Monomeric selectively S-alkylated derivatives of seminal ribonuclease: preparation and properties

Procedures are described for preparing monomeric selectively S-carboxamido-methylated and S-aminoethylated derivatives of seminal ribonuclease. The main properties of the derivatives, including their extinction coefficients, have been determined. Their catalytic activities and that of the S-carboxymethyl derivative have been tested. On double-stranded RNA as a substrate the monomeric derivatives are less active than the native dimeric enzyme, but much more active than pancreatic ribonuclease. On yeast RNA as a substrate the amino-ethyl derivative is found to be less active (80%) than the native enzyme, while the other two are over 30 percent more active. The monomers are stable in solution and when lyophilized from acetic acid solution do not associate to the same extent as pancreatic or native seminal ribonucleases.     

A role for the intersubunit disulfides of seminal RNase in the mechanism of its antitumor action.

The dimeric structure of seminal ribonuclease (BS-RNase) is maintained by noncovalent interactions and by two intersubunit disulfide bridges. Another unusual feature of this enzyme is its antitumour action, consisting in a cytotoxic activity selective for malignant cells. This cytotoxic action is exerted when the protein reaches the cytosol of the affected cells, where it degrades ribosomal RNA, thus blocking protein synthesis and leading cells to death. The current model proposed for the mechanism of antitumour action of BS-RNase is based on the ability of the protein to resist the neutralizing action of the cytosolic RNase inhibitor, a resistance due to the dimeric structure of the enzyme. Monomeric RNases, and monomeric derivatives of BS-RNase, are strongly bound by the inhibitor and inactive as antitumor agents. Here we report on monomeric derivatives of BS-RNase that, although strongly inhibited by the cytosolic RNase inhibitor, are cytotoxic towards malignant cells. These monomers are produced by reductive cleavage of the intersubunit disulfides of the native, dimeric protein followed by linking the exposed sulfhydryls to small thiols through formation of mixed disulfides. We found that sulfhydryls from cell monolayers and cell membranes can attack these mixed disulfides in the monomeric derivatives, and reconstitute, through sulfhydryl-disulfide interchange reactions, the native dimeric protein, which is internalized as such, and displays its antitumour action.     

Properties of matrix-bound dimer and monomer derivatives of immobilized creatine kinase from rabbit skeletal muscle.

Dimeric creatine kinase (EC 2.7.3.2) from rabbit skeletal muscle can be immobilized via a single subunit to CNBr-activated Sepharose 4B and subsequently treated with guanidine hydrochloride followed by renaturation to yield a catalytically active matrix-bound subunit derivative. The importance of the intact dimeric structure in the activation of the enzyme by acetate was demonstrated. Immobilization did not appear to alter the pH optimum of the enzyme, and the kinetic parameters fot the matrix-bound derivatives were generally similar to those for the soluble enzyme, but the matrix-bound derivatives showed higher thermal stability and greater resistance to denaturation than did the soluble enzyme. The rates of reaction of thiol groups of the matrix-bound derivatives with iodoacetamide in the absence and in the presence of combinations of substrates were similar to those of the soluble enzyme. Studies with 5,5'-dithiobis-(2-nitrobenzoic acid) and with iodoacetamide revealed the presence of an additional reactive thiol group in the matrix-bound subunit derivative, which is presumably masked in the dimeric derivatives.     

The elucidation of the microheterogeneity of highly purified p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by various biochemical techniques

Highly purified p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens can be separated into at least five fractions by anion-exchange chromatography. All fractions exhibit the same specific activity and the enzyme exists mainly in the dimeric form in solution. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of a mixture of the different fractions reveals two apparent forms of enzyme molecules, while isoelectric focusing experiments, on the other hand, reveal six apparently different forms of enzyme molecules. It is shown that the different forms of enzyme molecules are due to the (partial) oxidation of Cys-116 in the sequence of the enzyme. This interpretation of the data is supported by kinetic measurements of the formation of hybrid dimeric molecules monitored by fast protein liquid chromatography, using purified enzyme containing Cys-116 either in the native and or the fully oxidized (sulfonic acid) state. By chemical modification studies using maleimide derivatives, 5,5'-dithiobis(2-nitrobenzoate) and H2O2, it is shown that sulfenic, sulfinic and sulfonic acid derivatives of Cys-116 are products of oxidation. The results are briefly discussed with respect to the possibility that this isolation artifact might also be partially responsible for the appearance of multiple forms of enzyme molecules in other biochemical preparations.     

Cloning and expression of a putative cytochrome P450 gene that influences the colour of Phalaenopsis flowers

Anthocyanins are responsible for reds through blues in flowers. Blue and violet flowers generally contain derivatives of delphinidin, whereas red and pink flowers contain derivatives of cyanidin or pelargonidin. Differences in hydroxylation patterns of these three major classes of anthocyanidins are controlled by the cytochrome P450 enzymes. Flavonoid-3',5'-hydroxylase, a member of the cytochrome P450 family, is the key enzyme in the synthesis of 3',5'-hydroxylated anthocyanins, generally required for blue or purple flowers. Here we report on the isolation of a cDNA clone of a putative flavonoid-3',5'-hydroxylase gene from Phalaenopsis that was then cloned into a plant expression vector. Transient transformation was achieved by particle bombardment of Phalaenopsis petals. The transgenic petals changed from pink to magenta, indicating that the product of the putative flavonoid-3',5'-hydroxylase gene influences anthocyanin pigment synthesis.     

Active Dimeric Form of Inorganic Pyrophosphatase from Escherichia coli

A dimeric form can be obtained from native hexameric Escherichia coli inorganic pyrophosphatase (E-PPase) by destroying the hydrophobic intersubunit contacts, and it has been shown earlier to consist of the subunits of different trimers. The present paper is devoted to the kinetic characterization of such a "double-decked" dimer obtained by the dissociation of either the native enzyme or the mutant variant Glu145Gln. The dimeric form of the native inorganic pyrophosphatase was shown to retain high catalytic efficiency that is in sharp contrast to the dimers obtained as a result of the mutations at the intertrimeric interface. The dimeric enzymes described in the present paper, however, have lost the regulatory properties, in contrast to the hexameric and trimeric forms of the enzyme.     

Dissociation and reconstitution of bovine seminal RNAase: Construction of a hyperactive hybrid dimer

The quaternary structure of bovine seminal ribonuclease, the only dimeric protein in the superfamily of ribonucleases, is maintained both by noncovalent forces and by two intersubunit disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalytically active, but do not retain certain properties of the dimeric enzyme, such as: (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step; and (ii) the antitumor and immunosuppressive actions. In this report we describe the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers indistinguishable from the native protein. With this procedure a hybrid dimer was constructed, made up of a native subunit associated to a subunit catalytically inactivated by selective alkylation of the active site His-119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was one order-of-magnitude more active than the dimeric enzyme.     

Purification of hog liver isomerase. Mechanism of isomerization of 3-alkenyl and 3-alkynyl thioesters.

A hog liver enzyme that catalyzes the reversible conversion of 3-acetylenic fatty acyl thioester to (+)-2,3-dienoyl fatty acyl thioester has been purified to homogeneity. The enzyme is not inhibited by the allenic product that it generates. The same homogenous enzyme catalyzes the conversions of 3-cis- or 3-trans-acyl Coenzyme A derivatives to 2-trans-acyl-CoA derivatives. Four forms of the isomerase differing in charge (pI = 6.57, 6.83, 7.01, and 7.27) have been separated by isoelectric focusing. Ultracentrifugation and sodium dodecyl sulfate-gel electrophoresis indicate that each of these enzyme forms is dimeric and composed of two 45,000-dalton subunits. With 3-acetylenic substrates, all enzyme forms exhibit broad specificity for chain length (C6 to C12) and for the thioester moiety (N-acetylcysteamine (NAC), pantetheine, or CoA). The 3-cis and 3-trans olefinic substrates are active only in the form of their coenzyme A derivatives; their NAC thioesters inhibit competitively. Mechanistic studies favor an isomerization pathway by way of carbanion intermediates. The acetylene-allene isomerase described here and the reported crotonase-catalyzed hydration of allenic thioesters (Branchini, B.R., Miesowicz, F.M., and Bloch, K. (1977) Bioorg. Chem. 6, 49-52) may be responsible for the degradation of naturally occurring acetylenic and allenic acids.     

Purification, cloning and autoproteolytic processing of an aspartic proteinase from Centaurea calcitrapa.

Plant aspartic proteinases (APs) have been isolated from several seed and leaf sources but the only well characterized enzymes from flowers are cardosins and cyprosins from cardoon, Cynara cardunculus L. Here we report a full-length cDNA clone encoding an AP named cenprosin from the flowers of Centaurea calcitrapa L., a thistle related to cardoon. As found for all eukaryotic APs, the deduced primary sequence consists of a signal sequence, a propart and a mature enzyme. In addition, an internal sequence region of 104 residues typical only of plant APs (a plant-specific insert) is present in the primary structure. Northern analysis revealed that the strongest expression is in fresh flowers. The enzyme is also expressed in fairly high amounts in seeds and in leaves, a feature not detected for cardoon APs. The corresponding enzyme was purified in its precursor form from fresh flowers using ammonium-sulfate precipitation followed by ion-exchange and hydrophobic-interaction chromatography. The processing of the precursor into its mature form was studied in vitro. The enzyme underwent autocatalytic processing at pH 3.0 resulting in two chains of 16 and 30 kDa. When dried flowers were used as a starting material for purification, only 16- and 30-kDa chains were obtained, suggesting that autoproteolytic activation of procenprosin in vivo occurs mainly during drying of the flowers. This may indicate a specific degradative role for the enzyme during senescence of the flowers.     

Interaction of various nucleotide-dependent enzymes with bifunctional analogs of ATP, derivatives of polymethylene diamines

The interaction of bifunctional ATP derivatives, Appp5'[NH-(CH2) n-NH]ppp5'A (n = 0 or 2-8) with tyrosyl-, valyl-, lysyl-, tryptophanyl-tRNA synthetases and creatine kinase was investigated. ATP derivatives don't inhibit the tRNA aminoacylation catalyzed by tyrosyl-tRNA synthetase. These derivatives behave as mixed-type inhibitors with respect to ATP in the case of valyl- and lysyl-tRNA-synthetases. In the case of the other enzymes all analogs of ATP manifest competitive inhibition towards ATP. The affinity of all ATP derivatives to tryptophanyl-tRNA synthetase does not differ significantly (Ki = 0.2 divided by 0.6 mM). The Ki values for these derivatives in the case of creatine kinase are also very similar with the exception of A5'ppp-NH-(CH2)3-NH-ppp5'A. The Ki value for this derivative is one order of magnitude lower than for other ones. The affinity reagents received by periodate oxidation of bifunctional ATP analogs derivatives of di-, tetra- and heptamethylenediamine modify non-identical subunits of creatine kinase with different velocities, but modification of M- and M'-subunits proceeds independently. An analogues derivative of trimethylenediamine interacts simultaneously with two centers of the dimeric form of kinase forming non-equivalent complexes. The covalent attachment of the reagent to one subunit of creatine kinase does not except the complex formation and covalent binding of bifunctional ATP analogs with the other subunit of the dimer, but results in a one order of magnitude decrease in affinity of the ATP derivative to the nonmodified centre of the enzyme. These data permit to evaluate the distance between ATP binding sites of creatine kinase in its dimeric form as 5-6 A approximately. Such a distance between active sites may be the reason for the higher activity of the M- and M'-creatine kinase subunits taken separately as compared to the enzyme dimeric form.     

Tissue-specific expression of multiple forms of cyprosin (aspartic proteinase) in flowers of Cynara cardunculus

Three heterodimeric aspartic proteinases (cyprosin 1, 2 and 3) with milk-clotting activity have previously been purified from flowers of Cynara cardunculua and partly characterized (U. Heimgartner et al. 1990, Phytochemistry 29: 1405–1410). These proteinases have now been further studied. Isoelectric focusing has revealed a micro-heterogeneity of the apparently pure cyprosins. Three isozymes with close isoelectric points around 4.0 have been found. Reversed-phase high performance liquid chromatography of electrophoretically purified large subunits of cyprosin has also shown a microheterogeneity. Peptide mapping of cyprosins 2 and 3 by trypsin or BrCN cleavage indicate that they are derived from common procyprosin(s). Studies on the organspecific accumulation of the enzyme were carried out using flower buds and flowers at different stages of development and styles and corollas from open flowers, leaves and seeds. Immunostained western blots revealed the presence of cyprosin in very young flowers in low amounts. The amount of enzyme increased towards later stages of development and it was mostly present in the violet parts of styles and corollas. The enzyme could not be detected in leaves or seeds. Proteolytic and milk-clotting activities correlate well with these findings. The enzyme was localized by immunolabelling in the epidermal cell layer of styles. Mature flowers collected at 8 different locations in Portugal showed some variation in proteolytic activity while the milk-clotting activity was essentially the same for all extracts.     

Properties of S-adenosylmethionine decarboxylase from rabbit liver: effects of some hydrazone compounds on its activity

S-Adenosylmethionine decarboxylase (EC 4.1.1.50) has been partially purified from rabbit liver by ammonium sulphate fractionation and gel filtration and anion exchange chromatographies. Sodium dodecylsulphate-polyacrylamide disc gel electrophoresis analysis showed an approximate dimeric subunit mol. wt of 34,000. The enzyme showed a pH optimum at 7.5 (in phosphate buffer) and did not require bivalent cations for catalysis. The enzyme showed sigmoid kinetics to S-adenosylmethionine with a Hill coefficient of 1.7, which became michaelian with Km 70 microM in the presence of 2.5 mM putrescine. Methylglyoxal bis(guanylhydrazone) was an effective inhibitor of the enzyme, but phenylated derivatives of this compound as phenylglyoxal bis(guanylhydrazone) and diphenylglyoxal bis-(guanylhydrazone) inhibited less well.     

Isopycnic-centrifugation studies in caesium chloride and in caesium sulphate on dermatan sulphate proteoglycans from bovine sclera

1. Frog epidermis tyrosinase was coupled to Sepharose activated with low concentrations of CNBr. The tetrameric form of the enzyme was linked to the matrix via its subunits. Dissociation of the bound active enzyme with guanidinium chloride yielded an active immobilized dimeric derivative. 2. Immobilized dimeric derivative was able to interact with soluble subunits formed transiently during renaturation. An 85% recovery of the native dopa oxidase specific activity was achieved after hybridization. 3. Fluorescence spectra of different immobilized derivatives suggested that tryptophan residues were involved in the interactions between tyrosinase subunits. 4. It is suggested that the activation of the subunits of tyrosinase involves a conformational change towards a more unfolded state, which favours a reassociation to the dimeric active state.     

Enzymatic control of anthocyanin expression in the flowers of pea (Pisum sativum) mutants

Using enzymological and immunological methods we have investigated the relationship between chalcone synthase and the A locus, a major gene involved in the control of anthocyanin expression in pea (Pisum sativum L.) flowers. Pea plants containing the dominant allele A usually synthesize anthocyanins in the petal tissue, whereas plants homozygous for the a allele do not produce anthocyanins. We sought to determine whether or not the A locus also controlled the presence or absence of chalcone synthase, the first enzyme of the flavonoid pathway in the flowers of three genetic lines (A, purple-violet flowers; A,am, white flowers with sometimes pink edges; and a, white flowers). Chalcone synthase was found to be present in all three genetic lines by enzyme activity measurement, indirect enzyme-linked immunosorbent assay (ELISA), and Western blotting. Spectroscopic investigations showed that only the genetic lines A and A,am contained anthocyanins and flavonol glycosides, respectively, in the flowers; line a accumulated p-coumaric acid or its derivatives. These data suggest that the A locus in Pisum is not the structural gene for chalcone synthase and it does not appear to regulate the expression of this enzyme.This work was supported by a grant from the Cornell University Biotechnology Program, which is sponsored by the New York State Science and Technology Foundation and a consortium of industries.     

Cloning and characterization of Arabidopsis thaliana pyridoxal kinase

Pyridoxal kinase (PK; EC 2.7.1.35), a key enzyme in vitamin B(6) metabolism, was cloned from Arabidopsis thaliana (L.) Heynh. and characterized. The amino acid sequence of the A. thaliana PK was found to be similar to the mammalian enzyme, with a homology of more than 40%. Characterization studies showed that the kinase is a dimeric molecule consisting of two identical subunits, each subunit having a molecular mass of approximately 35 kDa. The enzyme exhibited maximal activity at pH 6.0. Similar to the mammalian enzyme, the enzyme from A. thaliana preferred Zn(2+) instead of the commonly used Mg(2+) as the divalent cation for catalysis. Under optimal conditions, the V(max) of the enzyme was 604 nmol pyridoxal 5'-phosphate (PLP) mg(-1) min(-1), and the K(m) values for pyridoxal and ATP were 688 micro M and 98 micro M, respectively. Examination of levels of enzyme expression showed that leaves, stems, roots and flowers can generate PLP independently at similar levels. Furthermore, expression of the PK gene in A. thaliana seeds was found to start 60 h after imbibition. Results from the present study suggest that plant tissues depend on PK for the production of PLP.     

肌酸激酶在低浓度脲溶液中失活也非二聚体解聚所致

本文用兔肌肌酸激酶(MM)和兔脑肌酸激酶(BB)杂化的方法;研究了肌酸激酶在脲溶液中的解聚情况.结果表明在低浓度的脲溶液中二聚体没有或者很少解聚,而此时酶已经失去大部分或全部活性.因此可以认为在低浓度脲溶液中酶的失活也非二聚体解聚所致,进一步支持了酶分子活性部位柔性的观点.     

Molecular cloning and characterization of a vacuolar class III peroxidase involved in the metabolism of anticancer alkaloids in Catharanthus roseus

Catharanthus roseus produces low levels of two dimeric terpenoid indole alkaloids, vinblastine and vincristine, which are widely used in cancer chemotherapy. The dimerization reaction leading to alpha-3',4'-anhydrovinblastine is a key regulatory step for the production of the anticancer alkaloids in planta and has potential application in the industrial production of two semisynthetic derivatives also used as anticancer drugs. In this work, we report the cloning, characterization, and subcellular localization of an enzyme with anhydrovinblastine synthase activity identified as the major class III peroxidase present in C. roseus leaves and named CrPrx1. The deduced amino acid sequence corresponds to a polypeptide of 363 amino acids including an N-terminal signal peptide showing the secretory nature of CrPrx1. CrPrx1 has a two-intron structure and is present as a single gene copy. Phylogenetic analysis indicates that CrPrx1 belongs to an evolutionary branch of vacuolar class III peroxidases whose members seem to have been recruited for different functions during evolution. Expression of a green fluorescent protein-CrPrx1 fusion confirmed the vacuolar localization of this peroxidase, the exact subcellular localization of the alkaloid monomeric precursors and dimeric products. Expression data further supports the role of CrPrx1 in alpha-3',4'-anhydrovinblastine biosynthesis, indicating the potential of CrPrx1 as a target to increase alkaloid levels in the plant.     

Dimeric enzyme IImtl of the E. coli phosphoenolpyruvate-dependent phosphotransferase system. Cross-linking studies with bifunctional sulfhydryl reagents

The occurrence of intermolecular dithiols on EIImtl has been studied with a number of thiol-specific cross-linking reagents. The reaction of EIImtl with bifunctional maleimide derivatives inactivates the enzyme. At the same time the enzyme is irreversibly cross-linked to a dimeric species. Under optimal conditions 50% of the protein is cross-linked upon reaction with the dimaleimides. The enzyme is also cross-linked under oxidizing conditions in the presence of CuCl2, presumably by oxidizing an intermolecular dithiol to a disulfide. This oxidation can be reversed by the addition of the reducing agent dithiothreitol. The reaction of phosphorylated EIImtl with the same sulfhydryl-specific bifunctional reagents does not lead to any cross-linked product. The results are discussed in terms of the association state of the purified protein and the distribution of its thiol groups.     

Docking studies on the binding of quinoline derivatives and hematin to Plasmodium falciparum lactate dehydrogenase

The literature has reported that ferriprotoporphyrin IX (hematin) intoxicates the malarial parasite through competition with NADH for the active site of the enzyme lactate dehydrogenase (LDH). In order to avoid this, the parasite polymerizes hematin to hemozoin. The quinoline derivatives are believed to form complexes with dimeric hematin, avoiding the formation of hemozoin and still inhibiting LDH. In order to investigate this hypothesis we calculated the docking energies of NADH and some quinoline derivatives (in the free forms and in complex with dimeric hematin) in the active site of the Plasmodium falciparum LDH (PfLDH). Ours results showed better docking score values to the complexes when compared to the free compounds, pointing them as more efficient inhibitors of Pf_LDH. Further we performed Molecular Dynamics (MD) simulations studies on the best docking conformation of the complex chloroquine-dimeric hematin with PfLDH. Our in silico results corroborate experimental data suggesting a possible action route for the quinoline derivatives in the inhibition of PfLDH.     

Hydrolysis of α-lactalbumin by cardosin A immobilized on highly activated supports

In the present research effort, production of derivatives of cardosin A (a plant protease) encompassing full stabilization of its dimeric structure has been achieved, via covalent, multi-subunit immobilization onto highly activated agarose-glutaraldehyde supports. Boiling such enzyme derivatives in the presence of sodium dodecyl sulfate and β-mercaptoethanol did not lead to leaching of enzyme, thus providing evidence for the effectiveness of the attachment procedure. Furthermore, the cardosin A derivatives prepared under optimal conditions presented ca. half the specific activity of the enzyme in soluble form, and were successfully employed at laboratory-scale trials to perform (selective) hydrolysis of α-lactalbumin (α-La), one of the major proteins in bovine whey. Hydrolysates of α-La were assayed for by the OPA method, as well as by FPLC, SDS–PAGE and HPLC. Thermal inactivation of the immobilized cardosin A was also assessed at 40, 50 and 55 °C; at these temperatures, no thermal denaturation took place during incubation for 48 h. The highest degree of hydrolysis was attained by 5 h reaction, at 55 °C and pH 5.2. SDS–PAGE of α-La hydrolysates displayed bands corresponding to low molecular weight peptides. Our results suggest that cardosin A in immobilized form is a good candidate to bring about proteolysis in the dairy industry, namely in whey processing.