Stabilization of (90)y-labeled DOTA-biomolecule conjugates using gentisic acid and ascorbic acid.

Radiolytic degradation of radiolabeled compounds is a major challenge for the development of new therapeutic radiopharmaceuticals. The goal of this study is to explore the factors influencing the solution stability of a (90)Y-labeled DOTA-peptide conjugate (RP697), including the amount of total activity, the activity concentration, the stabilizer concentration, and the storage temperature. In general, the rate of radiolytic decomposition of RP697 is much slower at the lower activity concentration (<4 mCi/mL) than that at the higher concentration (>10 mCi/mL). RP697 remains relatively stable at the 20 mCi level and room temperature while it decomposes rapidly at the 100 mCi level under the same storage conditions. Radical scavengers, such as gentisic acid (GA) and ascorbic acid (AA), were used in combination with the low temperature (-78 degrees C) to prevent the radiolytic decomposition of RP697. It was found that RP697 remains stable for at least 2 half-lives of (90)Y when GA or AA (10 mg for 20 mCi of (90)Y) is used as a stabilizer when the radiopharmaceutical composition is stored at -78 degrees C. The stabilizer (GA and AA) can be added into the formulation either before or after radiolabeling. The post-labeling approach is particularly useful when the use of a large amount of the stabilizer interferes with the radiolabeling. The radiopharmaceutical composition developed in this study can also apply to other (90)Y-labeled DOTA-biomolecule conjugates. The amount of the stabilizer used in the radiopharmaceutical composition and storage temperature should be adjusted according to the sensitivity of the radiolabeled DOTA-biomolecule conjugate toward radiolytic decomposition.     

99mTc-labeling of hydrazones of a hydrazinonicotinamide conjugated cyclic peptide.

Eight HYNICtide hydrazones (three with aliphatic substituents and five with aromatic groups) were studied for their potential use as the final intermediate for preparation of RP444, a new radiopharmaceutical under development for imaging thrombosis. The goal of this study is to screen various hydrazones through stability testing and radiolabeling and find those which are able to remain stable without significant degradation in the manufacturing process and at the same time are reactive to produce enough free hydrazine in situ for successful (99m)Tc-labeling. In an initial screening study, only hydrazones 6 and 8, which contain aliphatic substituents, gave satisfactory (>/=90%) yields of RP444 using 50 degrees C and 30 min of heating. However, their solution instability excludes them from being used as commercial reagents. Hydrazones 1 and 4 gave >/=90% yields when the reaction mixtures were heated at 80 degrees C for 30 min. Both hydrazone 1 and hydrazone 4 can be used as the final intermediate for preparation of RP444. The combination of 40 mg of tricine, 1-10 mg of TPPTS, 20-40 microg of hydrazone 1 or 4 for 50 mCi of [(99m)Tc]pertechnetate, 20-50 microg of stannous chloride, pH 4.5 +/- 0.5, and heating at 80 degrees C for 30 min gives the best yield for RP444. It is surprising that hydrazones 1 and 4 have both the solution stability with respect to decomposition and to reaction with aldehydes and ketones and yet are able to hydrolyze in situ to produce enough free HYNICtide for the (99m)Tc-labeling.     

99mTc-labeling of a hydrazinonicotinamide-conjugated vitronectin receptor antagonist useful for imaging tumors.

This report describes the (99m)Tc labeling of a HYNIC-conjugated vitronectin receptor antagonist (SQ168 = [2-[[[5-[carboonyl]-2-pyridinyl]hydrazono]methyl]benzenesulfonic acid]-Glu(cyclo[Lys-Arg-Gly-Asp-D-Phe])-cyclo[Lys-Arg-Gly-Asp-D-Phe]). The ternary ligand complex [(99m)Tc(SQ168)(tricine)(TPPTS)] (RP593) was prepared using a non-SnCl(2)-containing formulation. The corresponding (99)Tc analogue, [(99)Tc]RP593, was also prepared and characterized by HPLC and LC-MS. A HPLC concordance experiment using RP593 and [(99)Tc]RP593 showed that the same technetium complex was prepared at both the tracer and macroscopic levels. The LC-MS data is completely consistent with the 1:1:1:1 composition for Tc:SQ168:tricine:TPPTS and provides direct evidence that the two radiometric peaks in the radio-HPLC chromatogram of RP593 are indeed due to the resolution of diastereomers. In an in vitro receptor binding assay, [(99)Tc]RP593 was shown to have comparable binding affinity for the vitronectin receptor to that of SQ168 itself.     

Isomerism and solution dynamics of (90)Y-labeled DTPA--biomolecule conjugates

This report describes the synthesis of two DTPA-conjugated cyclic peptides, cyclo(Arg-Gly-Asp-D-Phe-Lys)DTPA (SQ169) and [cyclo(Arg-Gly-Asp-D-Phe-Lys)](2)DTPA (SQ170), and a chromatographic study of their (90)Y complexes (RP762 and RP763, respectively). The goal is to study the solution structure and the possible isomerism of (90)Y-labeled DTPA-biomolecule conjugates at the tracer level (approximately 10(-10) M). RP762 was prepared in high radiochemical purity (RCP > 95%) by reacting 2 microg of SQ169 with 20 mCi of (90)YCl(3) (corresponding to a SQ169:Y ratio of approximately 4:1) in the 0.5 M ammonium acetate buffer (pH 8.0) at room temperature. RP763 was prepared in a similar fashion using SQ170. In both cases, the (90)Y-chelation was instantaneous. By a reversed-phase HPLC method, it was found that RP762 exists in solution as a mixture of two detectable isomers (most likely cis and trans isomers), which interconvert at room temperature. The interconversion of different isomeric forms of RP762 involves a rapid exchange of "wrapping isomers" via the "wagging" of the diethylenetriamine backbone, "shuffling" of the two NO(2) donor sets, and inversion at the ternimal amine-nitrogen atom. The inversion at a terminal nitrogen atom requires simultaneous dissociation of the NO(2) donor set. For RP763, the interconversion of different isomers is much faster than that for RP762 due to the weak bonding of two carbonyl-oxygen donors. Therefore, RP763 shows only one broad radiometric peak in its HPLC chromatogram. The rapid interconversion of different isomers is intramolecular via a partial dissociative mechanism. The results obtained in this study are consistent with the lack of kinetic inertness of (90)Y- and (111)In-labeled DTPA-biomolecule conjugates. Thus, the design of new BFCs should be focused on those which form lanthanide complexes with high thermodynamic stability and more importantly kinetic inertness.     

Characterization of glycerophosphorylcholine, -ethanolamine, -serine, -inositol, and -glycerol hydrolytic activity in housefly larvae

Homogenates of Musca domestica (housefly) larvae contain glycerophosphodiesterase activity, which is found in the supernatant fluid after centrifugation at 88,000 g. The phosphodiesterase is inhibited by EDTA and is stimulated by Mg(2+), Ni(2+), Co(2+), and Mn(2+). The pH optimum is 7.2. The enzyme is stable to heating at 50 degrees C for 15 min and is insensitive to sulfhydryl inhibitors. Glycerophosphoryl diesters of choline, ethanolamine, inositol, serine, glycerol, and beta-methylcholine are hydrolyzed to the common product, l-alpha-glycerophosphate, and the appropriate free alcohol. The rate of glycerophosphorylcholine hydrolysis is 70% greater than the rate of hydrolysis of the other glycerophosphodiesters. Apparent K(m) values for glycerophosphorylcholine, glycerophosphorylethanolamine, and glycerophosphoryl-beta-methylcholine are 2-4 x 10(-4) m, and for glycerophosphorylinositol, 2 x 10(-3) m. Competitive studies using various pairs of substrates, as well as the exchange of free choline into both glycerophosphorylcholine and glycerophosphorylinositol, suggest that a single enzyme cleaves all substrates. Product inhibition and reversal of the reaction were not detected. Choline, but not l-alpha-glycerophosphate, exchanges into glycerophosphorylcholine and glycerophosphorylinositol.     

Hydrolysis of adenylyl(2'-5')adenosine in chick embryo sera

Similar to adult chick sera in chick embryo sera the enzyme activity hydrolyzing the core of the dimer of A-5A - adenylyl(2' -5')adenosine is present. The enzyme activity possesses the same properties in both sera. Heating of the serum to 60 degrees C for 30 minutes does not affect the enzyme activity. A marked decrease in activity occurs after heating of the serum to above 65 degrees C. Hydrolysis of A2' p5' A in the serum is more efficient under alkaline conditions (pH 9-10) than at physiological pH 7.2-7.4.     

Adenylate cyclase of the causative agent of plague: its purification and properties

Three forms of adenylate cyclase have been detected in Y. pestis: membrane-bound, cytoplasmic and extracellular. Extracellular adenylate cyclase has been purified so as to achieve a homogeneous state, and some of its physicochemical parameters have been investigated. In the process of purification the initial preparation of this enzyme has been subjected to heating at 100 degrees C for 15 minutes, fractionation with ammonium sulfate, and gel filtration on Sephadex G-100. The homogeneity of adenylate cyclase has been confirmed by electrophoresis in 7.5% polyacrylamide gel and precipitation by the plague agglutinating serum. The enzyme has been found to have a molecular weight of 30,000 daltons and to show the optimum activity at pH 7.0-7.2 and at a temperature between 37 and 40 degrees C. Monospecific rabbit serum to the homogeneous preparation of adenylate cyclase has been obtained.     

Tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase: guanidine. HCl-induced unfolding and a low temperature requirement for refolding.

Guanidine x HCl (GdnHCl)-induced unfolding of tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase (CEOS; 141,300 M(r)) from Lactococcus lactis at pH 7.2 and 25 degrees C occurred in several phases. The enzyme was inactivated at approximately 1 M GdnHCl. A time-, temperature-, and concentration-dependent formation of soluble protein aggregates occurred at 0.5-1.5 M GdnHCl due to an increased exposure of apolar surfaces. A transition from tetramer to unfolded monomer was observed between 2 and 3.5 M GdnHCl (without observable dimer or trimer intermediates), as evidenced by tyrosyl and tryptophanyl fluorescence changes, sulfhydryl group exposure, loss of secondary structure, size-exclusion chromatography, and sedimentation equilibrium data. GdnHCl-induced dissociation and unfolding of tetrameric CEOS was concerted, and yields of reactivated CEOS by dilution from 5 M GdnHCl were improved when unfolding took place on ice rather than at 25 degrees C. Refolding and reconstitution of the enzyme were optimal at 相似文献   

Purification and characterization of cathepsin L-like proteinase from goat brain

Cathepsin L-like proteinase was purified approximately 1708-fold with 40% activity yield to an apparent electrophoretic homogeneity from goat brain by homogenization, acid-autolysis at pH 4.2, 30-80% (NH4)2SO4 fractionation, Sephadex G-100 column chromatography and ion-exchange chromatography on CM-Sephadex C-50 at pH 5.0 and 5.6. The molecular weight of proteinase was found to be approximately 65,000 Da, by gel-filtration chromatography. The pH optima were 5.9 and 4.5 for the hydrolysis of Z-Phe-Arg-4mbetaNA (benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methoxy-beta-naphthylamide) and azocasein, respectively. Of the synthetic chromogenic substrates tested, Z-Phe-Arg-4mbetaNA was hydrolyzed maximally by the enzyme (Km value for hydrolysis was 0.06 mM), followed by Z-Val-Lys-Lys-Arg-4mbetaNA, Z-Phe-Val-Arg-4mbetaNA, Z-Arg-Arg-4mbetaNA and Z-Ala-Arg-Arg-4mbetaNA. The proteinase was activated maximally by glutathione in conjunction with EDTA, followed by cysteine, dithioerythritol, thioglycolic acid, dithiothreitol and beta-mercaptoethanol. It was strongly inhibited by p-hydroxymercuribenzenesulphonic acid, iodoacetic acid, iodoacetamide and microbial peptide inhibitors, leupeptin and antipain. Leupeptin inhibited the enzyme competitively with Ki value 44 x 10(-9) M. The enzyme was strongly inhibited by 4 M urea. Metal ions, Hg(2+), Ca(2+), Cu(2+), Li(2+), K(+), Cd(2+), Ni(2+), Ba(2+), Mn(2+), Co(2+) and Sn(2+) also inhibited the activity of the enzyme. The enzyme was stable between pH 4.0-6.0 and up to 40 degrees C. The optimum temperature for the hydrolysis of Z-Phe-Arg-4mbetaNA was approximately 50-55 degrees C with an activation energy Ea of approximately 6.34 KCal mole(-1).     

Covalent fixation of polymer-linked benzene hexacarboxylate onto human haemoglobin.

The reaction of human deoxy and oxyhaemoglobin with a macromolecular effector, monomethoxypolyoxyethylene-linked benzene hexacarboxylate, in the presence of a water soluble carbodiimide, produces under defined conditions, the same conjugates preferentially acylated at the two valines beta 1. The oxygen affinity of both these conjugates is decreased by approximately 5-fold compared with that of native Hb (at pH 7.2, in 0.05 M Tris buffer, 25 degrees C, P50: 20.1 and 20.7 Torr versus about 4 Torr for Hb). This difference appears to be due to an overstabilization of the T state probably together with a decrease of the oxygen affinity of the R state. Addition of IHP to the conjugate solutions does not influence the P50 but addition of IHP to the reaction mixtures before the coupling limits the substitution of Hb by the macromolecular effector, to 20% (instead of 100% in absence of IHP). The cooperativity curve is shifted to the right with an Nmax of 3 at about 90% oxygen saturation, which corresponds to a potential release of 48% of oxygen at pH 7.2, 25 degrees C, between 100 and 40 Torr, compared with 40% for blood. Such kinds of conjugates especially those obtained from oxyhaemoglobin which are easily prepared, could be of a great interest as non-diffusing oxygen carriers in transfusional and perfusional fluids.     

Ascorbic acid: useful as a buffer agent and radiolytic stabilizer for metalloradiopharmaceuticals

The goal of this study is to explore the use of ascorbic acid (AA) as a buffer agent and a radiolytic stabilizer for preparation and stabilization of radiolabeled DOTA-biomolecule conjugates. Results from a titration experiment show that 0.1 M AA solution has sufficient buffer capacity at pH 5.0 while 0.5 M AA solution is useful even at pH 6.0. The radiolabeling experiment using TA138, a DOTA-conjugated nonpeptide integrin alpha(v)beta(3) receptor antagonist, clearly demonstrates that AA is a good buffer agent for pH control and an excellent antioxidant for stabilization of metal-labeled diagnostic ((111)In) and therapeutic ((90)Y and (177)Lu) radiopharmaceuticals if the radiolabeling is performed at pH 5-6. There is no need for the additional stabilizer (e.g., gentisic acid) and buffer agent such as ammonium acetate. The anaerobic AA formulation described in this study is particularly useful for radiolabeling of small biomolecules, which are sensitive to the radiolytic degradation during radiolabeling.     

Synthesis of feralex a novel aluminum/iron chelating compound.

There is a need for in vivo applicable Fe(3+) and Al(3+) chelation compounds for use as medicines to treat toxic overload conditions of these ions. A novel compound, 2-deoxy-2-(N-carbamoylmethyl-[N'-2'-methyl-3'-hydroxypyrid-4'-one])-D-glucopyranose, designed to chelate Fe(3+) or Al(3+), has been synthesised utilising three naturally occurring products: maltol, glycine and glucosamine. The synthesis is a simple two step process. First, glycine is coupled to maltol by an aminolysis reaction, to yield the intermediate product 1-carboxymethyl -3-hdroxy-2-methylpyrid-4-one, which is joined with glucosamine using a dicyclohexylcarbodiimide promoted peptide coupling method to produce the desired end product, 2-deoxy-2-(N-carbamoylmethyl-[N'-2'-methyl-3'-hydroxypyrid-4'-one])-D-glucopyranose. The latter has been given the trivial name Feralex-G. NMR analysis permitted assignment of frequencies for all carbon and covalently bound hydrogen atoms and was consistent with the proposed structure of the compound. Electron spray Ionisation Mass Spectrometry (ESI-MS) yielded the expected molecular mass of 344. Proton displacement/pH titration analysis yielded three Feralex-G molecules bound to 1 Al(3+) or Fe(3+) over a measurable pH range of 3-10.5. A rapid TLC method to monitor progression of the synthetic procedures is also described.     

pH dependence of the donor side reactions in Ca2+-depleted photosystem II

We have studied how low pH affects the water-oxidizing complex in Photosystem II when depleted of the essential Ca(2+) ion cofactor. For these samples, it was found that the EPR signal from the Y(Z)(*) radical decays faster at low pH than at high pH. At 20 degrees C, Y(Z)(*) decays with biphasic kinetics. At pH 6.5, the fast phase encompasses about 65% of the amplitude and has a lifetime of approximately 0.8 s, while the slow phase has a lifetime of approximately 22 s. At pH 3.9, the kinetics become totally dominated by the fast phase, with more than 90% of the signal intensity operating with a lifetime of approximately 0.3 s. The kinetic changes occurred with an approximate pK(a) of 4.5. Low pH also affected the induction of the so-called split radical EPR signal from the S(2)Y(Z)(*) state that is induced in Ca(2+)-depleted PSII membranes because of an inability of Y(Z)(*) to oxidize the S(2) state. At pH 4.5, about 50% of the split signal was induced, as compared to the amplitude of the signal that was induced at pH 6.5-7, using similar illumination conditions. Thus, the split-signal induction decreased with an apparent pK(a) of 4.5. In the same samples, the stable multiline signal from the S(2) state, which is modified by the removal of Ca(2+), was decreased by the illumination to the same extent at all pHs. It is proposed that decreased induction of the S(2)Y(Z)(*) state at lower pH was not due to inability to oxidize the modified S(2) state induced by the Ca(2+) depletion. Instead, we propose that the low pH makes Y(Z)(*) able to oxidize the S(2) state, making the S(2) --> S(3) transition available in Ca(2+)-depleted PSII. Implications of these results for the catalytic role of Ca(2+) and the role of proton transfer between the Mn cluster and Y(Z) during oxygen evolution is discussed.     

Immunochemical studies on thermal denaturation of ovomucoid

The thermal denaturation of ovomucoid was investigated by immunochemical methods, namely immunoprecipitation analyses and antibody-Sepharose 4B column chromatography. In the immunoprecipitation analyses, heated ovomucoid (90 degrees C, 90 min, pH 7.2) required about twice the antigen addition of the native protein to approach maximal precipitation with specific antibody, and the maximal immunoprecipitation was decreased to 80% of that by native ovomucoid. However, heated protein inhibited the binding of antibody with native ovomucoid, and 100% inhibition was attained at about 4-times the antigen addition necessary for the native protein. Heated ovomucoid (100 degrees C, 120 min) showed little immunoprecipitation and inhibition. To ovomucoid antigenicity was diminished more slowly than the trypsin inhibitory activity by heating, e.g., heated ovomucoid (90 degrees C, 120 min) retained more than 30% of the antigenicity but little trypsin inhibitory activity. By passing through the immunoaffinity column, heated ovomucoid (90 degrees C, 90 min) was separated into two fractions, either with (fraction II) or without (fraction I) antigenicity. Fraction II contained smaller fractions of ordered secondary structure than native ovomucoid, and trypsin inhibitory activity of fraction II was only 24% of the native one. These results indicated that thermally denatured ovomucoid was heterogeneous regarding the conformational damage caused by heating, and the structure around some antigenic sites in an ovomucoid molecule was retained even after the backbone conformation was partially destroyed and trypsin inhibitory activity was lost.     

Purification and characterization of pectate lyase from banana (Musa acuminata) fruits

Pectate lyase (PEL) has been purified by hydrophobic, cation exchange and size exclusion column chromatographies from ripe banana fruit. The purified enzyme has specific activity of 680 +/- 50 pkat mg protein(-1). The molecular mass of the enzyme is 43 kDa by SDS-PAGE. The pI of the enzyme is 8 with optimum activity at pH 8.5. Analysis of the reaction products by paper and anion exchange chromatographies reveal that the enzyme releases several oligomers of unsaturated galacturonane from polygalacturonate. The K(m) values of the enzyme for polygalacturonate and citrus pectin (7.2% methylation) are 0.40 +/- 0.04 and 0.77 +/- 0.08 g l(-1), respectively. PEL is sensitive to inhibition by different phenolic compounds, thiols, reducing agents, iodoacetate and N-bromosuccinimide. The enzyme has a requirement for Ca(2+) ions. However, Mg(2+) and Mn(2+) can substitute equally well. Additive effect on the enzyme activity was observed when any two metal ions (out of Mg(2+), Ca(2+) and Mn(2+)) are present together. The banana PEL is a enzyme requiring Mg(2+), in addition to Ca(2+), for exhibiting maximum activity.     

-deltaG(AB) and pH dependence of the electron transfer from P(+)Q(A)(-)Q(B) toP(+)Q(A)Q(B)(-) in Rhodobacter sphaeroides reaction centers

The electron transfer from the reduced primary quinone (Q(A)(-)) to the secondary quinone (Q(B)) can occur in two phases with a well-characterized 100 micros component (tau(2)) and a faster process occurring in less than 10 micros (tau(1)). The fast reaction is clearly seen when the native ubiquinone-10 at Q(A) is replaced with naphthoquinones. The dependence of tau(1) on the free-energy difference between the P(+)Q(A)(-)Q(B) and P(+)Q(A)Q(B)(-) states (-) and on the pH was measured using naphthoquinones with different electrochemical midpoint potentials as Q(A) in Rhodobacter sphaeroides reaction centers (RCs) and in RCs where - is changed by mutation of M265 in the Q(A) site from Ile to Thr (M265IT). Q(B) was ubiquinone (UQ(B)) in all cases. Electron transfer was measured by using the absorption differences of the naphthosemiquinone at Q(A) and the ubisemiquinone at Q(B) between 390 and 500 nm. As - was changed from -90 to -250 meV tau(1) decreased from 29 to 0.2 micros. The free-energy dependence of tau(1) provides a reorganization energy of 850 +/- 100 meV for the electron transfer from Q(A)(-) to Q(B). The slower reaction at tau(2) is free-energy independent, so processes other than electron transfer determine the observed rate. The fraction of the reaction at tau(1) increases with increasing driving force and is 100% of the reaction when - is approximately 100 meV more favorable than in the native RCs with ubiquinone as Q(A). The fast phase, tau(1), is pH independent from pH 6 to 11 while tau(2) slows above pH 9. As the Q(A) isoprene tail length is increased from 2 to 10 isoprene units the fraction at tau(1) decreases. However, tau(1), tau(2), and the fraction of the reaction in each phase are independent of the tail length of UQ(B).     

Linkage isomerization reaction of intrastrand cross-links in trans-diamminedichloroplatinum(II)-modified single-stranded oligonucleotides.

The stability of trans-(Pt(NH3)2[d(CGAG)-N7-G,N7-G]) adducts, resulting from cross-links between two guanine residues at d(CGAG) sites within single-stranded oligonucleotides by trans-diamminedichloro-platinum(II), has been studied under various conditions of temperature, salt and pH. The trans-(Pt(NH3)2[d(C GAG)-N7-G,N7-G]) cross-links rearrange into trans-(Pt(NH3)2[d(CGAG)-N3-C,N7-G]) cross-links. The rate of rearrangement is independent of pH, in the range 5-9, and of the nature and concentration of the salt (NaCl or NaCIO4) in the range 10-400 mM. The reaction rate depends upon temperature, the t1/2 values for the disappearance of the (G,G) intrastrand cross-link ranging from 120 h at 30 degrees C to 70 min at 80 degrees C. The linkage isomerization reaction occurs in oligonucleotides as short as the platinated tetramer d(CGAG). Replacement of the intervening residue A by T has no major effect on the reaction. The C residue adjacent to the adduct on the 5' side plays a key-role in the reaction; its replacement by a G, A or T residue prevents the reaction occuring. No rearrangement was observed with the C residue adjacent to the adduct on the 3' side. It is proposed that the linkage isomerization reaction results from a direct attack of the base residue on the platinum(II) square complex.     

Expression and characterization of fumarase (FUMR) from Rhizopus oryzae

Fumarase catalyzes the reversible hydration of fumarate to l-malate in Rhizopus oryzae. A recombinant pET22b-fumR harboring a fumarase gene (fumR) from R. oryzae was constructed for high level expression in E. coli BL21 (DE3). The FUMR activity was optimal at 30°C and pH 7.2. The enzyme was stable below 45°C and at pH 3.0-9.0. No effects of Zn(2+), Fe(2+), or EDTA were observed on enzyme activity. A slight inhibition of FUMR activity was seen with Mg(2+), while Ca(2+) had a small stimulatory effect. The K(m) for l-malic acid and fumaric acid were 0.46 mM and 3.07 mM, respectively. The activity of FUMR catalyzing hydration of fumarate to l-malate was completely inhibited by 2mM fumaric acid. The unique enzymatic properties suggested that overexpression of FUMR could enhance fumaric acid accumulation in R. oryzae.     

Trifunctional conjugation reagents. Reagents that contain a biotin and a radiometal chelation moiety for application to extracorporeal affinity adsorption of radiolabeled antibodies

A method of removing radiolabeled monoclonal antibodies (mAbs) from blood using a device external to the body, termed extracorporeal affinity-adsorption (EAA), is being evaluated as a means of decreasing irradiation of noncancerous tissues in therapy protocols. The EAA device uses an avidin column to capture biotinylated-radiolabeled mAbs from circulated blood. In this investigation, three trifunctional reagents have been developed to minimize the potential deleterious effect on antigen binding brought about by the combination of radiolabeling and biotinylation of mAbs required in the EAA approach. The studies focused on radiolabeling with (111)In and (90)Y, so the chelates CHX-A' '-DTPA and DOTA, which form stable attachments to these radionuclides, were incorporated in the trifunctional reagents. The first trifunctional reagent prepared did not incorporate a group to block the biotin cleaving enzyme biotinidase, but the two subsequent reagents coupled aspartic acid to the biotin carboxylate for that purpose. All three reagents used 4,7,10-trioxa-1,13-tridecanediamine as water-soluble spacers between an aminoisophthalate core and the biotin or chelation group. The mAb conjugates were radioiodinated to evaluate cell binding as a function of substitution. Radioiodination was used so that a direct comparison with unmodified mAb could be made. Evaluation of the number of conjugates per antibody versus cell binding immunoreactivities indicated that minimizing the number of conjugates was best. Interestingly, a decrease of radioiodination yield as a function of the number of isothiocyanate containing conjugates per mAb was noted. The decreased yields were presumably due to the presence of thiourea functionality formed in the conjugation reaction. Radiolabeling with (111)In and (90)Y was facile at room temperature for conjugates containing the CHX-A' ', but elevated temperature (e.g., 45 degrees C) was required to obtain good yields with the DOTA chelate. Stability of (90)Y labeled mAb in serum, and when challenged with 10 mM EDTA, was high. However, challenging the (90)Y labeled mAb with 10 mM DTPA demonstrated high stability for the DOTA containing conjugate, but low stability for the CHX-A' ' containing conjugate. Thus, the choice between these two chelating moieties might be made on requirements for facile and gentle labeling versus very high in vivo stability. Application of the trifunctional biotinylation reagents to the blood clearance of labeled antibodies in EAA is under investigation. The new reagents may also be useful for other applications.     

Purification, characterization, and synergistic action of phytate-resistant alpha-amylase and alpha-glucosidase from Geobacillus thermodenitrificans HRO10

The alpha-amylase (1, 4-alpha-d-glucanohydrolase; EC 3.2.1.1) and alpha-glucosidase (alpha-d-glucoside glucohydrolase; EC 3.2.1.20) secreted by Geobacillus thermodenitrificans HRO10 were purified to homogeneity (13.6-fold; 11.5% yield and 25.4-fold; 32.0% yield, respectively) through a series of steps. The molecular weight of alpha-amylase was 58kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The alpha-amylase activity on potato starch was optimal at pH 5.5 and 80 degrees Celsius. In the presence of Ca(2+), the alpha-amylase had residual activity of more than 92% after 1h of incubation at 70 degrees Celsius. The alpha-amylase did not lose any activity in the presence of phytate (a selective alpha-amylase inhibitor) at concentrations as high as 10mM, rather it retained 90% maximal activity after 1h of incubation at 70 degrees Celsius. EGTA and EDTA were strong inhibitory substances of the enzyme. The alpha-amylase hydrolyzed soluble starch at 80 degrees Celsius, with a K(m) of 3.05mgml(-1) and a V(max) of 7.35Uml(-1). The molecular weight of alpha-glucosidase was approximately 45kDa, as determined by SDS-PAGE. The enzyme activity was optimal at pH 6.5-7.5 and 55 degrees Celsius. Phytate did not inhibit G. thermodenitrificans HRO10 alpha-glucosidase activity, whereas pCMB was a potent inhibitor of the enzyme. The alpha-glucosidase exhibited Michaelis-Menten kinetics with maltose at 55 degrees Celsius (K(m): 17mM; V(max): 23micromolmin(-1)mg(-1)). Thin-layer chromatography studies with G. thermodenitrificans HRO10 alpha-amylase and alpha-glucosidase showed an excellent synergistic action and did not reveal any transglycosylation catalyzed reaction by the alpha-glucosidase.