Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from chlorophyll a

Chlorophyll b is an ubiquitous accessory pigment in land plants, green algae, and prochlorophytes. Its biosynthesis plays a key role in the adaptation to various light environments. We isolated six chlorophyll b-less mutants by insertional mutagenesis by using the nitrate reductase or argininosuccinate lyase genes as tags and examined the rearrangement of mutant genomes. We found that an overlapping region of a nuclear genome was deleted in all mutants and that this encodes a protein whose sequence is similar to those of methyl monooxygenases. This coding sequence also contains putative binding domains for a [2Fe-2S] Rieske center and for a mononuclear iron. The results demonstrate that a chlorophyll a oxygenase is involved in chlorophyll b formation. The reaction mechanism of chlorophyll b formation is discussed.     

Unusual mechanism of oxygen atom transfer and product rearrangement in the catalytic reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase

The oxygenation reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase with the substrate, MHPC, was investigated. Two oxygenated flavin intermediates C(4a)-hydroperoxy flavin and C(4a)-hydroxy flavin were found, implying that the enzyme functions similarly to flavoprotein hydroxylases. This finding is supported by the results of independent oxygen-18 tracer experiments, which showed that one atom of oxygen from 18O2 and one atom of oxygen from H218O are incorporated in the product. MHPC oxygenase normally catalyzes both the oxygenation and the hydrolytic ring opening of the pyridine ring of MHPC to yield the acyclic compound, alpha-(N-acetylaminomethylene)succinic acid. Using 5-hydroxynicotinic acid (5HN), which has no 2-methyl group, we tested whether the hydrolytic reaction was due to the presence of the 2-methyl group on MHPC (that prevented rearomatization of the initial product) or to the specific properties of MHPC oxygenase. Product analysis of the enzymatic reaction of 5HN and MHPC oxygenase shows that the enzyme catalyzes the hydroxylation and subsequent hydrolysis of the hydroxylated substrate to yield an acyclic product. The investigation of the oxygenation reaction demonstrates that the enzyme uses the same mechanism to catalyze the 5HN reaction as it does in the MHPC reaction.     

Cloning and expression of cDNA for soluble form of rat heme oxygenase-1

Heme oxygenase catalyzes the oxidation of heme to biliverdin and carbon monoxide. The gene encoding the truncated soluble rat heme oxygenase-1 (Metl-Pro267) was cloned. The enzyme protein was expressed in E. coli JM109 and purified to homogeneity. The molecular weight of the recombinant enzyme was 30 kDa as assessed by SDS-polyacrylamide gel electrophoresis. From a 3-L culture, about 90 mg of the purified enzyme was routinely obtained. The dependency of the heme oxygenase reaction catalyzed by the soluble enzyme on the NADPH-cytochrome P-450 reductase concentrations and the effect of catalase on the reaction were examined to compare with the purified membrane-bound form of heme oxygenase-1 (Yoshida and Kikuchi, 1978b). The activity of the soluble enzyme was inhibited at high concentrations of NADPH-cytochrome P-450 reductase and the inhibition was not alleviated by addition of catalase unlike the membrane-bound form. The ferric iron of the heme-heme oxygenase complex was in a typical high spin state at acidic to neutral pH (pH 6.5-7.0) but conversion to low spin state was observed at basic pH (pH 9-10). The heme bound to heme oxygenase was converted to biliverdin at a stoichiometric ratio of unity in the presence of NADPH-cytochrome P-450 reductase system. During the heme degradation of the heme-heme oxygenase complex under atmospheric oxygen, several intermediates, that is, oxygenated heme and verdoheme, were spectrally discriminated.     

1,2-Dithiolan-3-one 1-oxides: a class of thiol-activated DNA-cleaving agents that are structurally related to the natural product leinamycin

Leinamycin is a recently discovered, thiol-dependent DNA-cleaving natural product. The mechanism of DNA cleavage by leinamycin is unknown. Inspired by this intriguing natural product, we have investigated the DNA-cleaving properties of three 1,2-dithiolan-3-one 1-oxides (1-3) that are structurally related to the suspected DNA-cleaving "core" of leinamycin. It was found that, similar to leinamycin, these three 1,2-dithiolan-3-one 1-oxides are thiol-dependent DNA-cleaving agents. At the concentrations of 1-3 used in these experiments (approximately 100 microM), efficient DNA cleavage is absolutely dependent on added thiol, with optimum cleavage occurring at 5-10 equiv (500 microM-1 mM) of added thiol. 2-Mercaptoethanol, glutathione, dithiothreitol, and thiophenol function with approximately equal efficiency as triggering agents for the cleavage reaction. DNA cleavage by 1-3 is not highly pH-dependent. Cleavage of DNA by these sulfur heterocycles is diminished by the removal of molecular oxygen from the reaction medium, by the radical scavengers methanol, ethanol, and mannitol, and by the enzyme catalase. Superoxide dismutase does not suppress DNA cleavage by these compounds. When diethylenetriaminepentaacetic acid is employed in these reactions as a chelator of adventitious trace metal ions, DNA cleavage is efficiently inhibited. The S-deoxy analog of 1 does not cleave DNA under conditions where 1 effects efficient thiol-mediated cleavage of DNA. These experiments indicate that, in concert with thiols, 1,2-dithiolan-3-one 1-oxides convert molecular oxygen to DNA-cleaving oxygen radicals. The marked effect of catalase further suggests that molecular oxygen is converted to hydrogen peroxide which ultimately cleaves DNA via a trace metal-dependent Fenton reaction. This work demonstrates that 1,2-dithiolan-3-one 1-oxides represent a general class of thiol-potentiated DNA-cleaving molecules.     

Sequence-alignment modelling and molecular docking studies of the epoxygenase component of alkene monooxygenase from Nocardia corallina B-276

Whole cells of Nocardia corallina B-276 catalyse the stereoselective epoxygenation of alkenes to chiral epoxides. The bacterium expresses an enzyme, alkene monooxygenase, which catalyses the epoxygenation reaction stereoselectively. The enzyme consists of a terminal oxygenase (epoxygenase), an NADH-dependent reductase (reductase) and a regulatory component (coupling protein). The epoxygenase component contains a bridged diiron centre similar to that found in the hydroxylase component of soluble methane monooxygenase. Sequence-alignment modelling, supported by chemical modification and fluorescence probing, identified a hydrophobic oxygen/substrate binding site within the epoxygenase. The diiron centre was coordinated by the two His and two Glu residues from two conserved Glu-Xaa-Xaa-His sequences and by two further Glu residues. Molecular docking of substrates and products into the proposed active-site model of the epoxygenase suggested that Ala91 and Ala185 were responsible for the stereoselectivity exerted by AMO. It is proposed that these residues clamped the intermediate and/or product of the reaction, thereby controlling the configuration of the epoxide produced. In soluble methane monooxygenase these residues are replaced by two Gly residues which do not provide sufficient steric hindrance to prevent rotation of the intermediate in the active site and, therefore, the product of the reaction catalysed by this enzyme is achiral.     

Molecular cloning and functional expression of a water-soluble chlorophyll protein, a putative carrier of chlorophyll molecules in cauliflower

A cDNA for a water-soluble chlorophyll (Chl) protein (WSCP) from cauliflower (Brassica oleracea L. var botrys) was cloned and sequenced. The cDNA contained an open reading frame encoding 19 residues for a signal peptide and 199 residues for the mature form of WSCP. The sequence showed extensive homology to drought-stress-related, 22-kDa proteins in some Brassicaceae plants. Functional WSCP was expressed in Escherichia coli as a fusion protein with a maltose-binding protein (MBP). When the recombinant MBP-WSCP was incubated with thylakoid membranes, the MBP-WSCP removed Chls from these membranes. During this process, the monomer of the apo-MBP-WSCP successfully bound Chls and was converted into tetrameric holo-MBP-WSCP. The reconstituted MBP-WSCP exhibited absorption and fluorescent spectra identical to those of the native WSCP purified from cauliflower leaves. The Chl a/b ratio in native WSCP indicates a high content of Chl a, which was mainly due to the higher affinity of MBP-WSCP for Chl a. WSCP is the first example of a hydrophilic protein that can transfer Chls from thylakoid hydrophobic proteins. Possible functions of WSCP are discussed.     

Type 1 Cu structure of blue nitrite reductase from Alcaligenes xylosoxidans GIFU 1051 at 2.05 A resolution: comparison of blue and green nitrite reductases

The crystal structure of the blue nitrite reductase from Alcaligenes xylosoxidans GIFU 1051 (AxgNIR) has been determined at 2.05 A resolution. AxgNIR contains both type 1 and 2 Cu sites, the geometry of the former being distorted tetrahedral. The superpositioning of the type 1 Cu sites in the blue enzyme and a green nitrite reductase revealed that the orientation of the Met150 side chain differed. The deviation of the Sdelta(Met150) atom from the axial position of the NNS plane formed by two Ndelta(His95 and His145) and one Sgamma(Cys136) atom caused the difference in the colors of the enzymes, i.e. blue and green.     

Calmodulin-dependent regulation of inducible and neuronal nitric-oxide synthase

Neuronal and endothelial nitric-oxide synthases depend upon Ca2+/calmodulin for activation, whereas the activity of the inducible nitric-oxide synthase is Ca2+-independent, presumably due to tightly bound calmodulin. To study these different mechanisms, a series of chimeras derived from neuronal and inducible nitric- oxide synthases were analyzed. Chimeras containing only the oxygenase domain, calmodulin-binding region, or reductase domain of inducible nitric-oxide synthase did not confer significant Ca2+-independent activity. However, each chimera was more sensitive to Ca2+ than the neuronal isoform. The calmodulin-binding region of inducible nitric-oxide synthase with either its oxygenase or reductase domains resulted in significant, but not total, Ca2+-independent activity. Co-immunoprecipitation experiments showed no calmodulin associated with the former chimera in the absence of Ca2+. Trifluoperazine also inhibited this chimera in the absence of Ca2+. The combined interactions of calmodulin bound to inducible nitric-oxide synthase calmodulin-binding region with the oxygenase domain may be weaker than with the reductase domain. Thus, Ca2+-independent activity of inducible nitric-oxide synthase appears to result from the concerted interactions of calmodulin with both the oxygenase and reductase domains in addition to the canonical calmodulin-binding region. The neuronal isoform is not regulated by a unique autoinhibitory element in its reductase domain.     

Fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase by reactive oxygen species occurs near Gly-329

The large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in the illuminated lysates of wheat (Triticum aestivum L.) chloroplasts is broken down by reactive oxygen radicals into 37- and 16-kDa polypeptides. Analysis of the terminal amino acid residues of both fragments revealed that the C terminus of the 37-kDa fragment was Ser-328 and the N terminus of the 16-kDa fragment was Thr-330. Gly-329, which links the two fragments, was missing, suggesting that the fragmentation of the LSU in the lysates driven by oxygen-free radicals occurs at Gly-329. Purified rubisco, exposed to a hydroxyl radical-generating system, was also cleaved at the same site of the LSU. The cleavage site was positioned at the N-terminal end of the flexible loop (loop 6) within the beta/alpha-barrel domain, constituting the catalytic site of rubisco. The binding of a reaction intermediate analogue, 2-carboxyarabinitol 1,5-bisphosphate, to the active form of rubisco completely protected the enzyme from the fragmentation. The fragmentation was differentially affected by CO2, Mg2+, ribulose 1, 5-bisphosphate, or 2-carboxyarabinitol 1,5-bisphosphate. All these results indicate that the conformation of the catalytic site of the enzyme is involved as an important factor determining the breakdown of rubisco by reactive oxygen species. Reactive oxygen species generated at its catalytic site by a Fenton-type reaction may trigger the site-specific degradation of the LSU in the lysates of chloroplasts in the light.     

Purification of the cyclooxygenase that forms prostaglandins. Demonstration of two forms of iron in the holoenzyme

The fatty acid cyclooxygenase (ec 1.14.99.1) that produces the prostaglandin and thromboxane precursor, 15-hydroperoxy-9 alpha, 11 alpha-peroxidoprosta-5, 13-dienoic acid (PGG2), has been purified from sheep vesicular glands to a specific activity of 46,000 units/mg of protein by combining detergent solubilization, (NH4)SO4 fractionation, chromatography on DEAE-cellulose and Flurbiprofen-Sepharose, isoelectric focusing, and gel filtration. The final enzyme preparation exhibited only one band of 70,000 molecular weight following sodium dodecyl sulfate gel electrophoresis and staining with Coomassie blue. Treatment of the purified oxygenase with [3H] acetylsalicylic acid yielded a radioactive product which co-electrophoresed with the protein of 70,000 molecular weight. Thus, the isolated protein appeared to be the same one which, in crude preparations, selectively binds acetyl groups in association with prostaglandin synthetic activity. Incubation of the purified oxygenase with [1-14C] arachidonic acid in the presence of stannous chloride yielded only 9 alpha, 11 alpha, 15-trihydroxy-prosta-5,13-dienoic acid (PGF2alpha). Without stannous chloride, a mixture of radioactive products was observed which was characteristic of nonenzymic breakdown of PGG2. Thus, the isolated enzyme catalyzed the insertion of both oxygen molecules required for the formation of prostaglandins and thromboxanes from polyunsaturated fatty acid substrates. The aerobic absorption spectrum of the isolated oxygenase showed a faint peak at 412 nm indicative of heme. The iron content indicated that a significant amount of nonheme iron was present. The purified oxygenase was activated by added hemin, which was readily bound to the protein. The subsequently isolated heme-protein complex showed a major absorption peak at 407 nm.     

Degradation of HMG-CoA reductase in vitro. Cleavage in the membrane domain by a membrane-bound cysteine protease

We have recently shown that the endoplasmic reticulum (ER) membrane protein, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is cleaved in isolated membrane fractions enriched for endoplasmic reticulum. Importantly, the cleavage rate is accelerated when the membranes are prepared from cells that have been pretreated with mevalonate or sterols, physiological regulators of the degradation process in vivo (McGee, T. P., Cheng, H. H., Kumagai, H., Omura, S., and Simoni, R. D. (1996) J. Biol. Chem. 271, 25630-25638). In the current study, we further characterize this in vitro cleavage of HMG-CoA reductase. E64, a specific inhibitor of cysteine-proteases, inhibits HMG-CoA reductase cleavage in vitro. In contrast, lactacystin, an inhibitor of the proteasome, inhibits HMG-CoA reductase degradation in vivo but does not inhibit the in vitro cleavage. Purified ER fractions contain lactacystin-sensitive and E64-insensitive proteasome activity as measured by succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin hydrolysis. We removed the proteasome from purified ER fractions by solubilization with heptylthioglucoside and observed that the detergent extracted, proteasome-depleted membrane fractions retain regulated cleavage of HMG-CoA reductase. This indicates that ER-associated proteasome is not involved in degradation of HMG-CoA reductase in vitro. In order to determine the site(s) of proteolysis of HMG-CoA reductase in vitro, four antisera were prepared against peptide sequences representing various domains of HMG-CoA reductase and used for detection of proteolytic intermediates. The sizes and antibody reactivity of the intermediates suggest that HMG-CoA reductase is cleaved in the in vitro degradation system near the span 8 membrane region, which links the N-terminal membrane domain to the C-terminal catalytic domain of the protein. We conclude that HMG-CoA reductase can be cleaved in the membrane-span 8 region by a cysteine protease(s) tightly associated with ER membranes.     

Kainic acid induction of heme oxygenase in vivo and in vitro

Heme oxygenase, catalyses oxidation of the heme molecule in concert with NADPH-cytochrome P450 reductase and then specifically cleaves heme into biliverdin, carbon monoxide, and iron. Biliverdin and its product, bilirubin, are known to be strong antioxidants. Kainic acid is a potent neurotoxin, and induces selective neuronal loss in the rat hippocampus. Kainic acid acts on the kainate receptors, and kainic acid neurotoxicity may be in part mediated by oxidative stress. In this study, we examined whether or not heme oxygenase was activated in kainic acid-induced neurotoxicity. After intracerebroventricular injection of kainic acid, the heme oxygenase-1 protein level was strongly enhanced, although the constitutive heme oxygenase (heme oxygenase-2) protein level was not changed. One day after treatment, the protein level of heme oxygenase-1 reached a maximum and then gradually decreased over a period of three to seven days. In the rat hippocampus, cells expressing heme oxygenase-1 in vivo were predominately microglia and only a few astrocytes. In addition, heme oxygenase-1 immunoreactivity was predominantly co-localized with major histocompatibility complex class II-, and partly co-localized with class I-immunoreactive microglia. In cultured glial cells in vitro, heme oxygenase- protein was expressed in the microglia even with the vehicle treatment, and was strongly induced in astrocytes by kainic acid treatment. These results suggest that ameboid microglia, which express both heme oxygenase-1 and major histocompatibility complex antigens, may play a key role in a delayed episode of kainic acid-induced microglial activation and neurodegeneration.     

NCC malonyltransferase catalyses the final step of chlorophyll breakdown in rape (Brassica napus)

Of the three final products of chlorophyll breakdown that in senescing cotyledons of oilseed rape are accumulated progressively, the nonfluorescent Bn-NCC-1 is the most abundant catabolite. It represents the malonylester of the minor catabolite Bn-NCC-3. The in vitro malonylation of Bn-NCC-3 into Bn-NCC-1 was investigated. Extracts from senescent as well as from presenescent cotyledons contained corresponding activities in the presence of malonyl-coenzyme A as the co-substrate. Malonyltransferase activity exhibited pH- and activation optima at 8 and 34 degrees, respectively, and it was saturable with an apparent Michaelis constant of 58 microM for Bn-NCC-3. The partially purified enzyme recognized chlorophyll catabolites as substrate specifically, provided that they had a free hydroxyl group in the ethyl side chain of pyrrole B.     

A sensitive microassay reveals marked regional differences in the capacity of rat brain to generate carbon monoxide

Heme oxygenase activity is the sole known physiological source for the production of carbon monoxide (CO), a gaseous messenger candidate. A sensitive radioenzymatic microassay was validated to study regional distribution of heme oxygenase activity within the rat brain. The assay utilized a 14,000 X g supernatant of brain homogenate and [14C]heme as the substrate. Thin layer chromatography revealed that incubation of cerebellar supernatant with (14C]heme yielded a single reaction product, indistinguishable from bilirubin, that was selectively extracted into toluene. Radioactivity in toluene increased linearly in respect to time and added protein, was totally dependent on NADPH and was not detected with boiled homogenate. The reaction was dose-dependently inhibited by Zn-protoporphyrin IX (IC50 0.3 microM) and by an antibody generated against rat NADPH-cytochrome P450 reductase indicating specific involvement of heme oxygenase. As little as 36 fmol [14C]bilirubin/min could be readily detected requiring only microgram-quantities of cerebellar homogenate. Heme oxygenase activity measurements from discrete brain regions revealed for the first time marked differences in enzyme activity with the increasing order: frontal cortex < cerebellum = caudate-putamen < hippocampus = hypothalamus = colliculi < trapezoid body. This activity pattern closely reflects the distribution of immunoreactivity and mRNA for heme oxygenase. The present microassay should offer a valuable tool for studies directly assessing a possible role for CO in neural signaling.     

A model for the catabolism of rhizopine in Rhizobium leguminosarum involves a ferredoxin oxygenase complex and the inositol degradative pathway

Rhizopines are nodule-specific compounds that confer an intraspecies competitive nodulation advantage to strains that can catabolize them. The rhizopine (3-O-methyl-scyllo-inosamine, 3-O-MSI) catabolic moc gene cluster mocCABRDE(F) in Rhizobium leguminosarum bv. viciae strain 1a is located on the Sym plasmid. MocCABR are homologous to the mocCABR gene products from Sinorhizobium meliloti. MocD and MocE contain motifs corresponding to a TOL-like oxygenase and a [2Fe-2S] Rieske-like ferredoxin, respectively. The mocF gene encodes a ferredoxin reductase that would complete the oxygenase system, but is not essential for rhizopine catabolism. We propose a rhizopine catabolic model whereby MocB transports rhizopine into the cell and MocDE and MocF (or a similar protein elsewhere in the genome), under the regulation of MocR, act in concert to form a ferredoxin oxygenase system that demethylates 3-O-MSI to form scyllo-inosamine (SI). MocA, an NAD(H)-dependent dehydrogenase, and MocC continue the catabolic process. Compounds formed then enter the inositol catabolic pathway.     

Ultrafast evolution of the excited states in the chlorophyll a/b complex CP29 from green plants studied by energy-selective pump-probe spectroscopy

The energy transfer process in the minor light-harvesting antenna complex CP29 of green plants was probed in multicolor transient absorption experiments at 77 K using selective subpicosecond excitation pulses at 640 and 650 nm. Energy flow from each of the chlorophyll (Chl) b molecules of the complex could thus be studied separately. The analysis of our data showed that the "blue" Chl b (absorption around 640 nm) transfers excitation to a "red" Chl a with a time constant of 350 +/- 100 fs, while the 'red' Chl b (absorption at 650 nm) transfers on a picosecond time scale (2.2 +/- 0.5 ps) toward a "blue" Chl a. Furthermore, both fast (280 +/- 50 fs) and slow (10-13 ps) equilibration processes among the Chl a molecules were observed, with rates and associated spectra very similar to those of the major antenna complex, LHC-II. Based on the protein sequence homology between CP29 and LHC-II, a basic modelling of the observed kinetics was performed using the LHC-II structure and the F?rster theory of energy transfer. Thus, an assignment for the spectral properties and orientation of the two Chl's b, as well as for their closest Chl a neighbors, is put forward, and a comparison is made with the previous assignments and models for LHC-II and CP29.     

Structure of nitric oxide synthase oxygenase dimer with pterin and substrate

Crystal structures of the murine cytokine-inducible nitric oxide synthase oxygenase dimer with active-center water molecules, the substrate L-arginine (L-Arg), or product analog thiocitrulline reveal how dimerization, cofactor tetrahydrobiopterin, and L-Arg binding complete the catalytic center for synthesis of the essential biological signal and cytotoxin nitric oxide. Pterin binding refolds the central interface region, recruits new structural elements, creates a 30 angstrom deep active-center channel, and causes a 35 degrees helical tilt to expose a heme edge and the adjacent residue tryptophan-366 for likely reductase domain interactions and caveolin inhibition. Heme propionate interactions with pterin and L-Arg suggest that pterin has electronic influences on heme-bound oxygen. L-Arginine binds to glutamic acid-371 and stacks with heme in an otherwise hydrophobic pocket to aid activation of heme-bound oxygen by direct proton donation and thereby differentiate the two chemical steps of nitric oxide synthesis.     

Meprin A, the major matrix degrading enzyme in renal tubules, produces a novel nidogen fragment in vitro and in vivo

We examined the effect of meprin A, the major matrix degrading metalloproteinase in rat kidney, on the laminin-nidogen complex. N-terminal sequence information from the most abundant 55 kDa fragment revealed that it was a breakdown product of nidogen rather than laminin. In comparison with over 50 nidogen cleavage sites produced by other proteases, the meprin A-induced nidogen cleavage site at amino acid position 899-900, a glutamine-glycine site in the G3 domain, is unique. In addition, these data demonstrate that meprin A degrades the G3 domain of nidogen even in the presence of laminin binding, which usually accords protection from proteolytic degradation. Meprin A also degraded purified nidogen into similar breakdown products. Given that the tubular basement membrane is located on the basilar side of the cell, the location of meprin A on the apical brush border makes it difficult to envision a role for meprin A in injury-induced basement membrane component breakdown. Thus, we examined the possibility that following renal tubular epithelial cell injury, meprin A undergoes a translocation to reach the underlying basement membrane. After renal ischemia-reperfusion there was a marked alteration in meprin A staining with meprin A now distributed throughout the renal tubular cell cytoplasm and directly adherent to the tubular basement membrane. This was in contrast to the usual linear staining of the brush border of tubules in the corticomedullary junction. These data provide unequivocal evidence that following injury, meprin A undergoes redistribution and/or adherence to the tubular basement membrane. Since in our in vitro studies, we identified a distinct meprin-induced 55 kDa nidogen breakdown product, the urine was also examined for the presence of nidogen degradation products after rat renal ischemia-reperfusion injury. Western blots showed a marked increase in the urinary 55 kDa nidogen fragment as early as the first day following ischemia-reperfusion injury and continuing for six days. Taken together, these in vivo data strongly support the notion that the nidogen breakdown products are the result of partial degradation of tubular basement membrane by meprin A following renal tubular ischemia-reperfusion injury.     

Immunoreactivity, stability, pharmacokinetics and biodistribution of a monoclonal antibody to human leukemic B cells after three different methods of radioiodination

Dal B02, a murine monoclonal antibody against human chronic lymphocytic leukemia (CLL) was radioiodinated using chloramine T (Chl.T), Bolton-Hunter (B-H) or N-succinimidyl-p-iodobenzoate (PIB). The preparations had comparable radiochemical purity (> 97%) and immunoreactive fraction (65-80%) but the Chl.T-based product was most susceptible to deiodination and loss of immunoreactivity. After i.v. injection into CLL-xenografted nude mice, the preparations had identical patterns of clearance from the blood but the PIB-based product led to more radioactivity in liver and spleen and less in the thyroid compared to the other preparations. The Chl.T-based product showed loss of immunoreactivity in circulation and less tumor-localized radioactivity 168 h after administration. The differences between the B-H-based and PIB-based products were less impressive than between PIB-based and Chl.T-based products.