A proteolytic enzyme, Php-B ( Photorhabdus protease B), was purified from the entomopathogenic bacterium, Photorhabdus luminescens. The enzyme is intracellular, and its molecular mass is 74 kDa. Tested on various peptide and oligopeptide substrates, Php-B hydrolysed only oligopeptides, with significant activity against bradykinin and a 2-furylacryloyl-blocked peptide, Fua-LGPA (2-furylacryloyl-Leu-Gly-Pro-Ala; kcat=3.6x10(2) s(-1), K(m)=5.8x10(-5) M(-1), pH optimum approx. 7.0). The p K(a1) and the p K(a2) values of the enzyme activity (6.1 and 7.9 respectively), as well as experiments with enzyme inhibitors and bivalent metal ions, suggest that the activity of Php-B is dependent on histidine and cysteine residues, but not on serine residues, and that it is a metalloprotease, which most probably uses Zn2+ as a catalytic ion. The enzyme's ability to cleave oligopeptides that contain a sequence similar to collagen repeat (-Pro-Xaa-Gly-), bradykinin and Fua-LGPA (a synthetic substrate for bacterial collagenases and collagen peptidases), but not native collagens (types I and IV) or denatured collagen (gelatin), indicates that Php-B is probably a collagen peptidase, the first enzyme of this type to be identified in an insect pathogen, that might have a role in the nutrition of P. luminescens by degrading small collagen fragments. For the determination of enzyme kinetic constants, we fitted a numerically integrated Michaelis-Menten model to the experimental progress curves. Since this approach has not been used before in the characterization of proteases that are specific for the P1'-P4' substrate sites (e.g. collagenolytic enzymes), we present a comparison of this method with more conventional ones. The results confirm the reliability of the numerical integration method in the kinetic analysis of collagen-peptide-hydrolysing enzymes.
his review will summarize available information on the ability of macromolecular conjugates containing no specific recognition motifs to deliver anthracyclines (daunomycin, adriamycin) or methotrexate to target cells such as tumour cells or macrophages. Conjugates with natural (proteins, DNA, carbohydrates) and synthetic macromolecules (linear and branched chain poly-alpha-amino acids, non-biodegradable DIVEMA, HPMA etc.) will be reviewed. Experimental data from several laboratories indicate that these conjugates are taken up by cells mainly by fluid-phase or adsorptive endocytosis. It is believed that these processes do not involve 'specific receptors'. Two examples of methotrexate and daunomycin conjugates will be discussed to show the effect of the chemical structure of branched chain polypeptides on the uptake and antitumour or antiparasitic (Leishmania donovani infection) efficacy of conjugates.
Methotrexate (MTX) has been coupled to various structurally related, polycationic (poly[Lys(DL-Ala m )] (AK), poly[Lys(Ser i -DL-Ala m )] (SAK), poly[Lys(DL-Ala m -Leu i )] (ALK)), or amphoteric (poly[Lys(Glu i -DLAla m )] (EAK)) synthetic branched polypeptides containing poly [L-Lys] backbone by the aid of BOP reagent. The average degree of MTX incorporation was found to be dependent on the charge properties of the polymer. Under the experimental conditions used, the molar substitution ratio achieved was higher for polycations (25%) than for the amphoteric polypeptide (10%). We have studied the effect of polycationic polypeptides on Leishmania donovani infection. Results demonstrated that MTX conjugates in which the drug is covalently attached to carrier have pronounced leishmanicid activity. In this communication we showed that (a) a branched polypeptide-methotrexate conjugate with a polycationic carrier (ALK) increases the effect of MTX against Leishmania donovani infection in mice; (b) the covalent bond between the carrier and methotrexate is essential for both in vivo and in vitro activity; and (c) the number of Leishmania donovani parasites in infected macrophages are markedly reduced in conjugate treated animals. In vitro observation might also indicate that the MTX conjugate exhibits an effect through an uptake by macrophages which is different from that of the free drug. INTRODUCTONMethotrexate (MTX, 1 L-4-amino-N 10 -methylpteroylglutamic acid), a folate antimetabolite, has been in clinical use for more than 35 years. It is a potent anticancer agent of proven benefit in the treatment of acute leukemia, osteogenic sarcoma (1), and of rheumatological disorders (2-4). Recently its inhibitory potential has been demonstrated against a group of intracellular parasites (Leishmania) of macrophages (5). Visceral leishmaniasis or kala-azar in man is caused by the protozoan parasite Leishmania donovani, which proliferates intracellularly within the mononuclear phagocytes of the infected host (6).Methotrexate has been linked to various macromolecules such as BSA, mannosylated-, galactosylated-, or glucosylated-BSA (7, 8), mannosylated-HSA (9), or maleylated-BSA (10), for delivery into cells containing L. donovani parasites or to polylysine for studying the mechanism of action of cellular uptake by pinocytosis in mice and human tumor cell lines (11)(12)(13)(14).The methods of conjugation of methotrexate to these carriers have involved predominantly the glutamic acid moiety. Activation of R-and γ-carboxyl groups has been achieved in situ in the presence of the carrier using water soluble carbodiimide like EDC (15) or by ester formation with N-hydroxysuccinimide (11). (None of the above methods reported have been used for selective derivatization of R-or γ-carboxyl groups of MTX (15).)MTX was also one of the first antitumor drug attached covalently to high molecular weight carriers to improve the therapeutic index by site-specific targeting and/or by changing the pharmacological properties of MTX to provide controlle...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.