Segregate families from the Euphorbiaceae

Segregate families from the Euphorbiaceae. Several families have been segregated from the Euphorbiaceae. Some of these are of long-standing, and are now generally accepted. Others are more recent, and somewhat controversial. Five such have recently been recognized at Kew, and are discussed here, as are two others which have not been so recognized.     

Acid phosphatases from latices of euphorbiaceae

Five phosphatases were isolated from the latices of three members of the Euphorbiaceae. From Euphorbia lathyris were obtained phosphatases 1₁ and 1₂; from E. trigona phosphatase t and from Elaeophorbia drupifera the enzymes d₁ and d₂. Phosphatases 1₁, 1₂ and t were purified to homogeneity. Amino acid compositions are reported and other properties of the enzymes are described. The two enzymes described from E. lathyris both have two pH maxima d(1₁ at 5.0 and 6.8,1₂ at 5.8 and 7.5) while t, d₁ and d₂ respectively have maxima at pHs of 5.6,5.6 and 5.0. On the basis of their responses to several residue-specific inhibitors the five phosphatases apparently comprise three groups: 1₂ and d₁, t and d₂, and 1₁.     

Biochemical properties of the proteasome from Thermoplasma acidophilum.

We have purified proteasomes to apparent homogeneity from the archaebacterium Thermoplasma acidophilum. This proteinase has a molecular mass of about 650 kDa and an isoelectric point of 5.6. The proteasome hydrolyses peptide substrates containing an aromatic residue adjacent to the reporter group, as well as [14C]methylated casein optimally at pH 8.5 and 90 degrees C. The enzyme activity is enhanced severalfold by Mg2+ and Ca2+ at 25-500 mM. This increase in activity results primarily from a change in Km. The serine-proteinase inhibitors diisopropylfluorophosphate and 3,4-dichloroisocoumarin irreversibly inhibit the enzyme, obviously by modification of both the alpha and beta subunits in the proteasome. The inhibition of proteasomal activity by the peptidylchloromethanes, Cbz-Leu-Leu-CH2Cl and Cbz-Ala-Ala-Phe-CH2Cl (Cbz, benzyloxycarbonyl), is reversible and predominantly of a competitive type. The enzyme is not activated by any of the compounds that typically stimulate the activities of the eukaryotic proteasome.     

Lectins from latices of Euphorbia and Elaeophorbia species

Crude latex sera from 17 members of the genus Euphorbia and from Elaeophorbia drupifera (Euphorbiaceae) contained a wide range of agglutinating abilities. Homogeneous lectins were isolated from latices of Euphorbia coerulescens, E. hermentiana, E. lactea, E. lactea cristata, E. lathyris, E. trigona and Elaeophorbia drupifera. The M,s of the lectins ranged from 60 to 67 000, and the unit weights from 27 to 38 000. pI measurements showed that each latex contained from five to 13 isolectins. The amino acid compositions of the seven lectins were determined: those from E. hermentiana, E. lactea, E. lactea cristata, E. trigona and Elaeophorbia drupifera are related.     

Biochemical properties of the major proteins from Rhodnius prolixus eggshell

Two proteins from the eggshell of Rhodnius prolixus were isolated, characterized and named Rp30 and Rp45 according to their molecular masses. Purified proteins were used to obtain specific antiserum which was later used for immunolocalization. The antiserum against Rp30 and Rp45 detected their presence inside the follicle cells, their secretion and their association with oocyte microvilli. Both proteins are expressed during the final stage of vitellogenesis, preserved during embryogenesis and discarded together with the eggshell. The amino terminals were sequenced and both proteins were further cloned using degenerated primers. The amino acid sequences appear to have a tripartite arrangement with a highly conserved central domain which presents a repetitive motif of valine-proline-valine (VPV) at intervals of 15 amino acid residues. Their amino acid sequence showed no similarity to any known eggshell protein. The expression of these proteins was also investigated; the results demonstrated that this occurred strictly in choriogenic follicles. Antifungal activity against Aspergillus niger was found to be associated with Rp45 but not with Rp30. A. niger exposed to Rp45 protein induced growth inhibition and several morphological changes such as large vacuoles, swollen mitochondria, multi-lamellar structures and a disorganized cell wall as demonstrated by electron microscopy analysis.     

Biochemical properties of lipoxygenase from opium poppy chloroplasts

Lipoxygenase (LOX) from opium poppy (Papaver somniferum L.) chloroplasts was isolated and 126.1-fold purified to electrophoretic homogeneity by combination of ion-exchange chromatography on HA-Ultragel column and affinity chromatography on a linoleyl-aminopropyl agarose column. The relative molecular mass of the LOX determined by SDS-PAGE was 92 kDa. Kinetic properties of purified LOX were determined in spectrophotometric assay by using of linoleic acid (K_M = 1.78 mM and V_max = 11.4 μmol mg⁻¹ min⁻¹) and linolenic acid (K_M = 1.27 mM and V_max = 10.2 μmol mg⁻¹ min⁻¹). The optimum pH was 6.0 for both linoleic and linolenic acid dioxygenation catalyzed by LOX. HPLC analysis of the products revealed a dual positional specificity of linoleic acid dioxygenation at pH 6.0 with ratio of 9- and 13-hydroperoxide products being about 1:1. The activity of purified LOX was stimulated by Mg²⁺ and Ca²⁺.     

Preliminary results on the ichthyocidal properties of Euphorbia ingens (Euphorbiaceae)

Euphorbia ingens belongs to the family Euphorbiaceae, which includes a variety of latex-producing plants, the majority of them having ichthyocidal properties (Coates-Palgrave 2000). Tests to determine the degree of virulence of E. ingens latex used Cyprinus carpio and Xenopus laevis as the main test organisms. Fish and frogs were exposed to various concentrations of latex to determine the concentration lethal only to the fish. The same concentrations were then used to determine the stability of the latex, by exposing fish in tanks with and without biological filtration. The breakdown period varied, with it breaking down quicker with biological filtration than without it. Once the stability of the latex within a system was determined, crabs, aquatic snails, frogs and fish were all simultaneously tested in a tank at the same concentration as before. Within 12 hours all the fish and half the frogs died, whereas the crabs and snails appeared not to suffer any detrimental effects. The poison degraded and became harmless to fish within 48 hours. Thus, the latex of Euphorbia ingens is a temporarily effective ichthyocide when applied in the correct concentrations.     

Biochemical properties of a beta-xylosidase from Clostridium cellulolyticum.

A 43-kDa beta-xylosidase from Clostridium cellulolyticum was purified to homogeneity. The enzyme releases xylose from p-nitrophenylxylose and xylodextrins with a degree of polymerization ranging between 2 and 5. The N-terminal amino acid sequence of the enzyme showed homologies with three other bacterial beta-xylosidases. By proton nuclear magnetic resonance spectroscopy, the enzyme was found to act by inverting the beta-anomeric configuration.     

Biochemical properties of penicillin amidohydrolase from Micrococcus luteus.

Some biochemical properties of whole-cell penicillin amidohydrolase from Micrococcus luteus have been studied. This whole-cell enzyme showed its maximal activity at 36 degrees C at pH 7.5. It was found that the activation energy of this enzyme was 8.03 kcal (ca. 33.6 kJ) per mol, and this amidohydrolase showed first-order decay at 36 degrees C. The penicillin amidohydrolase was deactivated rapidly at temperatures above 50 degrees C during storage or preincubation for 24 h. The Michaelis constant, Km, for penicillin G was determined as 2.26 mM, and the substrate inhibition constant, Kis, was 155 mM. The whole-cell penicillin amidohydrolase from M. luteus was capable of hydrolyzing penicillin G, penicillin V, ampicillin, and cephalexin, but not cephalosporin C and cloxacillin. This whole-cell enzyme also had synthetic activity for semisynthetic penicillins or cephalosporins from D-(--)-alpha-phenylglycine methyl ester and 6-alpha-aminopenicillanic acid or 7-amino-3-deacetoxycephalosporanic acid.     

Biochemical properties of penicillin amidohydrolase from Micrococcus luteus.

Some biochemical properties of whole-cell penicillin amidohydrolase from Micrococcus luteus have been studied. This whole-cell enzyme showed its maximal activity at 36 degrees C at pH 7.5. It was found that the activation energy of this enzyme was 8.03 kcal (ca. 33.6 kJ) per mol, and this amidohydrolase showed first-order decay at 36 degrees C. The penicillin amidohydrolase was deactivated rapidly at temperatures above 50 degrees C during storage or preincubation for 24 h. The Michaelis constant, Km, for penicillin G was determined as 2.26 mM, and the substrate inhibition constant, Kis, was 155 mM. The whole-cell penicillin amidohydrolase from M. luteus was capable of hydrolyzing penicillin G, penicillin V, ampicillin, and cephalexin, but not cephalosporin C and cloxacillin. This whole-cell enzyme also had synthetic activity for semisynthetic penicillins or cephalosporins from D-(--)-alpha-phenylglycine methyl ester and 6-alpha-aminopenicillanic acid or 7-amino-3-deacetoxycephalosporanic acid.     

Biochemical properties of alcohol dehydrogenase from Drosophila lebanonensis.

Purified Drosophila lebanonensis alcohol dehydrogenase (Adh) revealed one enzymically active zone in starch gel electrophoresis at pH 8.5. This zone was located on the cathode side of the origin. Incubation of D. lebanonensis Adh with NAD+ and acetone altered the electrophoretic pattern to more anodal migrating zones. D. lebanonensis Adh has an Mr of 56,000, a subunit of Mr of 28 000 and is a dimer with two active sites per enzyme molecule. This agrees with a polypeptide chain of 247 residues. Metal analysis by plasma emission spectroscopy indicated that this insect alcohol dehydrogenase is not a metalloenzyme. In studies of the substrate specificity and stereospecificity, D. lebanonensis Adh was more active with secondary than with primary alcohols. Both alkyl groups in the secondary alcohols interacted hydrophobically with the alcohol binding region of the active site. The catalytic centre activity for propan-2-ol was 7.4 s-1 and the maximum velocity of most secondary alcohols was approximately the same and indicative of rate-limiting enzyme-coenzyme dissociation. For primary alcohols the maximum velocity varied and was much lower than for secondary alcohols. The catalytic centre activity for ethanol was 2.4 s-1. With [2H6]ethanol a primary kinetic 2H isotope effect of 2.8 indicated that the interconversion of the ternary complexes was rate-limiting. Pyrazole was an ethanol-competitive inhibitor of the enzyme. The difference spectra of the enzyme-NAD+-pyrazole complex gave an absorption peak at 305 nm with epsilon 305 14.5 X 10(3) M-1 X cm-1. Concentrations and amounts of active enzyme can thus be determined. A kinetic rate assay to determine the concentration of enzyme active sites is also presented. This has been developed from active site concentrations established by titration at 305 nm of the enzyme and pyrazole with NAD+. In contrast with the amino acid composition, which indicated that D. lebanonensis Adh and the D. melanogaster alleloenzymes were not closely related, the enzymological studies showed that their active sites were similar although differing markedly from those of zinc alcohol dehydrogenases.     

Biochemical properties of alkaline phosphatase from endometrial cancer cells

The occurrence of alkaline phosphatase (AP) activity was examined in several human endometrial adenocarcinomas. Catalytic activities were detectable only in 10 out of 15 tumors, with no apparent correlation between elevated AP and histological type. The apparent molecular weight of the enzyme after partial purification was about 140,000 daltons. Kinetic activity, thermodynamic properties and the pH dependence of the activity were in the ranges reported for other subforms. Several other physicochemical properties were also investigated and compared with those displayed by enzymes obtained from normal human tissues. The inhibition studies show that the enzyme shares several properties with the placental form, particularly in resistance to zinc chloride and EDTA action. On the other hand, in sensitivity to uncompetitive inhibitors and to urea and ascorbic acid, it is closer to other non-Regan heat-sensitive forms. The results support the view that a polymorphism in the expression of AP in neoplastic tissues can occur. A wider spectrum of physicochemical properties is clearly needed to define better the characteristics of oncodevelopmental enzymes.     

Biochemical properties of a thermostable phytase from Neurospora crassa

A gene (Ncphy) encoding a putative phytase in Neurospora crassa was cloned and expressed in Pichia pastoris, and the biochemical properties of the recombinant protein were examined in relation to the phytic acid hydrolysis in animal feed. The recombinant phytase (rNcPhy) hydrolyzed phytic acid with a specific activity of 125 U mg-1, Km of 228 micromol L-1, Vmax of 0.31 nmol (phosphate) s-1 mg-1, a temperature optimum of 60 degrees C and a pH optimum of 5.5 and a second pH optimum of 3.5. The enzyme displayed pH stability around pH 3.5-9.5 and showed satisfactory thermostability at 80 degrees C. The phytase from N. crassa has potential for improving animal feed processing at higher temperatures.     

Biochemical properties of metalloproteinases from the hemolymph of the mussel Mytilus galloprovincialis Lam

The expression of matrix metalloproteinases (MMP) with gelatinase activity was found in the whole hemolymph of the marine mussel Mytilus galloprovincialis Lam. Cleavage activity was specific for gelatin; very little activity towards human type-IV collagen, and no activity for cold fish gelatin, casein or bovine serum albumin were detected. EDTA and 1,10-phenanthroline were inhibitory, suggesting that mussel MMPs require divalent cations for their proteolytic activity; in fact, the presence of exogenously added divalent ions significantly protected the MMPs from inhibition. No inhibition was detected with serine or cysteine proteinase inhibitors. The specific vertebrate inhibitors as well as the classical vertebrate activator of MMPs were without effect, whereas sulphydryl reducing agents had a strong inhibitory effect. Mussel MMPs showed an exponential curve of thermal-dependent decay that was not protected by the presence of metal ions. Overall the results indicate both similarities and differences between invertebrate and vertebrate gelatinases, providing information for understanding the biological role of these ancient proteinases.