Synthesis, chromatographic purification, and analysis of isomers of biliverdin IX and bilirubin IX.

Neutral solvent systems were developed to isolate the alpha, beta, gamma, and delta isomers of biliverdin IX dimethyl ester by TLC. The individual free acids of biliverdin IX were obtained by saponification of the corresponding dimethyl esters. The bilirubin IX isomers were prepared by reducing the corresponding biliverdin IX isomers with NaBH3CN. Starting from a pure biliverdin IX dimethyl ester, the corresponding free acid of biliverdin IX or bilirubin IX was available within 3-4 h. Preparation of spectrally pure bile pigment required final TLC on acid-cleaned neutral TLC plates. The absorption spectra of the free acids and dimethyl esters of biliverdin IX in methanol showed a broad band at about 650 nm and a sharp band at about 375 nm. The long-wave-length band was extremely sensitive to the presence of strong acid. A 10-fold molar excess of HCl caused a 35- to 50-nm shift of the absorption maximum to longer wavelengths and near doubling of the maximum absorption. The molar absorption coefficients of biliverdins were identical for each free acid and dimethyl ester pair. In each case, Beer's law was followed in both methanol and acidified methanol. Methanol also proved to be a suitable solvent for spectroscopic determination of the non-alpha isomers of bilirubin IX. The wavelength of maximum absorption and molar absorption coefficient of each dipyrrolic ethyl anthranilate azo pigment derived from the various bilirubin IX isomers are also reported.     

Biosynthesis of phorcabiline, blue-green biliary pigment of Actias selene (Lepidoptera, Attacidae)]

The blue-green bile pigments of Actias selene (Attacidae) have been investigated at different stages of its development. Coproporphyrinogen-14-C, protoporphyrin-IX3-H, and pterobilin-14-C, injected to larvae are metabolised into phorcabilin I, the main neopterobilin in this animal. It is concluded that phorcabilin I is a bile pigment of the IX gamma series and that pterobilin is its direct precursor. A method for the preparation of labelled protoporphyrin from quail egg-shell is reported.     

The chemical and enzymatic oxidation of hematohemin IX. Identification of hematobiliverdin IX alpha as the sole product of the enzymatic oxidation

Oxidative cleavage of hematohemin IX in pyridine solution in the presence of ascorbic acid (coupled oxidation), followed by esterification of the products with boron trifluoride/methanol produced the four possible hematobiliverdin dimethyl esters in 11.1% overall yield. Transetherifications took place simultaneously with the esterification reaction and resulted in the formation of the dimethyl ester of hematobiliverdin IX gamma 8a,13a-dimethyl ether (1.8%), the dimethyl ester of hematobiliverdin IX beta 13a,18a-dimethyl ether (1.9%), the dimethyl ester of hematobiliverdin IX delta 8a-monomethyl ether (1.4%), and the dimethyl ester of hematobiliverdin IX alpha 18a-monomethyl ether (0.4%). The latter was the sole product obtained after the enzymatic oxidation of hematohemin with heme oxygenase, after esterification of the reaction product with boron trifluoride/methanol. When the esterification step was omitted hematobiliverdin IX alpha was obtained from the enzymatic oxidation. The structures of the hematobiliverdin derivatives were secured by their NMR and mass spectra data. Saponification of the dimethyl esters afforded the hematobiliverdin methyl ethers, which were excellent substrates of biliverdin reductase and were readily reduced to the corresponding bilirubins. Hematobiliverdin IX alpha was also a good substrate of biliverdin reductase. It is concluded that the enzymatic oxidation of hematohemin IX by heme oxygenase is alpha-selective, while biliverdin reductase shows no selectivity in the reduction of the four hematobiliverdin isomers.     

UDP-glucuronosyltransferase activity and bilirubin conjugation in the bullfrog.

Bile pigments of bile and serum of Rana catesbeiana were investigated by means of high-pressure liquid chromatography. The major pigment in both bile and serum was bilirubin IX alpha. Bilirubin UDP-glucuronosyltransferase activity was found in the livers of all animals examined, but no conjugated bilirubin was detectable in the bile. Frog bile was found to contain large amounts of beta-glucuronidase. When the beta-glucuronidase inhibitor saccharo-1,4-lactone was introduced into the gall bladder followed by an exogenous bilirubin load, bilirubin glucuronide appeared in the bile.     

Isolation of N-phenylprotoporphyrin IX from the red cells and spleen of the phenylhydrazine-treated rat

Blood and spleens of phenylhydrazine-injected rats were treated with a solution of acidic methanol and zinc ion to isolate a green pigment. The pigment was resolved into two, I and II, by thin-layer chromatography. Pigment I was a mixture of two isomers of zinc complex of esterified N-phenylprotoporphyrin IX, in which vinyl-substituted pyrrole rings A and B were phenylated; and pigment II was a mixture of two isomers of the porphyrin complex with the N-phenyl group on propionic acid-substituted rings C and D. These pigments were also chemically prepared from the reaction of phenylhydrazine with oxyhemoglobin, independently characterized, and used to confirm the structures of the biological pigments. Determination revealed that the total amount of pigments found in the blood and spleen at 24 h after injection of phenylhydrazine corresponds to about 0.4% of the injected phenylhydrazine.     

Crystal structure of the calcium-stabilized human factor IX Gla domain bound to a conformation-specific anti-factor IX antibody

The binding of Factor IX to membranes during blood coagulation is mediated by the N-terminal gamma-carboxyglutamic acid-rich (Gla) domain, a membrane-anchoring domain found on vitamin K-dependent blood coagulation and regulatory proteins. Conformation-specific anti-Factor IX antibodies are directed at the calcium-stabilized Gla domain and interfere with Factor IX-membrane interaction. One such antibody, 10C12, recognizes the calcium-stabilized form of the Gla domain of Factor IX. We prepared the fully carboxylated Gla domain of Factor IX by solid phase peptide synthesis and crystallized Factor IX-(1-47) in complex with Fab fragments of the 10C12 antibody. The overall structure of the Gla domain in the Factor IX-(1-47)-antibody complex at 2.2 A is similar to the structure of the Factor IX Gla domain in the presence of calcium ions as determined by NMR spectroscopy (Freedman, S. J., Furie, B. C., Furie, B., and Baleja, J. D. (1995) Biochemistry 34, 12126-12137) and by x-ray crystallography (Shikamoto, Y., Morita, T., Fujimoto, Z., and Mizuno, H. (2003) J. Biol. Chem. 278, 24090-24094). The complex structure shows that the complementarity determining region loops of the 10C12 antibody form a hydrophobic pocket to accommodate the hydrophobic patch of the Gla domain consisting of Leu-6, Phe-9, and Val-10. Polar interactions also play an important role in the antibody-antigen recognition. Furthermore, the calcium coordination network of the Factor IX Gla domain is different than in Gla domain structures of other vitamin K-dependent proteins. We conclude that this antibody is directed at the membrane binding site in the omega loop of Factor IX and blocks Factor IX function by inhibiting its interaction with membranes.     

Spectrofluorometric estimation of intermediates of chlorophyll biosynthesis: protoporphyrin IX, Mg-protoporphyrin, and protochlorophyllide.

A highly sensitive spectrofluorometric method for quantitative estimation of certain precursors of chlorophyll biosynthesis from the mixtures of plant tetrapyrroles having overlapping fluorescence emission spectra is developed. At room temperature (293 degrees K) protoporphyrin IX is monitored from its emission maximum, 633 nm, when excited at 400 nm (E400/F633). Protochlorophyllide is estimated at 638 nm, while being excited at 440 nm (E440/F638). Mg-protoporphyrin+Mg-protoporphyrin monoester pool has emission around 589-592 nm. Therefore the integration value of the emission band that extends from 580 to 610 nm is taken to calibrate its concentration. This spectrofluorometric method designed for the determination of protoporphyrin IX, esterified and nonesterified Mg-protoporphyrin pool, and protochlorophyllide is far superior to available spectrophotometric methods and estimates as low as 1 nM concentration of plant pigments. As minute quantities of individual pigments can be quantitatively analyzed from their mixtures, this method eliminates analytical uncertainties due to recovery losses caused by chromatography. However, only dilute samples can be estimated by this spectrofluorometric method as the quantitative relation between fluorescence and concentration deviates from linearity at high, i.e., above 150 nM, concentrations of pigment to be quantified.     

Molecular cloning of rat and human type IX collagen cDNA and localization of the alpha 1(IX) gene on the human chromosome 6

Type IX collagen is found in hyaline cartilage, where it is associated with type II collagen in quarter-staggered collagen fibrils. Chicken type IX collagen has been extensively characterized and shown to contain molecules with three triple-helical domains, interspersed with non-triple-helical sequences. The molecule contains three, genetically distinct, subunits and one of these subunits carries a covalently bound glycosaminoglycan side chain. In the present report, we describe for the first time the primary structure of mammalian type IX collagen chains, based on cloning and sequencing of cDNA from rat and human cDNA libraries. The results suggest that mammalian alpha 1(IX) chains have the same multi-domain structure as the avian protein. We also demonstrate, by in situ hybridization of chromosome spreads, that the human alpha 1(IX) collagen gene is located on the long arm of chromosome 6. The cloning of human type IX collagen cDNA provides a probe for molecular studies of human chondrodysplasias that may involve abnormalities in this extracellular collagen-proteoglycan.     

Formation of delta 2- and delta 3-cholenoic acids from bile acid 3-sulfates by a human intestinal Fusobacterium strain

We isolated two strains of an unnamed Fusobacterium species from human intestinal microflora, which stereospecifically transformed bile acid 3-sulfates into C-3-unsubstituted, ring A-unsaturated bile acids. Both 3 alpha- and 3 beta-sulfates of 5 beta-bile acids were metabolized to delta 3-5 beta-cholenoic acids; 3 beta-sulfates of 5 alpha-bile acids were converted into a mixture of delta 2-5 alpha-bile acids and 3 alpha-hydroxy-5 alpha-bile acids, whereas 3 alpha-sulfates of 5 alpha-bile acids were left intact. Unsulfated bile acids were not transformed into unsaturated derivatives. These strains differ from previously isolated intestinal bacteria, which desulfated bile acid sulfates without further transformation.     

Formation of delta 2- and delta 3-cholenoic acids from bile acid 3-sulfates by a human intestinal Fusobacterium strain.

We isolated two strains of an unnamed Fusobacterium species from human intestinal microflora, which stereospecifically transformed bile acid 3-sulfates into C-3-unsubstituted, ring A-unsaturated bile acids. Both 3 alpha- and 3 beta-sulfates of 5 beta-bile acids were metabolized to delta 3-5 beta-cholenoic acids; 3 beta-sulfates of 5 alpha-bile acids were converted into a mixture of delta 2-5 alpha-bile acids and 3 alpha-hydroxy-5 alpha-bile acids, whereas 3 alpha-sulfates of 5 alpha-bile acids were left intact. Unsulfated bile acids were not transformed into unsaturated derivatives. These strains differ from previously isolated intestinal bacteria, which desulfated bile acid sulfates without further transformation.     

Cloning,expression, and crystallization of the V delta domain of a human gamma delta T-cell receptor.

T-lymphocytes recognize a wide variety of antigens through highly diverse cell-surface glycoproteins known as T-cell receptors (TCRs). These disulfide-linked heterodimers are composed of alpha and beta or gamma and delta polypeptide chains consisting of variable (V) and constant (C) domains non-covalently associated with at least four invariant chains to form the TCR-CD3 complex. It is well established that alpha beta TCRs recognize antigen in the form of peptides bound to molecules of the major histocompatibility complex (MHC); furthermore, information on the three-dimensional structure of alpha beta TCRs has recently become available through X-ray crystallography. In contrast, the antigen specificity of gamma delta TCRs is much less well understood and their three-dimensional structure is unknown. We have cloned the delta chain of a human TCR specific for the MHC class I HLA-A2 molecule and expressed the V domain as a secreted protein in the periplasmic space of Escherichia coli. Following affinity purification using a nickel chelate adsorbent, the recombinant V delta domain was crystallized in a form suitable for X-ray diffraction analysis. The crystals are orthorhombic, space group P2(1)2(1)2 with unit cell dimensions a = 69.9, b = 49.0, c = 61.6 A. and diffract to beyond 2.3 A resolution. The ability of a V delta domain produced in bacteria to form well-ordered crystals strongly suggests that the periplasmic space can provide a suitable environment for the correct in vivo folding of gamma delta TCRs.     

Ferriprotoporphyrin IX, phospholipids, and the antimalarial actions of quinoline drugs

Two subclasses of quinoline antimalarial drugs are used clinically. Both act on the endolysosomal system of malaria parasites, but in different ways. Treatment with 4-aminoquinoline drugs, such as chloroquine, causes morphologic changes and hemoglobin accumulation in endocytic vesicles. Treatment with quinoline-4-methanol drugs, such as quinine and mefloquine, also causes morphologic changes, but does not cause hemoglobin accumulation. In addition, chloroquine causes undimerized ferriprotoporphyrin IX (ferric heme) to accumulate whereas quinine and mefloquine do not. On the contrary, treatment with quinine or mefloquine prevents and reverses chloroquine-induced accumulation of hemoglobin and undimerized ferriprotoporphyrin IX. This difference is of particular interest since there is convincing evidence that undimerized ferriprotoporphyrin IX in malaria parasites would interact with and serve as a target for chloroquine. According to the ferriprotoporphyrin IX interaction hypothesis, chloroquine would bind to undimerized ferriprotoporphyrin IX, delay its detoxification, cause it to accumulate, and allow it to exert its intrinsic biological toxicities. The ferriprotoporphyrin IX interaction hypothesis appears to explain the antimalarial action of chloroquine, but a drug target in addition to ferriprotoporphyrin IX is suggested by the antimalarial actions of quinine and mefloquine. This article summarizes current knowledge of the role of ferriprotoporphyrin IX in the antimalarial actions of quinoline drugs and evaluates the currently available evidence in support of phospholipids as a second target for quinine, mefloquine and, possibly, the chloroquine-ferriprotoporphyrin IX complex.     

Photogeneration of singlet molecular oxygen by the components of hematoporphyrin IX derivative

The photosensitized luminescence of singlet molecular oxygen has been studied in aqueous and alcoholic solutions of hematoporphyrin IX (HP) and di- and oligomeric components of "hematoporphyrin derivative" (photofrin II) which is known to be used as a drug in photodynamic tumor therapy. The quantum yields of 1O2 generation (gamma delta) by these compounds have been determined. It was found that the highest gamma delta values are characteristic of alcoholic and micellar detergent aqueous solutions. In detergent-free aqueous solutions containing mainly associated porphyrin molecules, gamma delta is much lower (5-30%), polymeric photofrin components being considerably less active than HP. Both localization of porphyrins in hydrophobic loci and high photosensitizing activity in lipid phase are supposed to play the key role in tumor photodestruction.     

Monoclonal antibody against chicken type IX collagen: preparation, characterization, and recognition of the intact form of type IX collagen secreted by chondrocytes

A series of monoclonal antibodies was prepared against the pepsin-resistant fragment of type IX collagen designated HMW. One of these antibodies (called 2C2) was selected for further analysis. Antibody 2C2 showed no cross-reactivity with other collagen types by inhibition enzyme-linked immunosorbent assays. It recognized an epitope present in native HMW, but failed to recognize any of the three chains of HMW fractionated after denaturation followed by reduction and alkylation of interchain disulfide bridges. Electron microscopic observations after rotary shadowing showed that the location of the epitope for antibody 2C2 was close to the carboxy-terminus of HMW. Immunofluorescent staining of sections of embryonic and adult cartilage with antibody 2C2 after removal of proteoglycans by testicular hyaluronidase digestion showed that type IX collagen is distributed throughout the cartilage matrix, and is not present in other connective tissues or skeletal muscle. The intact type IX collagen molecule, which was secreted by a suspension culture of freshly isolated embryonic chick chondrocytes, was recognized by rotary shadowing in the presence of antibody 2C2 after first precipitating the procollagens from the culture medium with ammonium sulfate (30%). Two different collagenous molecules were present in the precipitate: a longer molecule of type II procollagen (average length, 335 nm) with both amino- and carboxy-propeptides still remaining uncleaved, and a shorter molecule (average length, 190 nm) which was identified as type IX collagen. Antibody 2C2 consistently bound to the shorter molecules at a site located 136 nm from a distinctive knob at one end of the molecule, and did not bind to any specific site on the type II procollagen molecules. The structure of the intact type IX collagen molecule with the location of both collagenous and noncollagenous domains was as predicted after converting the nucleotide sequence of a cDNA clone encoding for one of the chains of type IX collagen to an amino acid sequence (Ninomiya, Y., and B. R. Olsen, 1984, Proc. Natl. Acad. Sci. USA, 81:3014-3018).     

Oxidation of the capsular polysaccharide of pneumoccal type IX by periodate

1. The pneumococcal type IX polysaccharide (polysaccharide S IX) has been oxidized by sodium metaperiodate and reduced by sodium borohydride. Of the constituent monosaccharides, N-acetylglucosamine and N-acetylmannosamine remain unaltered, whereas 40% of the glucose and 90% of the glucuronic acid are oxidized. 2. The effect of oxidation and subsequent reduction on the precipitation of polysaccharide S IX in anti-(pneumococcal) sera is described and interpreted in structural terms. 3. Oligosaccharides produced by oxidation, reduction and hydrolysis with dilute acid have been isolated and partially characterized. 4. The results in this paper and the preceding one (Higginbotham et al., 1972) are used to postulate a possible structure for polysaccharide S IX.     

Carbonic anhydrase IX: Biochemical and crystallographic characterization of a novel antitumor target

Isoform IX of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), CA IX, is a transmembrane protein involved in solid tumor acidification through the HIF-1α activation cascade. CA IX has a very high catalytic activity for the hydration of carbon dioxide to bicarbonate and protons, even at acidic pH values (of around 6.5), typical of solid, hypoxic tumors, which are largely unresponsive to classical chemo- and radiotherapy. Thus, CA IX is used as a marker of tumor hypoxia and as a prognostic factor for many human cancers. CA IX is involved in tumorigenesis through many pathways, such as pH regulation and cell adhesion control. The X-ray structure of the catalytic domain of CA IX has been recently reported, being shown that CA IX has a typical α-CA fold. However, the CA IX structure differs significantly from the other CA isozymes when the protein quaternary structure is considered. Thus, two catalytic domains of CA IX associate to form a dimer, which is stabilized by the formation of an intermolecular disulfide bond. The active site clefts and the proteoglycan (PG) domains are located on one face of the dimer, while the C-termini are located on the opposite face to facilitate protein anchoring to the cell membrane. As all mammalian CAs, CA IX is inhibited by several main classes of inhibitors, such as the inorganic anions, the sulfonamides and their bioisosteres (sulfamates, sulfamides, etc.), the phenols, and the coumarins. The mechanism of inhibition with all these classes of compounds is understood at the molecular level, but the sulfonamides and their congeners have important applications. It has been recently shown that both in vitro, in cell cultures, as well as in animals with transplanted tumors, CA IX inhibition with sulfonamides lead to a return of the extracellular pH to more normal values, which leads to a delay in tumor growth. As a consequence, CA IX represents a promising antitumor target for the development of anticancer agents with an alternative mechanism of action.     

Degradation of hemin IX and synthetic hemins to α-bilirubins and their conjugates in the isolated perfused rat liver

Hemin IX was perfused through rat liver of a normal, untreated animal. Its degradation products, collected in the bile fluid over a period of 90 min, were found to consist of the bilirubin IX-α diglucuronide (56%), the mixture of bilirubin IX-α monoglucuronides (42%), and free bilirubin IX-α (2%). When the synthetic hemin XIII $\text{2}$ was perfused with the same technique, it was found to be degraded in the same way. The bile fluid contained the diglucuronide of bilirubin XIII-α $\text{10}$ (55%), the monoglucuronide of bilirubin XIII-α $\text{9}$ (43%) and the free bilirubin XIII-α 8 (2%). Similar results were obtained when the iron 1,4-di(β-hydroxyethyl)-2,3,5,8-tetramethyl-6,7-di(β-carboxyethyl) porphyrin $\text{3}$ was perfused; the diglucuronide of the α-bilirubin $\text{11}$ comprised 65% of the excreted bile bilirubins, the monoglucuronide was 25% of the total and the free α-bilirubin $\text{11}$ 10% of the total. Perfusion of hematohemin gave 58% of the diglucuronide of α-hematobilirubin, as well as 40% of the monoglucuronides, and 2% of the free α-hematobilirubin. The simultaneous perfusion of hematohemin and of hemin IX produced an inhibition of the degradation of the hemin IX, while hematohemin was degraded as described above. It was concluded that the normal rat liver is prepared to dispose of exogenously added hemins by their oxidation to α-biliverdins, reduction of the latter to the corresponding α-bilirubin and excretion of their conjugated derivatives through the bile duct.     

Carbonic anhydrase IX

Cell migration can be principally viewed as a chain of well-orchestrated morphological events that lead to dynamic reshaping of the cell body. However, behind the scene of such a “morphological theater” there are very complex, interrelated molecular and physiological processes that drive the cell movement. Among them, ion transport and pH regulation play a key role, with carbonic anhydrase IX (CA IX) emerging as one of the important “molecular actors.” CA IX is a highly active cell surface enzyme expressed in a broad range of solid tumors in response to hypoxia and explored as a clinically useful biomarker of hypoxia and as a therapeutic target. Its biological role is to protect tumor cells from hypoxia and acidosis in the tumor microenvironment. The study published recently by our group showed that CA IX actively contributes to cell migration and invasion. For the first time, we demonstrated CA IX accumulation in lamellipodia of migrating cells and its direct in situ interaction with bicarbonate transporters. Our findings indicate that tumor cells need CA IX not only as a pro-survival factor in hypoxia and acidosis, but also as a pro-migratory component of the cellular apparatus driving epithelial-mesenchymal transition.     

Structure of the glycoprotein Ib.IX complex from platelet membranes

The glycoprotein Ib.IX complex is a major component of the platelet membrane. It mediates the adhesion of platelets to exposed subendothelium and provides an attachment site for the membrane skeleton on the plasma membrane. The present study was designed to characterize the structure of the glycoprotein Ib.IX complex. Electron microscopy of purified glycoprotein Ib.IX complex in detergent showed that each complex existed as a flexible rod with a globular domain on either end. The overall length of the complex was approximately 59.5 nm. The smaller globular domain had a diameter of approximately 8.9 nm; the larger, a diameter of approximately 15.9 nm. In the absence of detergent, the glycoprotein Ib.IX complexes tended to self-associate through the larger globular domain, suggesting that this domain contained the hydrophobic region that inserts into the membrane. Proteases known to cleave glycoprotein Ib alpha close to its membrane-insertion site released the larger globular domain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that this domain was composed of glycoprotein Ib beta, glycoprotein IX, and a Mr = 25,000 fragment of glycoprotein Ib alpha. Proteolysis at the external end of glycoprotein Ib alpha reduced the size of the smaller globular domain. This study shows that the glycoprotein Ib.IX complex has an elongated shape, with a globular domain on the end that inserts into the membrane and a smaller globular domain on the end of glycoprotein Ib alpha that is oriented external to the plasma membrane.     

Carbonic anhydrase inhibitors: the first selective, membrane-impermeant inhibitors targeting the tumor-associated isozyme IX

The inhibition of the tumor-associated transmembrane carbonic anhydrase IX (CA IX) isozyme possessing an extracellular active site has been investigated with a series of positively-charged, pyridinium derivatives of sulfanilamide, homosulfanilamide and 4-aminoethylbenzenesulfonamide. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and IV (membrane-bound) were also provided for comparison. A very interesting inhibition profile against CA IX with these sulfonamides has been observed. Several nanomolar (K(i)'s in the range of 6-54 nM) CA IX inhibitors have also been detected. Because CA IX is a highly active isozyme predominantly expressed in tumor tissues with bad prognosis of disease progression, this finding is very promising for the potential design of CA IX-specific inhibitors with applications as anti-tumor agents. This is the first report of inhibitors that may selectively target CA IX, due to their membrane-impermeability and high affinity for this clinically relevant isozyme.