A novel collagenase from Vibrio Alginolyticus: experimental study for Dupuytren's disease.

OBJECTIVE: Dupuytren contracture (DC) is a highly prevalent hand affection in which contracted fingers compromise hand function. It is a benign fibroproliferative condition affecting the hand palmar fascia with a deposition of excess matrix proteins in the extracellular space of the palmar aponeurosis. In particular type III over type I collagen V. Alginolyticus collagenase (CVA), is a new enzyme that is fully active on the collagen filaments and inactive on other components of the dermal extracellular matrix. The aim of this study is to evaluate the safety and effectiveness of an intra-lesional injection of CVA on an animal model of subcutaneous fibrosis mimicking the pathological anatomy of the cord of Dupuytren's disease. MATERIALS AND METHODS: We performed an in vivo study on 27 rats that were randomized into four groups, and we evaluated macroscopic and microscopic analysis examining the inflamed cell population and the extracellular matrix. RESULTS: In all cases, no skin necrosis, skin tears or wound dehiscence were recorded, demonstrating the safety of the CVA in contrast to group D which had full-thickness skin necrosis, and this is confirmed by the microscopic analysis of the samples treated with CVA, where no hematomas are found around the fibrotic area with the absence of leukocyte infiltrates and macrophages. CONCLUSIONS: CVA is confirmed to be selective for collagens I and III, reducing the risk of vascular lesions or skin ulcerations.

Combined use of WEB2170 and HBO therapy can reduce ischemia and reperfusion injury to the skeletal muscle in a rabbit model.

Ischemia/reperfusion injury is regarded as the main cause of failure in revascularization of limbs and transfer of free flaps in the so called nonreflow phenomenon. This type of damage is caused by the production of free radicals, above all, of neutrophils that release great quantities of extracellular superoxide through the action of a membrane enzyme. In our study we used 40 white rabbits. Rabbit rectus femoris muscle is perfused by a single artery and vein and is therefore a valuable model for study of ischemia-induced reperfusion injury of skeletal muscle. The objective of this study was to individualize a valid method of protection for the muscle from damage by ischemia-induced reperfusion injury. We have tested the effectiveness of WEB2170, a PAF antagonist, of hyperbaric oxygen therapy one (HBO), and of combined employment of WEB2170 and HBO. The results show that both PAF and HBO play important protective roles against damage from ischemia/reperfusion injury, and that the combined employment of both therapies has a synergistic effect. We propose therefore a new therapeutic protocol for the prevention of damage resulting from ischemia/reperfusion injury with the simultaneous employment of this PAF and HBO.

Simultaneous determination of naphazoline, diphenhydramine and phenylephrine in nasal solutions by capillary electrophoresis.

A capillary zone electrophoresis (CZE) method has been developed to separate and quantitate naphazoline (NAPH), dyphenhydramine (DIP) and phenylephrine (PHE) in nasal solutions. Samples were diluted 1:25 in ultrapure water and injected at the anodic end. A central composite design has been used to optimise the experimental conditions for a complete and fast separation of the active ingredients studied. Critical parameters such as voltage, pH and buffer concentration have been studied to evaluate how they affect responses such as resolution and migration times. Separation was performed on a silica capillary with 75 microm I.D. and 70 cm total length at an applied voltage of 17.7 kV with a phosphate run buffer of pH 3.72 and 0.063 mol l(-1). Calibration curves were prepared for NAPH, DIP and PHE. For each analyte, the correlation coefficients were >0.999 (n=15). The RSD% of six replicate injections for each analyte were reasonably good. The method was applied to the quantitation of the three components in a commercial dosage form. The proposed method has the advantage of needing a very simple sample pretreatment and being faster than a typical HPLC chromatographic method.

[Reporting echocardiography exams with the G8-Cardio ANMCO software]. / Refertazione dell'esame ecocardiografico con il software G8-Cardio ANMCO.

BACKGROUND: The availability of a common computerized program for echocardiographic study archiving and reporting at national and/or international level could make it possible to standardize the echo reports of different echocardiographic laboratories, and to use the wealth of data thus obtainable with echocardiography, and to exploit its capillary territorial distribution, with the aim of collecting echocardiographic data in a standard format for epidemiological, scientific and administrative purposes. METHODS: To develop such a software, an ad hoc joint National Association of Hospital Cardiologists and Italian Society of Echocardiography task force worked in conjunction with the Italian Branch of Agilent Technologies to standardize the phraseology of accepted echocardiographic terms and of the quantitative parameters derived from transthoracic and transesophageal echocardiographic examination at rest as well as during exercise and pharmacological stress, and to develop an ad hoc software. This echocardiographic study archiving and reporting program is part of the whole G8-Cardio ANMCO software developed to computerize the whole cardiological chart. The software has been developed by Agilent Technologies to provide a fast, easy-access and easy to use report generator for the non-computer specialist using DBMS Oracle 7.3 database and Power Builder 5.0 to develop a user-friendly interface. RESULTS: The number of qualitative and quantitative variables contained in the program is 733 for echocardiography at rest, while it depends on the stressor and on the length of the examination for the stress echo (dipyridamole 214-384, dobutamine 236-406, exercise 198-392). The program was tested and refined in our laboratory between November 1999 and May 2000. During this time period, 291 resting and 56 stress echocardiographic studies were reported and recorded in a database. On average, each resting echocardiographic study lasting 10 +/- 4 (range 5-17) min was recorded using 50 +/- 11 (range 33-67) variables and 41,566 bytes of hard-disk memory space. Stress echocardiographic studies, each lasting 7 +/- 5 (range 5-21) min, were recorded using 143 +/- 74 (range 38-194) variables and 38,531 bytes of hard-disk memory space. CONCLUSIONS: To our knowledge this software represents the first experience of a common computerized program for echo archiving and reporting carried out at national level.

Redox properties and acid-base equilibria of zucchini mavicyanin.

The reduction potential of mavicyanin isolated from zucchini peelings, which is a blue copper protein belonging to the subclass of the phytocyanins, has been determined through direct electrochemistry as a function of temperature and pH. The enthalpy and entropy changes accompanying protein reduction were found to be very similar with those determined previously for other phytocyanins and to differ remarkably from those of azurins and plastocyanins. This finding contributes to further characterize phytocyanins as a distinct cupredoxins family also on thermodynamic grounds and improves our understanding of how the reduction potential of these metal centers in proteins is modulated by coordinative and solvation properties. The E degrees' of mavicyanin is found to be sensitive to two acid-base equilibria at the extremes of pH. One occurs below pH 4, and is related to the protonation and detachment from the Cu(I) center of a histidine ligand. The other, observed above pH 8, causes a remarkable change in the electrostatic potential and/or the field strength around the copper.

Triterpenoid p-aminobenzoates from the seeds of zucchini.

An investigation of the triterpenoid fraction of zucchini seeds afforded two novel multiflorane p-aminobenzoates, identified as 7-epi zucchini factor A and debenzoyl zucchini factor B. Multiflorane p-aminobenzoates could not be detected in zucchini sprouts, which contained bryonolic acid as the only multiflorane constituent. No compound of this type could be obtained from adult plant parts (roots, stems, leaves).

Azide binding to the trinuclear copper center in laccase and ascorbate oxidase.

Azide binding to the blue copper oxidases laccase and ascorbate oxidase (AO) was investigated by electron paramagnetic resonance (EPR) and pulsed electron-nuclear double resonance (ENDOR) spectroscopies. As the laccase : azide molar ratio decreases from 1:1 to 1:7, the intensity of the type 2 (T2) Cu(II) EPR signal decreases and a signal at g approximately 1.9 appears. Temperature and microwave power dependent EPR measurements showed that this signal has a relatively short relaxation time and is therefore observed only below 40 K. A g approximately 1.97 signal, with similar saturation characteristics was found in the AO : azide (1:7) sample. The g < 2 signals in both proteins are assigned to an S = 1 dipolar coupled Cu(II) pair whereby the azide binding disrupts the anti-ferromagnetic coupling of the type 3 (T3) Cu(II) pair. Analysis of the position of the g < 2 signals suggests that the distance between the dipolar coupled Cu(II) pair is shorter in laccase than in AO. The proximity of T2 Cu(II) to the S = 1 Cu(II) pair enhances its relaxation rate, reducing its signal intensity relative to that of native protein. The disruption of the T3 anti-ferromagnetic coupling occurs only in part of the protein molecules, and in the remaining part a different azide binding mode is observed. The 130 K EPR spectra of AO and laccase with azide (1:7) exhibit, in addition to an unperturbed T2 Cu(II) signal, new features in the g parallel region that are attributed to a perturbed T2 in protein molecules where the anti-ferromagnetic coupling of T3 has not been disrupted. While these features are also apparent in the AO : azide sample at 10 K, they are absent in the EPR spectra of the laccase : azide sample measured in the range of 6-90 K. Moreover, pulsed ENDOR measurements carried out at 4.2 K on the latter exhibited only a reduction in the intensity of the 20 MHz peak of the 14N histidine coordinated to the T2 Cu(II) but did not resolve any significant changes that could indicate azide binding to this ion. The lack of T2 Cu(II) signal perturbation below 90 K in laccase may be due to temperature dependence of the coupling within the trinuclear : azide complex.

Inhibition of ascorbate oxidase by phenolic compounds. Enzymatic and spectroscopic studies.

Competitive inhibition by phenolic compounds of the ascorbic acid oxidation reaction catalyzed by ascorbate oxidase was investigated at pH 7.0 and 23.0 degrees C. Inhibition of p-nitrophenol is pH dependent over the range 5.0-8.0, with inhibitor binding favored at higher pH. Bulky substituents on the phenol nucleus reduce or prevent the inhibitory effect. The presence of phenol affects the binding characteristics of azide to the trinuclear cluster of the enzyme. In particular, binding of azide to type 2 copper is prevented, and the affinity of azide to type 3 copper is reduced. In addition, reduction of type 1 copper is observed upon prolonged incubation of ascorbate oxidase with excess phenol and azide, but not with phenol alone. It is proposed that binding of phenolic inhibitors occurs at or near the site where the substrate (ascorbate) binds. NMR relaxation measurements of the protons of phenols in the presence of ascorbate oxidase show paramagnetic effects due to the proximity of the bound inhibitor to a copper center, likely type 1 copper. Copper-proton distance estimates between this paramagnetic center and p-cresol or p-nitrophenol bound to ascorbate oxidase are between 4.4 and 5.9 A.

Laser flash photolysis experiments on the effects of freezing and salt addition on intramolecular electron transfer within one-electron reduced ascorbate oxidase.

Laser flash photolysis has been used to investigate the effects of freezing protein solutions and of adding various salts on the kinetics of one-electron photoreduction by 5-deazariboflavin semiquinone (5-DRFH.) of oxidized ascorbate oxidase (AO) from zucchini in 100 mM phosphate buffer (pH 7.0). The initial reaction between oxidized AO and 5-DRFH. is quite rapid (k approximately 10(8) M-1 s-1) and occurs at the blue Type I Cu center. Subsequent to this, a slower, protein concentration-independent intramolecular reoxidation of the Type I Cu is observed, with kET approximately 150 s-1, resulting in 40-50% reoxidation of the blue Cu center and the establishment of an electron transfer (ET) equilibrium between the various Cu centers in AO. When such a sample of AO was frozen overnight at -30 degrees C, flash photolysis of the thawed sample showed no effect on the kinetics of reduction of the Type I Cu by 5-DRFH. However, the rate constant for intramolecular ET decreased to a value of 2.7 s-1, with only 20% reoxidation of the Type I center. Reduction of the enzyme with ascorbic acid, followed by O2 oxidation, resulted in restoration of rapid intramolecular reoxidation (kET = 130 s-1), with 33% of the Type I Cu reduced by 5-DRFH. being reoxidized. These results are consistent with previous work which showed that samples of AO with initially low activity can be reactivated by ascorbic acid turnover in the presence of O2. When AO was frozen in the presence of ascorbic acid, similar inhibition of intramolecular ET was obtained, whereas upon turnover of this sample by further addition of ascorbic acid and exposure to O2, activity was not restored. The effects of addition of (NH4)2SO4, Na2SO4, NH4Cl, NaCl, KCl, and KF on the kinetics of Type I Cu reduction by 5-deazariboflavin semiquinone and on the subsequent intramolecular ET were also examined. A twofold increase in the bimolecular rate constant for reduction of the Type I Cu was observed for the two sodium salts at high concentrations (500 mM). Intramolecular ET was also significantly affected upon addition of all three chloride salts. Although the intramolecular ET rate constant was not altered, the fraction of reduced Type I Cu reoxidized by the trinuclear cluster decreased with increasing Cl- concentration, regardless of the cation. Total inhibition of intramolecular ET was observed at a significantly lower concentration of KF than observed with the Cl- salts. Sulfate ion had no effect on either parameter. These changes are thus ion specific, suggesting that they are related to ion binding by the protein, possibly at one of the coppers of the trinuclear cluster.

The type 2 copper of ascorbate oxidase.

Ascorbate oxidase, dissolved in Hepes or sodium phosphate buffers, was analyzed by EPR and activity measurements before and after storage at -30 degrees C and 77 K. The specific activity was somewhat higher in the phosphate buffer, about 3500-3700 Dawson units compared to about 3100 units of the enzyme dissolved in Hepes buffer. After storage at -30 degrees C the activity fell to 1400-2000 units in the phosphate buffer but only to 2600-2800 units in the Hepes buffer. Large changes occurred in the EPR spectrum of enzyme dissolved in the phosphate buffer after storing at -30 degrees C suggesting an alteration of the type 2 copper site. These changes were, however, reverted when the samples were thawed and rapidly frozen at 77 K. Copper analysis showed that about 50% of the total copper was EPR detected. The type 2 Cu2+ EPR intensity was in most samples close to 25% of the total EPR intensity. This low contribution of type 2 Cu2+ could not be changed if the enzyme was completely reduced and reoxidized, treated with Fe(CN)6(3), large excess of NaF, addition of 50% (v/v) ethylene glycol or dialyzed against 0.1 M Mes buffer (pH 5.5). Since the crystal structure shows that there are one each of types 1 and 2 copper in the monomers there must be another species with an EPR signal rather different from these two copper species. This signal is proposed to originate from some trinuclear centers. The EPR simulations show that it is possible to house a broad unresolved signal under the resolved type 1 and 2 signals so that the total integral becomes 50% of the total copper in the molecule.

Mavicyanin, a stellacyanin-like protein from zucchini peelings: primary structure and comparison with other cupredoxins.

The complete amino-acid sequence of mavicyanin, a small blue copper-containing glycoprotein isolated from zucchini peelings, is presented. The sequence of this cupredoxin was deduced from analysis of peptides obtained after cleavage of the protein with trypsin or Asp-N endoproteinase. Mavicyanin consists of a single polypeptide chain of 108 amino-acid residues. Accurate molecular weight determination by electrospray mass spectrometry (12 752 Da) indicates a mass difference of approx. 1005 Da with respect to the mass of the protein, as determined on the basis of the amino-acid sequence (11747 Da). This difference was tentatively assigned to the carbohydrate moiety, not yet characterized, attached to the protein via an N-linkage to Asn-58 and O-linkages to unidentified Ser/Thr residues. The comparison of the primary structure of mavicyanin with those of other cupredoxins shows that three copper ligands (His-44, Cys-57 and His-90) are conserved, while a glutamine residue (Gln-95), as in stellacyanin, is possibly the fourth ligand. An amino-acid sequence alignment of mavicyanin with copper proteins currently identified as phytocyanins is also proposed, showing same invariant residues in key positions related to the maintenance of the beta-barrel fold and to the active site.

Direct measurement by laser flash photolysis of intramolecular electron transfer in the three-electron reduced form of ascorbate oxidase from zucchini.

Ascorbate oxidase, which has been fully reduced by its substrate, can rapidly transfer a single electron to the laser-generated triplet state of 5-deazariboflavin. Subsequent to this, intramolecular electron transfer occurs resulting in the oxidation of the blue type I copper center. This latter process proceeds via biphasic kinetics, with observed rate constants of 9500 s-1 and 1400 s-1, both of which are protein concentration independent. This indicates that the initial oxidation reaction involves the type II, III trinuclear center, probably occurring via parallel reactions of two of the three copper atoms. The rate constants for intramolecular electron transfer in the three-electron reduced enzyme are one to two orders of magnitude larger than previously observed for the one-electron reduced enzyme, indicating a dramatic effect of the redox state of the enzyme on the intramolecular communication between the copper centers.

The chloroperoxidase-catalyzed oxidation of phenols. Mechanism, selectivity, and characterization of enzyme-substrate complexes.

The reactivity of a series of para-substituted phenolic compounds in the peroxidation catalyzed by chloroperoxidase was investigated, and the results were interpreted on the basis of the binding characteristics of the substrates to the active site of the enzyme. Marked selectivity effects are observed. These operate through charge, preventing phenolic compounds carrying amino groups on the substituent chain to act as substrates for the enzyme, and through size, excluding potential substrates containing bulky substituents to the phenol nucleus. Also, chiral recognition is exhibited by chloroperoxidase in the oxidation of N-acetyltyrosine, where only the L isomer is oxidized. Kinetic measurements show that, in general, the efficiency of chloroperoxidase in the oxidation of phenols is lower than that of horseradish peroxidase. Paramagnetic NMR spectra and relaxation rate measurements of chloroperoxidase-phenol complexes are consistent with binding of the substrates close to the heme, in the distal pocket, with the phenol group pointing toward the iron atom. On the other hand, phenolic compounds which are not substrates for chloroperoxidase bind to the enzyme with a much different disposition, with the phenol group very distant from the iron and probably actually outside the active-site cavity.

Oxidative turnover increases the rate constant and extent of intramolecular electron transfer in the multicopper enzymes, ascorbate oxidase and laccase.

Using laser flash photolysis of lumiflavin/EDTA solutions containing ascorbate oxidase, we find that the rate constant for intramolecular electron transfer varies from one enzyme preparation to another and is generally a more sensitive measure of the state of the active site than are steady-state assays. Thus, type I copper is initially reduced in a second-order reaction followed by first-order reoxidation by the type II-III trinuclear copper center. The observed rate constant for this intramolecular process in presumably native enzyme is 160 s-1 at pH 7, whereas an enzyme preparation which had less than 20% activity had a rate constant of 2.6 s-1. Other samples of relatively active enzyme showed biphasic intramolecular kinetics intermediate between the above values. The inactive enzyme sample could be reactivated by dialysis against ascorbate or by treatment with ferricyanide, resulting in a corresponding increase in the intramolecular rate constant for type I copper reoxidation to a value comparable to that of native enzyme. Using this same methodology, we have determined that the type I copper in Japanese lacquer tree laccase is reoxidized by the type II-III trinuclear copper center in a first-order (intramolecular) process with rate constants of 1 s-1 at pH 7.0 and 4.9 s-1 at pH 6.0, values which are approximately two orders of magnitude smaller than for ascorbate oxidase. The intramolecular rate constant and enzyme activity for laccase also increased, but only by a factor of 2-6, when the enzyme was treated with ascorbate or ferricyanide, respectively. We further found that intramolecular electron transfer in laccase was completely inhibited by fluoride ion, in contrast to ascorbate oxidase which is unaffected by this ion. These differences in behavior for these two very similar enzymes are rather remarkable, when it is considered that the distance between copper atoms is constrained by the location of the protein-derived copper ligands in the three-dimensional structure, and that the redox potentials of the enzymes are similar. Our results may be interpreted in terms of an interconversion between active and inactive enzyme in which there is a rearrangement of the type II-III trinuclear copper center, resulting in a lowering of the redox potential and a block in electron transfer. Turnover restores the active enzyme conformation.(ABSTRACT TRUNCATED AT 400 WORDS)

Spectroscopic and binding studies on the stereoselective interaction of tyrosine with horseradish peroxidase and lactoperoxidase.

The interaction of a series of derivatives of tyrosine with horseradish peroxidase (HRP) and lactoperoxidase (LPO) was studied by using optical difference spectroscopy, c.d. and proton n.m.r. spectroscopy in order to reveal differences in the mode of binding of L-tyrosine and D-tyrosine, which are substrates of but react at different rates with the two peroxidases, to HRP and LPO. All the donor molecules form 1:1 complexes with HRP and LPO, but they display a range of affinities for the enzymes. Whereas D-tyrosine binds to HRP more strongly than does L-tyrosine, the opposite holds for the binding to LPO. The distances of the protons of bound tyrosine molecules from the haem iron atoms of HRP and LPO indicate that the site of binding of these substrates is the same as that of simple phenols. This involves the interaction of the phenol nucleus with a protein tyrosine residue [Sakurada, Takahashi & Hosoya (1986) J. Biol. Chem. 261, 9657-9662; Modi, Behere & Mitra (1989) Biochim. Biophys. Acta 996, 214-225]. However, for the present substrates the additional interaction of the carboxylate group with a protein residue (probably an arginine residue) provides further stabilization for the adducts HRP-D-tyrosine and LPO-L-tyrosine with respect to the corresponding complexes with the opposite enantiomers. The differences in the mode of binding of L-tyrosine and D-tyrosine to HRP and LPO is thus determined by the fact that the spatial arrangement of the interacting protein residues can recognize the chirality of the C(alpha)-CO2- and C(beta)-C6H4OH attachment bonds of the substrates.

A new insight into the histogenesis of 'mesodermomas'--malignant mesotheliomas.

A myogenic phenotype was induced in cultures of human mesothelial cells treated for 72 h with atrazine, a triazine derivative. Immunoreactivity for both myosin and myoglobin was detected in a large number of these cells, irrespective of their polygonal or spindle morphology, whereas no expression of desmin was observed. These findings support the embryological identity of mesothelium and mesoderm, the former being, in the post-embryonic stage, potentially capable of differentiation along the same lineages which the latter normally displays during embryogenesis. In the light of this concept it can be assumed that primary malignancies arising from the mesothelium have the competence to express the pluripotent nature of embryonic mesoderm, and hence the term mesodermoma is appropriate for this group of tumours, including mesotheliomas in a classical sense. A postulated mechanism for the phenotypic change of mesothelial cells is also outlined, involving atrazine conversion to 5-aza-chloro-cytidine, a probable DNA hypomethylating and gene activating agent, like its analogue 5-azacytidine.

Direct measurement of intramolecular electron transfer between type I and type III copper centers in the multi-copper enzyme ascorbate oxidase and its type II copper-depleted and cyanide-inhibited forms.

Transient kinetics of reduction of zucchini squash ascorbate oxidase (AO) by lumiflavin semiquinone have been studied by using laser flash photolysis. Second-order kinetics were obtained for reduction of the type I copper with a rate constant of 2.7 X 10(7) M-1 s-1, which is comparable to that obtained with other blue copper proteins such as plastocyanin. Following reduction, the type I copper was reoxidized in a protein concentration independent (i.e., intramolecular) reaction (kobs = 160 s-1). Comparison with literature values for limiting rate constants in transient single-turnover kinetic experiments suggests that intramolecular electron transfer probably is the rate-limiting step in enzyme catalysis. The extent of reoxidation of type I copper was approximately 55%, which is consistent with the approximately equal redox potentials of the type I and type III copper centers. Neither azide nor fluoride caused any significant changes in kinetics, although they are enzyme inhibitors and are thought to bind to the type II copper. In contrast, cyanide caused a concentration-dependent decrease in the extent of intramolecular electron transfer (with no change in rate constant), and decreased the rate constant for reduction of the type I copper by a factor of 2. The apparent dissociation constant for cyanide (0.2-0.4 mM) is similar to that reported for inhibition of enzyme activity. Removal of the type II copper from AO only marginally affected the kinetics of electron transfer to type I copper (k = 3.2 x 10(7) M-1 s-1) and slightly increased the extent but did not alter the rate constant of intramolecular electron transfer.(ABSTRACT TRUNCATED AT 250 WORDS)

Preventing ascorbate interference in ion-chromatographic determinations of urinary oxalate: four methods compared.

In an attempt to decrease ascorbate interference on the ion-chromatographic determination of urinary oxalate, we compared the effectiveness of four different methods for ascorbate elimination by analyzing a representative urine pool supplemented with successive ascorbate additions. Two of the methods--treatment with ferric ions or boric acid--have been described elsewhere; treatments with nitrites or ascorbate oxidase (EC 1.10.3.3) are investigated here as possible alternatives. Consideration of the main features, advantages, and drawbacks of the four procedures leads us to conclude that boric acid dilution is a good routine method and that pre-incubation with ascorbate oxidase reliably prevents ascorbate interference in assays of urinary oxalate.

Flavonoid Accumulation Is Correlated with Adventitious Roots Formation in Eucalyptus gunnii Hook Micropropagated through Axillary Bud Stimulation.

Eucalyptus gunnii Hook microcuttings, obtained in vitro through axillary bud stimulation, show different rooting responses on the same rooting medium depending on the physiological state induced by cytokinins used in the previous multiplication medium. 6-Furfurylamino purine and 6-(4-hydroxy-3-methylbut-2-enylamino)purine induced a physiological state characterized by high sensitivity of microcuttings to the rooting stimulus exerted by the auxin 3-indolebutyric acid, but N(6)-benzyladenine did not produce the same effect. The former physiological state was characterized by an increased accumulation of two endogenous flavonoids (identified as quercetin glycosides) which may be markers of a well defined physiological state. They could have some direct influence on the rooting processes of the explants cultivated in vitro.

X-ray crystal structure of the blue oxidase ascorbate oxidase from zucchini. Analysis of the polypeptide fold and a model of the copper sites and ligands.

Two crystal forms of the multi-copper protein ascorbate oxidase from Zucchini have been analysed at 2.5 A (1 A = 0.1 nm) resolution and a model of the polypeptide chain and the copper ions and their ligands has been built. Crystal forms M2 and M1 contain a dimer of 140,000 Mr and a tetramer of 280,000 Mr, respectively, in the asymmetric unit. The crystallographic analysis proceeded by multiple isomorphous replacement in M2 followed by solvent flattening and averaging about the local dyad axis. M1 was solved by Patterson search techniques using the M2 electron density. M1 was fourfold averaged. M1 and M2 were combined and the process of averaging repeated in cycles. An atomic model was built into the resulting electron density map and refinement initiated. The current R values of M2 and M1 are 24.5% and 32.6%, respectively. Excellent stereo chemistry was maintained, with root-mean-square deviations of bond lengths and bond angles from average values of 0.02 A and 3.1 degrees, respectively. Each subunit of about 550 amino acid residues has a globular shape with dimensions of 49 A x 53 A x 65 A. It is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type. It is distantly related to plastocyanin. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine, and a methionine ligand and represents the type-1 copper. It is located in the third domain. The trinuclear cluster has eight histidine ligands. It may be subdivided into a pair of copper atoms with six histidine ligands arranged trigonal prismatic. The pair probably represents the type-3 copper. The remaining copper has two histidine ligands. Its third site of co-ordination is formed by the pair of copper atoms. The fourth ligand may be OH- represented by a small protrusion of electron density. This copper probably is the type-2 copper. The symmetry of the trinuclear cluster is C2 and the ligands are supplied symmetrically by domains 1 and 3. However, domain 1 does not contain a type-1 copper and lacks the characteristic ligands. The unprecedented trinuclear cluster probably represents the oxygen binding and electron storage site.