologous cysteine proteinase from Plasmodium falciparum. The modulation of the cruzipain and falcipain activity by NO may be relevant in developing new strategies against T. cruzi and P. falciparum in human host. As a whole, the NO-mediated S-nitrosylation of pathogenic viral, bacterial, fungal, and parasitic cysteine proteinases may represent a general mechanism of antimicrobial and antiparasitic host defences.
The sequence of 8734 nucleotides (nt) from the 5'-end of the beet yellows closterovirus (BYV) RNA was determined to complete the 15,480-nt sequence of the virus genome. The 5'-terminal two-thirds of the sequence are occupied by two overlapping open reading frames (ORFs) 1a and 1b, encoding products with calculated M(r) of 295K and 48K, respectively. The RNA sequence surrounding the stop codon in ORF 1a shows structural elements typical of ribosomal frameshifting signals in a number of animal and plant viruses. It is predicted that the ORF 1b product is expressed via a +1 ribosomal frameshifting as the 348K ORF 1a/1b fusion protein. This putative protein contains the array of methyltransferase, RNA helicase, and RNA-dependent RNA polymerase domains that is conserved in the Sindbis-like supergroup of positive-strand RNA viruses. The 348K protein of BYV is longer than the putative replicases of the most closely related viruses (tobra- and tobamoviruses) by about 1300 amino acids distributed between two unique regions, one at the N-terminus, and the other in the central portion. The N-terminal domain showed sequence similarity to the helper component papain-like protease of potyviruses. By using in vitro translation of the T7 transcripts encoding the N-terminal 92K peptide of the BYV ORF 1a product, we found that the N-terminal fragment of 588 amino acids is released from the translation product by cleavage at the Gly-Gly dipeptide. Site-directed mutagenesis of either of the predicted catalytic residues Cys-509 and His-569 or of the Gly-588 at the cleavage site completely abolished the cleavage. The central unique region of the 348K protein contains a domain distantly resembling the aspartic protease of HIV and other lentiviruses. As shown previously, the 3'-terminal portion of the BYV genome encompasses seven more ORFs, one of which codes for a protein related to the HSP70 cell heat shock proteins, whereas two others encode the capsid protein and its diverged copy. Thus, despite the apparent evolutionary relationship with Sindbis-like viruses, BYV comprises a collection of genomic modules absorbed from different sources and has a unique expression strategy.
Using quasielastic light scattering we have previously shown an increase in high frequency internal motion of isolated thick filament upon activation. This we have attributed to cross-bridge motion. Here we show that after cleavage of the S1 moiety of myosin from isolated filaments with papain, calcium ions no longer activate the isolated filaments to produce high-frequency motions.
The effect of LC1 on cardiac myosin structure and activity was investigated using as a model S1 prepared by papain digestion in the presence of Mg2+. The resulting S1 contained LC2 but a part of the N-terminal region of LC1 was cleaved. Sequencing the N-terminal part of the band migrating below LC1 on SDS gels revealed it to consist of alternating alanyl and prolyl residues thus establishing LC1 as the origin of this band. However, Western blots did not reveal any LC1 while radioimmunoassays indicated it to be present at the 5% level suggesting the anti-LC1 antibody used in these experiments did not recognize the C-terminal portion of LC1 still attached to Mg.S1. Mixing a 10-15 M excess of isolated light chains with Mg.S1 in the presence of 10 mM ATP, 12 mM MgCl2, 4.7 M NH4Cl allowed LC1 to recombine with LC1-deficient Mg.S1. Equilibrium ultracentrifugation analysis revealed a highly heterogeneous LC1-deficient S1 which upon recombination with intact LC1 became monodisperse as indicated by the superimposition of molecular weight averages all across the centrifuge cell. LC1-deficient Mg.S1 had a Vm of 0.4 s-1, Ka of 30 microM and a Kbind of 28 microM. In the presence of intact LC1, Vm rose to 0.8 s-1 while Ka and Kbind were reduced to 7.5 and 12 microM, respectively. The fourfold decrease in Ka strongly indicated an increased affinity for actin by Mg.S1 in the presence of uncleaved LC1. Also, Ca(2+)-regulation of dog heart myofibrils was suppressed when Ca(2+)-activated MgATPase assays, as a function of Ca2+, were performed in the presence of anti-LC1 antibodies. These observations suggest the presence of intact, uncleaved LC1 in S1 is required for the stability of S1 heavy chains and proper Ca(2+)-regulation.
The present study describes a method for isolating single smooth muscle cells from pig urinary bladder using a continuous resuspension device. Low concentrations of collagenase and papain were sufficient to obtain a high yield of viable smooth muscle cells, which remained viable for about 3-4 h as tested with fluorescein diacetate. Addition of fetal calf serum increased the lifespan of the isolated cells and the percentage of contractile smooth muscle cells, but caused spontaneous shortening of the cells. The length and volume of the isolated smooth muscle cells depended on the calcium concentration used in the isolation buffer solution. The isolated muscle cells were apparently relaxed if a calcium concentration less than 1.0 mmol/l was used in the isolation medium. In higher calcium concentrations the isolated cells were significantly shorter, probably as a result of a contraction caused by mechanical stimulation of the cells during the isolation procedure.
A low-molecular-weight proteinase inhibitor was isolated from human epidermal extracts by means of ultrafiltration, gel filtration and ion-exchange chromatography. On polyacrylamide gel electrophoresis (PAGE), the isolated fraction exhibited a single band of activity at pH 9.4, and inhibitory activity against papain and trypsin was detected in this band. Using sodium-dodecyl-sulphate PAGE, the molecular mass of the inhibitor was estimated to be 2,200 daltons.
The influence of papain, neuraminidase and AET to lymphocytes is reported. All three substances strengthen the lymphocytotoxic reaction in low concentrations or by short incubation time and lower it in higher concentrations or longer incubation since. Membrane changes by these three substances are reversible under defined conditions.
Extracts of the mountain holly, Nemopanthus mucronatus, contained an agglutinin to papain treated porcine erythrocytes but not untreated or neuraminidase treated cells. Human erythrocytes similarly treated did not react.
Treatment of Cholera toxin with pancreatic elastase and papain in vitro showed a high resistance of the toxin molecule to these enzymes, under non-denaturing conditions or in the presence of 2 M urea. These experiments support the hypothesis of a particularly stable molecular structure of the toxin, as an explanation of its activity in the intestinal lumen where the pancreatic proteases are active.
The absorption-elution technique with low ionic strength solution (LISS) and papain-treated test cells previously used for bloodstains was employed for the detection of ABO antigens on human hair. Antigen identification was always possible, with good intensity of agglutination, even in those cases where classic techniques had given false-negative results. It was possible to obtain positive results with fragments of human hair as small as 0.2 cm.
Crude papain was administered intravenously to young rabbits and the cartilage of the collapsed ear was examined electron-microscopically. Degeneration and recovery of chondrocytes, and decrease in and recovery of the electron-density of elastic fibers, were observed during the collapse and restoration of the ear. Some samples were stained with ruthenium red. In the collapsed ear, with a marked decrease of proteoglycan in the cartilage, loss of ruthenium red-positive granules was observed in the extracellular matrix. Collagen fibrils in the cartilage appeared to be somewhat increased in number, some of their diameters became slightly greater, and a part were assembled into bundles, occasionally accompanied by periodic cross-striation. Decrease of proteoglycan in the cartilage matrix probably brought about the unmasking and the assembly of collagen fibrils. In one of the experimental animals, collagen fibrous segments of an atypical fibrous long spacing (FLS-)type with symmetrical cross-striation were found around the chondrocytes in the ear cartilage, during the period of recovery. Some kind of the endogenous sulfated carbohydrate may have acted to affect the arrangement of type II collagen or procollagen molecules newly produced by the recovering chondrocytes.
Beet necrotic yellow vein virus RNA 1 contains a single long ORF corresponding to the theoretical translation product of 237 kDa which contains the information necessary for replication of the viral genome. This ORF contains a putative papain-like proteinase domain which has been localized, on the basis of sequence alignments, between the helicase and polymerase domains. Here we show that the RNA 1 primary translation product can be cleaved autocatalytically in vitro into two species of 150 kDa and 66 kDa, the latter of which probably contains the entire polymerase domain. A 66 kDa protein was detected immunologically in infected C. quinoa protoplasts using an antiserum specific for the C-terminal region of the RNA 1 primary translation product, confirming that processing also occurs in vivo.
The hydrolysis of 32 X-phenyl-N-methanesulfonyl glycinates by papain was investigated. It was found that the variation in the Michaelis constants could be rationalized by the following correlation equation: log 1/Km = 0.61 pi '3 + 0.46 MR4 + 0.55 sigma + 2.00 with a correlation coefficient of 0.945. In this expression, pi '3 is the hydrophobic constant for the more lipophilic of the two possible meta substituents, MR4 is the molar refractivity of 4-substituents, and sigma is the Hammett constant summed for all substituents. Using this equation, we designed, synthesized, and successfully predicted Km for a new congener intended to maximize binding (1/Km). The interactions involved in enzyme-substrate binding, as characterized by the correlation equation, are interpreted using a computer-constructed color three-dimensional-graphics molecular model of the enzyme active site. The nonenzymatic hydrolysis (both acid and basic) of phenyl hippurates yield rate constants which are well correlated by Hammett equations; however, log k for both acid and alkaline hydrolysis are not linearly related to log 1/Km or log kcat/Km.