Biomedical subjects

Mohamed Azarkan

Publications and source records attributed to Mohamed Azarkan.

9 recordsLinked to original sources

PubMed2006

Crystallization and preliminary X-ray analysis of a protease inhibitor from the latex of Carica papaya.

A Kunitz-type protease inhibitor purified from the latex of green papaya (Carica papaya) fruits was crystallized in the presence and absence of divalent metal ions. Crystal form I, which is devoid of divalent cations, diffracts to a resolution of 2.6 A and belongs to space group P3(1) or P3(2). This crystal form is a merohedral twin with two molecules in the asymmetric unit and unit-cell parameters a = b = 74.70, c = 78.97 A. Crystal form II, which was grown in the presence of Co2+, diffracts to a resolution of 1.7 A and belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 44.26, b = 81.99, c = 140.89 A.

Carica↗

PubMed2006

Affinity chromatography: a useful tool in proteomics studies.

Separation or fractionation of a biological sample in order to reduce its complexity is often a prerequisite to qualitative or quantitative proteomic approaches. Affinity chromatography is an efficient protein separation method based on the interaction between target proteins and specific immobilized ligands. The large range of available ligands allows to separate a complex biological extract in different protein classes or to isolate the low abundance species such as post-translationally modified proteins. This method plays an essential role in the isolation of protein complexes and in the identification of protein-protein interaction networks. Affinity chromatography is also required for quantification of protein expression by using isotope-coded affinity tags.

Chromatography, Affinity↗

PubMed2006

The papaya Kunitz-type trypsin inhibitor is a highly stable beta-sheet glycoprotein.

The papaya Kunitz-type trypsin inhibitor, a 24-kDa glycoprotein, was purified to homogeneity. The purified inhibitor stoichiometrically inhibits bovine trypsin in a 1:1 molar ratio. Circular dichroism and infrared spectroscopy analyses demonstrated that the inhibitor contains extensive beta-sheet structures. The inhibitor was found to retain its full inhibitory activity over a broad pH range (1.5-11.0) and temperature (up to 80 degrees C), besides being stable at very high concentrations of strong chemical denaturants (e.g., 5.5 M guanidine hydrochloride). The inhibitor retained its compact structure over the pH range analyzed as shown by 8-anilino-1-naphtalenesulfonic acid binding characteristics, excluding the formation of some relaxed or molten state. Exposure to 2.5 mM dithiothreitol for 120 min caused a 33% loss of the inhibitory activity, while a loss of 75% was obtained in the presence of 20 mM of dithiothreitol during the same time period. A complete loss of the inhibitory activity was observed after incubation with 50 mM dithiothreitol for 5 min. Incubation of the inhibitor with general proteases belonging to different families revealed its extraordinary resistance to proteolysis in comparison with the soybean trypsin inhibitor, the archetypal member of the Kunitz-type inhibitors family. The inhibitor also exhibited a remarkable resistance to proteolytic degradation against pepsin for at least a 24-h incubation period. Instead, the soybean inhibitor was completely degraded after 2 h incubation with this aspartic protease. All these data demonstrated the high stability of the papaya trypsin inhibitor.

Anilino Naphthalenesulfonates↗

PubMed2006

Effects of mechanical wounding on Carica papaya cysteine endopeptidases accumulation and activity.

The mechanical wounding impact on the Carica papaya latex protein pattern was investigated by analyzing three latexes. A first one commercially available, a second harvested from unripe but fully grown fruits, both obtained from regularly tapped fruits. A third one was collected from similar fruits but wounded for the first time. The results demonstrated both quantitative and qualitative changes in the protein content and in the enzymatic activity. Repeated wounding results in either, accumulation or activation (or both of them) of papain, chymopapain and caricain. Furthermore, new cysteine protease activity was found to transiently accumulate in the latex collected from newly wounded fruits. The possible implication of this enzymatic material in the papaya cysteine endopeptidases pro-forms activation is discussed.

Carica↗

PubMed2006

Crystal structure of papaya glutaminyl cyclase, an archetype for plant and bacterial glutaminyl cyclases.

Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the intramolecular cyclization of protein N-terminal glutamine residues into pyroglutamic acid with the concomitant liberation of ammonia. QCs may be classified in two groups containing, respectively, the mammalian enzymes, and the enzymes from plants, bacteria, and parasites. The crystal structure of the QC from the latex of Carica papaya (PQC) has been determined at 1.7A resolution. The structure was solved by the single wavelength anomalous diffraction technique using sulfur and zinc as anomalous scatterers. The enzyme folds into a five-bladed beta-propeller, with two additional alpha-helices and one beta hairpin. The propeller closure is achieved via an original molecular velcro, which links the last two blades into a large eight stranded beta-sheet. The zinc ion present in the PQC is bound via an octahedral coordination into an elongated cavity located along the pseudo 5-fold axis of the beta-propeller fold. This zinc ion presumably plays a structural role and may contribute to the exceptional stability of PQC, along with an extended hydrophobic packing, the absence of long loops, the three-joint molecular velcro and the overall folding itself. Multiple sequence alignments combined with structural analyses have allowed us to tentatively locate the active site, which is filled in the crystal structure either by a Tris molecule or an acetate ion. These analyses are further supported by the experimental evidence that Tris is a competitive inhibitor of PQC. The active site is located at the C-terminal entrance of the PQC central tunnel. W83, W110, W169, Q24, E69, N155, K225, F22 and F67 are highly conserved residues in the C-terminal entrance, and their putative role in catalysis is discussed. The PQC structure is representative of the plants, bacterial and parasite enzymes and contrasts with that of mammalian enzymes, that may possibly share a conserved scaffold of the bacterial aminopeptidase.

Amino Acid Sequence↗

PubMed2004

Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex.

In living systems, the intramolecular cyclization of N-terminal glutamine residues is accomplished by glutaminyl cyclase enzymes (EC 2.3.2.5). While in mammals these enzymes are involved in the synthesis of hormonal and neurotransmitter peptides, the physiological role played by the corresponding plant enzymes still remains to be unravelled. Papaya glutaminyl cyclase (PQC), a 33 kDa enzyme found in the latex of the tropical tree Carica papaya, displays an exceptional resistance to chemical and thermal denaturation as well as to proteolysis. In order to elucidate its enzymatic mechanism and to gain insights into the structural determinants underlying its remarkable stability, PQC was isolated from papaya latex, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.82, b = 81.23, c = 108.17 A and two molecules per asymmetric unit. Diffraction data have been collected at ESRF beamline BM14 and processed to a resolution of 1.7 A.

Aminoacyltransferases↗

PubMed2004

Detection of three wound-induced proteins in papaya latex.

The effects of routine mechanical wounding for latex collection from unripe fruits of the tropical Carica papaya tree were investigated. For that purpose, the protein composition of three different latexes was analyzed. The first one, commercially available, was provided in the form of a spray-dried powder, the second one was harvested from fully grown but unripe papaya fruits that are regularly tapped for latex production and the last one, was obtained from similar fruits wounded for the first time. Repeated mechanical wounding was found to profoundly affect the protein content of the latex inducing, among others, activation of papain. Regularly tapped latexes also accumulated several low molecular weight proteins not yet identified, as well as three proteins identified as a trypsin inhibitor, a class-II chitinase and a glutaminyl cyclase on the basis of their enzymatic or inhibitory activities and chromatographic elution profiles. This latter was found here, for the first time, to be a wound-induced protein. The roles of these proteins in the plant defense mechanism are discussed.

Amidohydrolases↗

PubMed2003

Fractionation and purification of the enzymes stored in the latex of Carica papaya.

The latex of the tropical species Carica papaya is well known for being a rich source of the four cysteine endopeptidases papain, chymopapain, glycyl endopeptidase and caricain. Altogether, these enzymes are present in the laticifers at a concentration higher than 1 mM. The proteinases are synthesized as inactive precursors that convert into mature enzymes within 2 min after wounding the plant when the latex is abruptly expelled. Papaya latex also contains other enzymes as minor constituents. Several of these enzymes namely a class-II and a class-III chitinase, an inhibitor of serine proteinases and a glutaminyl cyclotransferase have already been purified up to apparent homogeneity and characterized. The presence of a beta-1,3-glucanase and of a cystatin is also suspected but they have not yet been isolated. Purification of these papaya enzymes calls on the use of ion-exchange supports (such as SP-Sepharose Fast Flow) and hydrophobic supports [such as Fractogel TSK Butyl 650(M), Fractogel EMD Propyl 650(S) or Thiophilic gels]. The use of covalent or affinity gels is recommended to provide preparations of cysteine endopeptidases with a high free thiol content (ideally 1 mol of essential free thiol function per mol of enzyme). The selective grafting of activated methoxypoly(ethylene glycol) chains (with M(r) of 5000) on the free thiol functions of the proteinases provides an interesting alternative to the use of covalent and affinity chromatographies especially in the case of enzymes such as chymopapain that contains, in its native state, two thiol functions.

Carica↗

PubMed2002

Evidence that thermodynamic stability of papaya glutamine cyclase is only marginal.

Papaya glutamine cyclase (PQC), a glycoprotein with a molecular mass of 32,980 Da, is a minor constituent of the papaya latex protein fraction. In neutral aqueous solutions, PQC adopts an all-beta conformation and exhibits high resistance to both proteolysis and denaturation. Complete unfolding of PQC requires a combination of an acidic medium and chemical denaturant such as urea or guanidine hydrochloride. The unfolding process takes place through formation of an intermediate A state that accumulates in the absence of chemical denaturants and displays all the features of a molten globule state. The different conformational states-N (native), A (acid-inactivated), and U (unfolded)-have been characterized by means of circular dichroism measurements, fluorescence spectroscopies, Stokes radii determinations, and 8-anilino-1-naphtalenesulfonic acid (ANS) binding characteristics. The unfolding pathways of the enzyme was further studied to estimate thermodynamic parameters characterizing both transitions N if A and A if U. In its A state, PQC is catalytically inefficient and highly susceptible to proteolysis. Also, its thermodynamic stability is decreased by some 3-5 kcal/mol. Conversion of the native to the A state involves digging up of five amino functions together with protonation of four to five acidic groups with pK(a)s, in the native state, around 2.7. It proceeds both cooperatively and reversibly although, in vitro, the refolding process is slow. Unfolding of the A state, on the other hand, occurs with a low degree of cooperativity. The intermediate A state thus seems to be only marginally more stable than the unfolded state. The role of suspected internal ion pairs in the stabilization of the native state of this enzyme is discussed.

Aminoacyltransferases↗