Thermogravimetric kinetics of corn stalk pretreated by oleaginous fungi Cunninghamella echinulata

The thermogravimetric and composition of corn stalk pretreated by oleaginous fungi Cunninghamella echinulata had been studied in this paper. Results indicated that pretreatment by oleaginous fungi C. echinulata could decrease the activation energy and make the pyrolysis more efficient and energy-saving. By bio-pretreatment, the contents of elements agreed with the weight loss, sugar content, and oil contents, especially the sulfur content was greatly decreased, greatly eliminating the inventory of gas contamination such as the emission of SOx and making the pyrolysis more environmentally friendly. Therefor, corn stalk with sugar pretreated by oleaginous fungi C. echinulata should be a good pyrolysis material to obtain high quality bio-oil.     

Bioconversion of Stemodia maritima diterpenes and derivatives by Cunninghamella echinulata var. elegans and Phanerochaete chrysosporium

Stemodane and stemarane diterpenes isolated from the plant Stemodia maritima and their dimethylcarbamate derivatives were fed to growing cultures of the fungi Cunninghamella echinulata var. elegans ATCC 8688a and Phanerochaete chrysosporium ATCC 24725. C. echinulata transformed stemodin (1) to its 7alpha-hydroxy- (2), 7beta-hydroxy- (3) and 3beta-hydroxy- (4) analogues. 2alpha-(N,N-Dimethylcarbamoxy)-13-hydroxystemodane (6) gave 2alpha-(N,N-dimethylcarbamoxy)-6alpha,13-dihydroxystemodane (7) and 2alpha-(N,N-dimethylcarbamoxy)-7alpha,13-dihydroxystemodane (8). Stemodinone (9) yielded 14-hydroxy-(10) and 7beta-hydroxy- (11) congeners along with 1, 2 and 3. Stemarin (13) was converted to the hitherto unreported 6alpha,13-dihydroxystemaran-19-oic acid (18). 19-(N,N-Dimethylcarbamoxy)-13-hydroxystemarane (14) yielded 13-hydroxystemaran-19-oic acid (17) along with the two metabolites: 19-(N,N-dimethylcarbamoxy)-2beta,13-dihydroxystemarane (15) and 19-(N,N-dimethylcarbamoxy)-2beta,8,13-trihydroxystemarane (16). P. chrysosporium converted 1 into 3, 4 and 2alpha,11beta,13-trihydroxystemodane (5). The dimethylcarbamate (6) was not transformed by this microorganism. Stemodinone (9) was hydroxylated at C-19 to give 12. Both stemarin (13) and its dimethylcarbamate (14) were recovered unchanged after incubation with Phanerochaete.     

Differential stereoselective pharmacokinetics of pantoprazole,a proton pump inhibitor in extensive and poor metabolizers of pantoprazole—a preliminary study

Pantoprazole (PAN) is a proton pump inhibitor that is administered as a racemic mixture. The pharmacokinetics of PAN enantiomers were investigated in extensive metabolizers (EMs) and apparent poor metabolizers (PMs) of PAN who received a single 40, 60, or 80 mg oral dose of racemic PAN as enteric-coated formulation. In the EMs, the serum concentrations of (−)-PAN were slightly higher than those of (+)-PAN at each dose level. The (+)/(−) ratios for the area under the concentration-time curve (AUC) and the half-life were 0.58–0.89 and 0.62–0.88, respectively. In the PMs, the serum concentrations of both enantiomers were much higher than those in the EMs at each dose level and significant differences in pharmacokinetics of (+)- and (−)-PAN were observed. The half-lives for (+)-PAN were 2.67–3.77 times longer than those for (−)-PAN. The AUCs for (+)-PAN were 2.65–3.45 times greater than those for (−)-PAN. Therefore, the metabolism of (+)-PAN is impaired to a greater extent than (−)-PAN in the PMs, which resulted in the stereoselective disposition of PAN in the PMs. It has been suggested that the EMs and the PMs of PAN could be differentiated by determining the (+)/(−) enantiomer ratio in serum at one time point, possibly 2–6 h after oral dosing, because the (+)/(−) enantiomer ratios in the PMs were opposite those in the EM subjects. Chirality 9:17–21, 1997 © 1997 Wiley-Liss, Inc.     

Biotransformation of albendazole by Cunninghamella blakesleeana: effect of carbon and nitrogen source

A filamentous fungus Cunninghamella blakesleeana was screened for its ability to biotransform the anthelmintic drug albendazole. The fungus produced three metabolites in the presence of the carbon and nitrogen sources studied. The transformation was identified by HPLC and the structures of the transformation products were assigned by LC-MS-MS analysis and on the basis of previous reports. The results indicated that the fungus metabolized albendazole into albendazole sulfoxide (M1), albendazole sulfone (M2) and an N-methyl metabolite of albendazole sulfoxide (M3). The effect of carbon and nitrogen source on the biotransformation of albendazole was significant. Among the carbon and nitrogen sources studied, fructose and urea respectively produced maximum extent of biotransformation in terms of substrate depletion. Among the carbon sources studied, maltose produced a higher percentage of M1 whereas M2 and M3 were produced to their maximum extent in presence of d-fructose in terms of metabolite per unit quantity of biomass. In the case of nitrogen sources, ammonium acetate, calcium nitrate and barium nitrate produced maximum percentage of M1, M2 and M3 respectively. The results reveal that the carbon and nitrogen source significantly influence the microbial transformation both qualitatively and quantitatively.     

Characterization of a novel cellulose synthesis inhibitor

The physiological effects of an experimental herbicide and cellulose synthesis inhibitor, N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine, called AE F150944, are described. In the aminotriazine molecular class, AE F150944 is structurally distinct from other known cellulose synthesis inhibitors. It specifically inhibits crystalline cellulose synthesis in plants without affecting other processes that were tested. The effects of AE F150944 on dicotyledonous plants were tested on cultured mesophyll cells of Zinnia elegans L. cv. Envy, which can be selectively induced to expand via primary wall synthesis or to differentiate into tracheary elements via secondary wall synthesis. The IC₅₀ values during primary and secondary wall synthesis in Z. elegans were 3.91×10^–8 M and 3.67×10^–9 M, respectively. The IC₅₀ in suspension cultures of the monocot Sorghum halapense (L.) Pers., which were dividing and synthesizing primary walls, was 1.67×10^–10 M. At maximally inhibitory concentrations, 18–33% residual crystalline cellulose synthesis activity remained, with the most residual activity observed during primary wall synthesis in Z. elegans. Addition to Z. elegans cells of two other cellulose synthesis inhibitors, 1 M 2,6-dichlorobenzonitrile and isoxaben, along with AE F150944 did not eliminate the residual cellulose synthesis, indicating little synergy between the three inhibitors. In differentiating tracheary elements, AE F150944 inhibited the deposition of detectable cellulose into patterned secondary wall thickenings, which was correlated with delocalization of lignin as described previously for 2, 6-dichlorobenzonitrile. Freeze-fracture electron microscopy showed that the plasma membrane below the patterned thickenings of AE F150944-treated tracheary elements was depleted of cellulose-synthase-containing rosettes, which appeared to be inserted intact into the plasma membrane followed by their rapid disaggregation. AE F150944 also inhibited cellulose-dependent growth in the rosette-containing alga, Spirogyra pratensis, but it did not inhibit cellulose synthesis in Acetobacter xylinum or Dictyostelium discoideum, both of which synthesize cellulose via linear terminal complexes. Therefore, AE F150944 may inhibit crystalline cellulose synthesis by destabilizing plasma membrane rosettes.Abbreviations AE F150944 N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine - CBI cellulose biosynthesis inhibiting - CGA CGA 325615, 1-cyclohexyl-5-(2,3,4,5,6-pentafluorophenoxy)-14,2,4,6-thiatriazin-3-amine - DCB 2,6-dichlorobenzonitrile - TE tracheary element     

Rapid oxidation of ring methyl groups is the primary mechanism of biotransformation of gemfibrozil by the fungus <Emphasis Type="Italic">Cunninghamella elegans</Emphasis>

The hypolipidemic agent gemfibrozil (GEM), which has been studied for its metabolism in humans and animals, was investigated to elucidate its primary metabolism by Cunninghamella elegans. The fungus produced ten metabolites (FM1–FM9 and FM6′) from the biotransformation of GEM. Based on LC/MS/MS and NMR analyses, a major metabolite, FM7, was identified as 2′-hydroxymethyl GEM. FM6 was considered to be 5′-hydroxymethyl GEM, after comparison of results LC/MS, LC/MS/MS, and UV absorption spectra to FM7. The combined concentration of FM6 and FM7 was found to increase up to 0.83 mM by day 2, and then decreased gradually with incubation time, followed by a noticeable increase in the biotransformation product, FM1, up to 0.86 mM by day 15. NMR analyses confirmed that FM1 was 2′,5′-dihydroxymethyl GEM. Further minor oxidations of the aromatic ring and carboxylic acid intermediates were also detected. Based upon these findings, the major fungal metabolic pathway for GEM is likely to occur via production of 2′,5′-dihydroxymethyl GEM from 2′-hydroxymethyl GEM. These relatively rapid and diverse biotransformations of GEM by C. elegans suggest that depending upon conditions, it may also follow a similar biodegradation fate when released into the natural environment.     

Role of cooperative H(+)/e(-) linkage (redox bohr effect) at heme a/Cu(A) and heme a(3)/Cu(B) in the proton pump of cytochrome c oxidase

It is a pleasure to contribute to the special issue published in honor of Vladimir Skulachev, a distinguished scientist who greatly contributes to maintain a high standard of biochemical research in Russia. A more particular reason can be found in his work (Artzabanov, V. Y., Konstantinov, A. A., and Skulachev, V. P. (1978) FEBS Lett., 87, 180–185), where observations anticipating some ideas presented in my article were reported. Cytochrome c oxidase exhibits protonmotive, redox linked allosteric cooperativity. Experimental observations on soluble bovine cytochrome c oxidase are presented showing that oxido-reduction of heme a/Cu_A and heme a ₃/Cu_B is linked to deprotonation/protonation of two clusters of protolytic groups, A₁ and A₂, respectively. This cooperative linkage (redox Bohr effect) results in the translocation of 1 H⁺/oxidase molecule upon oxido-reduction of heme a/Cu_A and heme a ₃/Cu_B, respectively. Results on liposome-reconstituted oxidase show that upon oxidation of heme a/Cu_A and heme a ₃/Cu_B protons from A₁ and A₂ are released in the outer aqueous phase. A₁ but not A₂ appears to take up protons from the inner aqueous space upon reduction of the respective redox center. A cooperative model is presented in which the A₁ and A₂ clusters, operating in close sequence, constitute together the gate of the proton pump in cytochrome c oxidase.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 220–230.Original Russian Text Copyright © 2005 by Papa.This revised version was published online in April 2005 with corrections to the post codes.     

A reaction cycle for cytochrome c oxidase as an electron-transport-driven proton pump: The effect of electrochemical potential and slips

A detailed reaction cycle for cytochrome oxidase, an electron-transport-driven proton pump, has been presented earlier by our research group. The essential feature of the model is that both cytochrome a and Cu_A must be reduced in order to allow the transition from the electron and proton input state to the output state. The model is thus based on an indirect coupling between electron transfer and proton translocation.In this study, the same model is examined with respect to (1) intrinsic electron and proton leaks and (2) the effect of applying an electrochemical potential gradient on the pump incorporated in a membrane, both with respect to the electrical and chemical components.The model is successfully used to simulate various experimental results. Comparisons of experimental results with simulations based on the model support the existence of electron and proton leaks. The analysis of electron leaks suggests that electron gating is best achieved by varying the reorganization energy rather than by varying the reduction potentials.It is also suggested that both the electrical and chemical components of the electrochemical potential gradient are responsible for the regulation of the enzyme activity. Furthermore, an attempt is made to interpret the seemingly contradictory results obtained when measuring the pH dependence of the reduction potential of cytochrome a. In addition, the simulations support the assumption that protons are pumped by a mechanism that combines a membrane Bohr effect with the transition-state mechanism.Abbreviations R molar gas constant - k _B Boltzmann contant - F Faraday constant - e elementary charge - T absolute temperature - transmembrane electrochemical potential gradient - pH transmembrane pH difference - pH₁ and pH₂ inside (matrix) and outside (cytosol) pH, respectively - transmembrane electrical potential - E _m midpoint potential     

Inhibition of microbiological sulfide oxidation by methanethiol and dimethyl polysulfides at natron-alkaline conditions

To avoid problems related to the discharge of sulfidic spent caustics, a biotechnological process is developed for the treatment of gases containing both hydrogen sulfide and methanethiol. The process operates at natron-alkaline conditions (>1 mol L⁻¹ of sodium- and potassium carbonates and a pH of 8.5–10) to enable the treatment of gases with a high partial CO₂ pressure. In the process, methanethiol reacts with biologically produced sulfur particles to form a complex mixture predominantly consisting of inorganic polysulfides, dimethyl disulfide (DMDS), and dimethyl trisulfide (DMTS). The effect of these organic sulfur compounds on the biological oxidation of sulfide to elemental sulfur was studied with natron-alkaliphilic bacteria belonging to the genus Thioalkalivibrio. Biological oxidation rates were reduced by 50% at 0.05 mM methanethiol, while for DMDS and DMTS, this was estimated to occur at 1.5 and 1.0 mM, respectively. The inhibiting effect of methanethiol on biological sulfide oxidation diminished due to its reaction with biologically produced sulfur particles. This reaction increases the feasibility of biotechnological treatment of gases containing both hydrogen sulfide and methanethiol at natron-alkaline conditions.     

The influence of acetate on the oxidation of sulfide by Rhodopseudomonas capsulata

The capacity to oxidize sulfide and the influence of the simultaneous presence of acetate in heterotrophically (acetate) and autotrophically (sulfide/CO₂) grown Rhodopseudomonas capsulata was investigated.Sulfide oxidation of acetate-limited cultures was found inversely related to the specific growth rate. Upon acetate deprevation (metering pump stopped) increased rates of sulfide oxidation were observed. This points to the existence of a constitutive acceptor for the electrons from sulfide. It is suggested that a carrier functional in the light-induced cyclic electron flow operates as such. The rate of sulfide oxidation, however, is low when compared to autotrophically-grown cells. This is probably due to the low levels of Calvin cycle enzymes present in the acetate-grown cells.In cells growing on sulfide/CO₂, the addition of acetate resulted in less sulfide being oxidized. Upon depletion of the acetate, the rate of sulfide oxidation again increased, however, insufficiently to maintain the accelerated growth rate. This indicates that under mixotrophic conditions the enzymes of the Calvin cycle are being synthesized to a far lesser extent.Non-Standurd Abbreviations PHB poly--hydroxybutyric acid - D dilution rate - TCA Tri carboxylic acid cycle - RubPcase ribulose 1,5-bisphosphate carboxylase - RP reducing power     

来源于西梅的(R)-醇腈酶促立体选择性转氰合成(R)-酮醇腈

在水/有机溶剂双相反应体系中,研究了来源于西梅的(R)-醇腈酶催化酮与丙酮醇腈合成(R)-酮醇腈的立体选择性转氰反应．系统探讨了不同酶源、酶粉颗粒大小、底物浓度、两底物配比、酶浓度和底物结构对转氰反应的影响．结果发现西梅醇腈酶能高效催化三甲基硅酮与丙酮醇腈的立体选择性转氰．酶粉颗粒大小以直径0.3～0.45 mm为优,底物浓度以21 mmol/L左右为佳,底物丙酮醇腈与三甲基硅酮摩尔浓度比以2∶1为宜,酶浓度以60.9 g/L左右为好．西梅醇腈酶对3, 3-二甲基-2-丁酮几乎没有催化活性,而对其硅结构类似物三甲基硅酮却具有非常高的立体选择性和催化活性,在上述优化反应条件下反应24 h的底物转化率和产物光学纯度均高达99%以上,表明底物中的硅原子对西梅醇腈酶的催化活性有非常显著的促进作用．     

The expression of a mammalian proteinase inhibitor,bovine spleen trypsin inhibitor in tobacco and its effects on Helicoverpa armigera larvae

The cDNA for bovine spleen trypsin inhibitor (SI), a homologue of bovine pancreatic trypsin inhibitor (BPTI), including the natural mammalian presequence was expressed in tobacco using Agrobacterium tumefaciens-mediated transformation. Stable expression required the N-terminal targeting signal presequence although subcellular localization was not proven. SI was found to exist as two forms, one coinciding with authentic BPTI on western blots and the second marginally larger due to retention of the C-terminal peptide. Both were retained on a trypsin-agarose affinity gel and had inhibitory activity. Newly emergent leaves contained predominantly the large form whereas senescent leaves had little except the fully processed form present. Intermediate-aged leaves showed a gradual change indicating that a slow processing of the inhibitor peptide was occurring. The stability of SI was shown by the presence of protein at high levels in completely senescent leaves. Modifications to the cDNA (3 and 5 changes and minor codon changes) resulted in a 20-fold variation in expression. Expression of modified SI in transgenic tobacco leaves at 0.5% total soluble protein reduced both survival and growth of Helicoverpa armigera larvae feeding on leaves from the late first instar. In larvae surviving for 8 days, midgut trypsin activity was reduced in SI-tobacco fed larvae, while chymotrypsin activity was increased. Activities of leucine aminopeptidase and elastase-like chymotrypsin remained unaltered. The use of SI as an insect resistance factor is discussed.     

Identification of a V-type proton pump in the outer mantle epithelium of Anodonta cygnea

The dissected outer mantle epithelium (OME) of Anodonta cygnea, when mounted in Ussing type chambers, generated a spontaneous potential difference of 22.0±12.6 mV and, when short-circuited, a positive current (I_sc) of 30.0±11 μA/cm² towards the shell side and a conductance of 1.1±0.4 mS/cm². When in contact with the shell side, Bafilomycin A₁ and Concanamycin A, specific inhibitors of V-proton pumps, induced 90% inhibition of I_sc in 5 and 35 min, respectively. They had no effect in the current from the hemolymph side. Both drugs induced a dramatic fall in conductance from the shell side. Tributyltin oxide (TBTO) inhibited 90% of the I_sc in 3 min and induced a fall in conductance only from the shell side. Two observations suggest a direct effect on the proton pump: it was effective only from the shell side and the time course of the effect was identical to that of Bafilomicyn A₁. Dicyclohexycarbodiimide (DCCD) and N-ethylmaleimide (NEM) inhibited I_sc from both sides but very slowly and there was a delay of the effect from the hemolymph side in relation to the shell side. Taken together, the results suggest the presence of a V-type proton pump located at the apical (shell side) barrier of the OME.     

Determination of hydrogen sulfide in a yeast culture solution by flow analysis utilising methylene blue spectrophotometric detection

Gas diffusion and flow injection analysis with a spectrophotometric detector has been developed for the determination of sulfide in a yeast culture solution. A detection limit (signal-to-noise ratio = 3) of 0.2 M sulfide was achieved. A relative standard deviation of 3.4% (n=12) was also achieved for 0.5 M sulfide. The technique is highly sensitive, accurate and precise, with low susceptibility to interferences, and allows significant reagent and instrument economy.     

Microbial conversion of n-alkanes into glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma (Candida antarctica)

n-Alkanes ranging from C₁₂ to C₁₈ were converted into glycolipid biosurfactants, mannosylerythritol lipids (MEL), by resting cells of Pseudozyma (Candida) antarctica T-34. The highest yield (0.87 g g^–1 substrate) was obtained from 6% (v/v) of n-octadecane after 7 days reaction. The amount of MEL reached 140 g l^–1 by intermittent feeding of the substrate.     

Identification of a Kunitz inhibitor from Albizzia kalkora and its inhibitory effect against pest midgut proteases

A purification protocol, involving water extraction, ammonium sulfate precipitation, Sepharose 4B-trypsin affinity and FPLC Superdex G-75 chromatography, was employed to isolate a trypsin inhibitor from Albizzia kalkora seeds. The inhibitor, which had a molecular mass of 19,768.23 Da, consisted of two disulfide-linked polypeptide chains with approximate molecular mass of 15.5 and 4.5 kDa, respectively. It was stable from pH 2-12 for 24 h, whereas it was unstable either above 80 degrees C for 10 min or under reduced condition over 60 min. The inhibitor, which inhibited trypsin activity with an apparent K (i) of 2.5 x 10(-7) M, had one reactive site involved with a lysine residue. Disulfide linkage and lysine residue were important in maintaining its active conformation. Partial amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz inhibitor family. Moreover, trypsin-like proteases from larval Helicoverpa armigera, Spodoptera exigua, and Pieris rapae were inhibited for 85, 57, and 68% respectively, by the inhibitor at 45 microg ml(-1).     

Protein synthesis by Beggiatoa alba B18LD in the presence and absence of sulfide

The interaction of sulfide oxidation and protein synthesis by Beggiatoa alba B18LD was investigated using the incorporation of radiolabeled leucine to estimate protein synthesis. Leucine was assimilated into whole cells in the presence of 6.1 mM acetate at a rate of 0.6 nmol · min^-1 · mg protein^-1, 43% of which was incorporated into the protein fraction. Protein synthesis by B. alba was unaffected by 1 mM sulfide, whether or not the cells had been preincubated with sulfide. B. alba oxidized radioactive sulfide to sulfur within 30 s of addition of the label, whether or not the organism was preinduced by sulfide. Furthermore, chloramphenicol, which inhibited protein synthesis, did not significantly inhibit sulfide oxidation by sulfide-induced or uninduced B. alba. This indicates that sulfide oxidation is a constitutive process. Enrichments of sulfur inclusions from B. alba B18LD that were analyzed by polyacrylamide gel electrophoresis demonstrated two enriched peptides with M_r values of 13,000 and 15,000. The 13,000 and 15,000 M_r peptide bands were more evident in cells grown in a medium containing sulfide than in cells from a medium lacking sulfide. Although sulfide did not increase the rate of overall protein synthesis, the synthesis of a few peptides was increased by the addition of sulfide to the growth medium. Among those, the 15,000 M_r peptide was one of the most distinctive.Non-standard abbreviations SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis - PPO 2,5-diphenyloxazole - POPOP 1,4-bis [5-phenyl-2-oxazolyl]-benzene - BSS basal salts solution - BH Beggiatoa heterotrophic (medium) - BSO Beggiatoa sulfide oxidation (medium) - CM chloramphenicol - TCA trichloroacetic acid - M_r molecular mass     

Association of a 19- and a 21-kDa GTP-binding protein to pancreatic microsomal vesicles is regulated by the intravesicular pH established by a vacuolar-type H+-ATPase

Summary Evidence suggests that certain ras-related small molecular weight GTP-binding proteins (smg-proteins) are involved in intracellular membrane trafficking and vesicle fusion. We have previously shown that intravesicular acidification due to a vacuolar-type H⁺-ATPase, which is Cl^– dependent and highly sensitive to the specific inhibitor bafilomycin, enhances GTP-induced fusion of pancreatic microsomal vesicles (Hampe, W., Zimmermann, P., Schulz, I. 1990. FEBS Lett. 271:62–66). This process may involve function of smg-proteins. The present study shows that MgATP (2 mm), but neither MgATPS nor ATP in the absence of Mg²⁺, increases association of 19- and 21-kDa smg-proteins to the vesicle membrane as monitored by their [-³²P]GTP binding. The affinity of smg-proteins for [-³²P]GTP was not altered by MgATP. Bafilomycin B₁ (10^–8 m), the protonophore CCCP (10^–5 m), and replacement of Cl^– in the incubation buffer by CH₃COO^– or NO ₃ ^– resulted in an almost complete inhibition of the MgATP-dependent association of the 19- and 21-kDa smg-proteins to the vesicle membranes. Furthermore, the MgATP effect on both smg-proteins was found to be due to the intravesicular pH and not to the H⁺ gradient over the vesicle membrane. We conclude that association of a 19-kDa (immunologically identified as the ADP-ribosylation factor, arf) and a yet unidentified 21-kDa GTP-binding protein to vesicle membranes is regulated by the intravesicular pH established by a vacuolar-type H⁺-ATPase.The arf-antibodies were kindly supplied by Dr. R.A. Kahn. We thank Prof. Dr. D. Gallwitz, Dr. R. Jahn, and Dr. E.G. Lapetina for kindly providing the ypt 1-, rab 3-, and rap 1-antibodies, respectively. ADP-ribosyltransferase C₃ from Clostridium botulinum was kindly supplied by Prof. Dr. K. Aktories. This work was supported by the Jung-Stiftung für Wissenschaft und Forschung. S.Z. was supported by a grant of the Deutsche Forschungsgemeinschaft (Ze 237/3-1).     

Localization of a metalloproteinase and its inhibitor in the protein bodies of buckwheat seeds

Cotyledons of dry buckwheat (Fagopyrum esculentum Moench) seeds were used to study the cellular localization of a metalloproteinase which performs in vitro the initial limited proteolysis of the main storage protein of the seed, and of its proteinaceous inhibitor. Fractions of complex protein bodies (PB 1) and of the cytoplasm and membrane material (CMM) were obtained by fractionating cotyledons in a mixture of acetone and CCl₄. The greater part of the metalloproteinase activity was found to be localized in the PB 1 fraction, with a lesser amount in the CMM fraction, whereas the metalloproteinase inhibitor was localized almost entirely in the PB 1 fraction. The data obtained indicate that the complex protein bodies of dry buckwheat seeds contain the components of the proteolytic system responsible for the initial degradation of the main storage protein — the 13S globulin — of buckwheat seeds, i.e. 13S globulin, the metalloproteinase, and its inhibitor. This confirms that it is possibile for the metalloproteinase to perform a controlled proteolysis of the 13S globulin in vivo. The effect of divalent cations on the degradation of the 13S globulin was also studied. A mechanism is discussed whereby the proteolysis of 13S globulin is initiated by divalent cations released as a result of phytin decationization during seedling growth.Abbreviations CMM cytoplasm and membrane material - PAGE polyacrylamide gel electrophoresis - PB 1 complex protein bodies with globoids