Structure of the Ss blood group antigens, II: a methionine/threonine polymorphism within the N-terminal sequence of the Ss glycoprotein

The N-terminal amino acid sequence (residues 1--35) of the Ss sialoglycoprotein (or glycophorin B) from human erythrocyte membranes of defined Ss blood group activity was determined by manual sequencing methods, using N-terminal tryptic or chymotryptic glycopeptides and various secondary peptides. The proposed structure differs considerably from that suggested on the basis of work with glucopeptides of unknown Ss blood group activity (Furthmayr, Nature 271, 519--523, 1978). Only one difference between glycopeptides from Ss and ss erythrocytes was found, i.e. a methionine/threonine polymorphism at position 29. On the basis of previous work (Dahr et al., Hoppe-Seyler's Z. Physiol. Chem. 361, 145--152, 1980), it is concluded that this amino acid heterogeneity represents the Ss polymorphism rather than the UX or UZ polymorphisms, which are in strong genetic linkage disequilibrium with the Ss antigens. A part of the sequence (residues 9--30) of the major (MN) red cell membrane sialoglycoprotein (glycophorin A) was re-investigated and revised at positions 11 and 17. As judged from the present data, the first 26 residues of the Ss and the blood group N-specific MN glycoprotein are identical. The sequence 27--35 of the Ss glycoprotein shows a homology with the residues 56--64 and 59--67 of the MN glycoprotein. Data on the partial N-terminal sequence of glycopeptides from a third erythrocyte membrane sialoglycoprotein (component D or glycophorin C) indicate that its structure is different from those of the two other glycoproteins.     

Primary structure of horse erythrocyte glycophorin HA. its amino acid sequence has a unique homology with those of human and porcine erythrocyte glycophorins

Summary The complete amino acid sequence of the major sialoglycoproteins of horse erythrocyte membranes, glycophorin HA, was determined by manual sequencing methods, using tryptic, chymotryptic, and cyanogen bromide fragments. Glycophorin HA is a polypeptide chain of 120 amino acid residues and contains 10 oligosaccharide units attached to the amino-terminal side of the molecule. Its amino terminus is pyroglutamic acid. All of the oligosaccharides are linked O-glycosidically to threonine or serine residues. The amino acid sequence is consistent with the transmembrane orientation of glycophorins.There is no significant homology between the glycosylated domains of horse, human, and porcine glycophorins, but there is a considerable homology between the hydrophobic domains of the three glycophorins, which interact with the lipid bilayer of the erythrocyte membrane.     

Glycophorins B and C from human erythrocyte membranes. Purification and sequence analysis

We have developed methods for the preparative purification of two sialoglycoproteins (glycophorins B and C) from human erythrocyte membranes by high-performance ion exchange and gel permeation chromatography in the presence of Triton X-100. Glycophorin B was obtained without any detectable contaminants, and glycophorin C exhibited a purity of about 90-95%. The amino acid sequence of the intramembranous domain (residues 36-71) of glycophorin B was determined and found to be similar to that of the hydrophobic region of the major sialoglycoprotein (glycophorin A). The amino acid sequence of the hydrophobic domain (residues 49-88) of glycophorin C, that was also determined, agreed completely with the structure recently deduced from cDNA sequencing.     

Improved method for the isolation and preliminary characterization of human DAF (decay-accelerating factor)

DAF (decay-accelerating factor) is one of the integral membrane proteins of erythrocytes, and is considered to play an important role in the regulation of complement activation. The purification of DAF has been impeded by the difficulty in removing glycophorin. We devised an effective method for removing glycophorin. Through the limited trypsinization of stromata prior to the extraction of DAF, glycophorin was readily digested so that the DAF could be purified free of glycophorin by DEAE-Sephacel and Bio-Gel A 0.5 m chromatographies. On SDS-PAGE, DAF from trypsinized stromata showed the same mobility as that from native stromata: its molecular weight was estimated to be about 70 kDa. Amino acid analysis of DAF showed high contents of serine and glutamic acid. The amino-terminal sequence of DAF prepared by the present method, determined for the 29 residues, did not show significant homology with that of glycophorin.     

Amino acid sequence of an alpha-delta-glycophorin hybrid. A structure reciprocal to Sta delta-alpha-glycophorin hybrid

In this report we examine the primary sequence of a variant glycophorin obtained from erythrocytes of an individual who exhibits an unusual MNSs blood group phenotype. We show that this protein is a hybrid molecule constructed from sequences of alpha- and delta-glycophorins (glycophorins A and B) in a alpha-delta arrangement. Serological typing revealed that the donor's phenotype was M+N+S+s+U+; yet his erythrocytes reacted with some but not all examples of anti-S antisera. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a variant glycophorin band, and immunoblotting and reaction with N-glycanase suggested that its amino terminus resembled that of M-alpha-glycophorin but that its carboxyl terminus did not. A preparation highly enriched in the variant was obtained and used to generate peptide fragments for sequencing. The sequence revealed that the variant was a hybrid molecule whose amino terminus corresponded to M-alpha-glycophorin and whose carboxyl terminus corresponded to S-delta-glycophorin. CNBr cleavage of the variant glycophorin yielded four peptides. The sequence of the amino-terminal CNBr peptide (residues 1-8) was identical to the amino-terminal octapeptide of M-alpha-glycophorin. The proceeding peptide (residues 9-61) contained a segment identical to residues 9-58 of alpha glycophorin, but its carboxyl-terminal sequence had the Gly-Glu-Met sequence from S-delta-glycophorin (residues 27-29). The other two peptides, insoluble in aqueous solutions, contained highly hydrophobic sequences, identical to residues 30-52 and 53-68 of delta-glycophorin. Sequences of overlapping peptides generated by trypsin and V8 protease confirmed the hybrid nature of the variant glycophorin: residues 1-58 were identical to residues 1-58 of M-alpha-glycophorin, and residues 59-100 were entirely identical to residues 27-68 of S-delta-glycophorin. The variant glycophorin is expected to have 4 additional residues at its carboxyl terminus that correspond to the carboxyl-terminal residues 69-72 of delta-glycophorin. The amino acid sequence arrangement of the variant alpha-delta-glycophorin is an exact reciprocal of that found in another hybrid glycophorin, Sta, that is a delta-alpha hybrid. We propose that the two hybrid glycophorins represent the two possible products resulting from a reciprocal recombination event.     

Molecular cloning of a ubiquitously distributed microtubule-associated protein with Mr 190,000

A heat-stable microtubule-associated protein (MAP) with apparent molecular weight of 190,000 is a major non-neural MAP which distributes ubiquitously among bovine tissues (termed here MAP-U). Previously we reported that microtubule-binding chymotryptic fragments of MAP-U and tau contain a common assembly-promoting (AP) sequence of 22 amino acid residues (Aizawa, H., Kawasaki, H., Murofushi, H., Kotani, S., Suzuki, K., and Sakai, H. (1989) J. Biol. Chem. 264, 5885-5890). We isolated cDNA clones for MAP-U containing the whole coding sequence. Northern blot analysis revealed that a major species of MAP-U mRNA is 5 kilobases in length and is expressed ubiquitously among bovine tissues. Nucleotide sequence analysis revealed the complete amino acid sequence of MAP-U which consists of 1,072 amino acid residues. Analysis of the deduced amino acid sequence of MAP-U indicated that this molecule is clearly divided into two domains in terms of electrostatic charge distribution: an amino-terminal acidic domain (residues 1-640) and a carboxyl-terminal basic domain (residues 641-1072). The amino-terminal domain of MAP-U shows no significant sequence homology with other known protein sequences including neural MAPs, tau, and MAP-2. The amino-terminal domain of MAP-U contains unique 18 1/2 repeats of 14-amino acid motif which have not been observed in other MAPs. The carboxyl-terminal domain of MAP-U is further divided into three regions: a Pro-rich region (residues 641-880), an AP sequence region (residues 881-1003), and a short hydrophobic tail (residues 1004-1072). The Pro-rich region is mainly composed of five species of amino acid residues, Pro, Ala, Lys, Ser, and Thr. The AP sequence region contains four tandem repeats of AP sequences, and thus, this region is considered to play a leading role in the interaction of MAP-U with microtubules.     

Purification of glycopeptides of human ceruloplasmin and immunoglobulin D by high-pressure liquid chromatography

To facilitate structural studies of glycoproteins, reverse-phase high-pressure liquid chromatography (HPLC) methods have been developed for preparative isolation of glycopeptides and have been applied to human ceruloplasmin as an example of glycopeptides containing glucosamine (GlcN) and to human immunoglobulin D (IgD) for glycopeptides containing galactosamine (GalN). The use of RP-P columns and of trifluoroacetic acid and heptafluorobutyric acid as counterions was investigated. Various elution systems (both isocratic and programmed gradient) were used with n-propanol to assess the relative hydrophilicity of the peptides. The procedure developed for the GlcN glycopeptides of ceruloplasmin enabled purification of nine major chymotryptic peptides (ranging in size from 15 to 29 residues) and also of many minor peaks. These were characterized by amino acid and endgroup analysis, and the complete sequence of five was determined. These represent three different sites of GlcN attachment in the amino-terminal half of the ceruloplasmin chain. The procedures developed have enabled isolation of glycopeptides from ceruloplasmin having a single GlcN oligosaccharide attached; the latter are valuable for study of the structure and function of the carbohydrate groups. Separation of GalN glycopeptides from IgD was more difficult because of the high content of GalN in the hinge. Purification and sequence analysis was aided by partial removal of sugar by treatment with HF and by other methods. Four (or five) GalN oligosaccharides are attached to serine or threonine residues in the IgD hinge region, and all but one are in close proximity in the repeating sequence Ala-Thr-Thr-Ala-Pro-Ala-Thr-Thr.     

The primary structure of mu-chain-disease protein BOT. Peculiar amino-acid sequence of the N-terminal 42 positions

The complete primary structure of the mu heavy-chain disease (mu-HCD) protein BOT has been determined. The monomeric HCD-mu-chain consists of 391 amino-acid residues, lacking the VH and mu CH1 domains but including the entire CH2, CH3 and CH4 domains (349 residues). The sequence of the preceding 42 N-terminal residues which we designate as the "pre-C-part" presents no homology to any known variable or constant immunoglobulin sequence, but contains an internal homology of positions 10-19 to positions 20-29. The origin of the "pre-C-part" structure and the deletion of the mu CH1 domain of protein BOT are discussed.     

Properties of the arginine esterases from Bitis nasicornis (horned adder) venom

Five forms of arginine esterase (DE-2 to DE-6) were purified from Bitis nasicornis venom by gel filtration on Sephadex G-50, followed by ion exchange chromatography on CM-cellulose and DEAE-sepharose. They contain 17.6 to 23.1% of carbohydrate, 242 to 244 amino acids including 14 half-cystine residues and have molecular masses of about 38 kDa. The enzymes have a high esterolytic activity towards N alpha-benzoyl-L-arginine ethyl ester but show no proteolytic activity against Azocoll and no clotting activity against fibrinogen. Their sequences of the first 19 amino-terminal residues are the same, but their carbohydrate content shows some variation. Furthermore, sequence studies on the N-terminal regions of the arginine esterases from B. nasicornis venom indicate that they share a significant degree of sequence homology with the kallikrein-like enzymes of Crotalus adamanteus and C. atrox venoms and also with porcine pancreatic kallikrein. Studies on tryptic glycopeptides of the arginine esterases show that carbohydrate occurs at the N-terminal region of the molecule and also towards the center.     

The primary structure of two molecular species of porcine organ-common type acylphosphatase.

Electrophoretically homogeneous preparations of organ-common type acylphosphatase from porcine testis and brain were separated into two molecular species by reversed-phase liquid chromatography. From tryptic peptide map analysis, it was inferred that each of the two testis proteins is the same as the corresponding one of the two brain proteins. The complete primary structures of the two acylphosphatases from testis were then determined. The one molecular species consists of 100 amino acid residues: [sequence; see text] The other consists of 98 amino acid residues identical to the 3rd-100th residues of the above sequence and is also acetylated at the amino-terminal alanine. The 98-residue sequence has only 59% homology with porcine muscle acylphosphatase, but has 92% homology with human erythrocyte acylphosphatase. It was thus confirmed that the major acylphosphatases in testis, brain, and erythrocyte belong to the same organ-common type isoenzyme, distinct from the muscle type isoenzyme.     

Membrane glycophorins in Sta blood group erythrocytes

Structural and immunochemical studies of glycophorins isolated from erythrocytes of an individual homozygous for the M Sta blood group phenotype are described. Reactivities with specific monoclonal antibodies indicated that two major M and N glycophorins were present. The M and N Sta glycophorins were resolved by Lens culinaris lectin affinity chromatography. The N species was not held on the lectin but the M species, like control alpha glycophorins, was retained and could be eluted with alpha-methylmannoside. The two proteins were present in almost equimolar amounts. Studies of the CNBr fragments provided evidence that the structure of M Sta glycophorin is the same as that of the usual M alpha glycophorin but that the N Sta glycophorin is a variant. The amino-terminal octapeptides of the M and N species were similar in amino acid and carbohydrate composition to those isolated, respectively, from M and N alpha glycophorins. The studies focused on CNBr glycopeptide B that, in control alpha glycophorins, extends from amino acid residues 9 to 81. The fragment from the M species exhibited properties identical to those of the corresponding fragment of control alpha glycophorins in terms of size, chromatographic behavior, amino acid and carbohydrate contents and compositions, the presence of O-glycosidically linked saccharides and a single Asn-linked carbohydrate unit. The structures of the O-linked units were inferred experimentally to be NeuAc(alpha 2,3)Gal-(beta 1,3)GalNAc and NeuAc(alpha 2,3)Gal(beta 1,3) [NeuAc(alpha 2,6)]GalNAc, present in a ratio similar to that found in controls; and the Asn-linked unit also appeared to be as in the control. The tryptic glycopeptide pattern of the M Sta glycophorin CNBr fragment B was identical to the pattern of the corresponding control fragment, and the composition of the tryptic peptides suggested sequence identity with the control fragment. In contrast, the N Sta glycophorin yielded two CNBr glycopeptides B; both contained fewer amino acid residues and virtually lacked Man and GlcNAc, indicating the absence of the Asn-linked carbohydrate. The much decreased levels of these carbohydrates in the intact N protein, corroborated the latter finding. The O-glycosidic saccharides appeared similar to those found in control alpha glycophorins. However, the tryptic glycopeptide pattern of the variant differed from control M or N alpha glycophorins, suggesting a deletion of a large segment of the molecule near residues 40-61 and/or a substitution of methionine for a residue upstream from residue 40.(ABSTRACT TRUNCATED AT 400 WORDS)     

High-sensitivity FAB-MS strategies for O-GlcNAc characterization.

In this paper we report the first application of fast atom bombardment mass spectrometry (FAB-MS) to O-linked N-acetylglucosamine (O-GlcNAc)-bearing glycopeptides. Using N-acetylgalactosamine (GalNAc)- and Gal-GalNAc-containing glycopeptides (isolated from Tn glycophorin and desialylated normal glycophorin, respectively) as readily available model compounds, rapid and sensitive derivatization/FAB-MS strategies applicable to serine/threonine-rich glycopeptides have been devised. Peptides and glycopeptides were propionylated in a 1 min reaction using a mixture of trifluoroacetic anhydride and propionic acid, and the product mixtures were analysed directly by FAB-MS. Glycopeptides and peptides rich in hydroxylated residues afforded characteristic clusters of molecular ions at high sensitivity. Additional sensitivity enhancement was achieved by prior esterification of carboxyl groups. These methods were used in a study of O-GlcNAc glycopeptides produced by purified O-GlcNAc transferase addition of GlcNAc to the synthetic peptides YSDSPSTST and YSGSPSTST in which Y is tyrosine, S is serine, D is aspartic acid, P is proline, T is threonine and G is glycine. The propionyl derivatives afforded high-quality spectra which unequivocally showed that the majority of the glycopeptides were substituted with a single GlcNAc residue. Low pmol quantities of material gave detectable signals. The propionylation/FAB-MS procedure has been combined with gas-phase sequencing strategies and shows promise for defining the sites of glycosylation of O-GlcNAc glycopeptides that are available in limited quantities.     

The beta-mannanase from "Caldocellum saccharolyticum" is part of a multidomain enzyme.

The complete sequence of a beta-mannanase gene from an anaerobic extreme thermophile was determined, and it shows that the expressed protein consists of two catalytic domains and two binding domains separated by spacer regions rich in proline and threonine residues. The amino-terminal catalytic domain has beta-mannanase activity, and the carboxy-terminal domain acts as an endoglucanase. Neither domain shows homology with any other cellulase or hemicellulase sequence at the nucleic acid or protein level.     

Oligosaccharide units of lysosomal cathepsin D from porcine spleen. Amino acid sequence and carbohydrate structure of the glycopeptides

The amino acid sequences near the glycosylation sites and the oligosaccharide structures have been determined for the lysosomal protease cathepsin D from porcine spleen. Cathepsin D light and heavy chains were separately digested with proteases and the glycopeptides were purified. A single sequence was constructed from the amino acid sequence of the light chain glycopeptides which is: Tyr-Asn-Ser-Gly-Lys-Ser-Ser-Thr-Tyr-Val-Lys-Asn(CH2O)-Gly-Thr-Thr-Phe. A single glycopeptide sequence was also obtained for the heavy chain: Lys-Gly-Ser-Leu-Asp-Tyr-His-Asn(CH2O)-Val-Thr-Arg-Lys-Ala-Tyr. The light chain sequence is homologous with the sequence of porcine pepsin from residues 56 to 71. The heavy chain sequence is homologous with the pepsin sequence from residues 176 to 189. Thus, the 2 oligosaccharide-linked asparagines in cathepsin D correspond to residues 67 and 183 in pepsin and other homologous aspartyl proteases. These positions are located on the surface of the crystal structures of aspartyl proteases. Five oligosaccharides linked to Asn-67 were separated and their structures determined with proton NMR. Four major oligosaccharides are structural variants from the high mannose-type having 3, 5, 6, and 7 mannoses, respectively. A minor structure contained a third GlcNAc. Three oligosaccharide structures were found linked to Asn-183. Two major oligosaccharides are of the high mannose-type each with 5 mannose residues. One of the two contains a fucose linked to a GlcNAc. A third, very minor oligosaccharide contains galactose.     

Complete amino acid sequence of a papain-solubilized human histocompatibility antigen, HLA-B7. 2. Sequence determination and search for homologies

The complete amino acid sequence of the papain-solubilized heavy chain of a human histocompatibility antigen (HLA-B7) has been elucidated. It consists of a polypeptide of 271 residues (31 333 daltons). A single glycan moiety is attached to an asparagine residue at position 86 by an N-glycosidic bond. Two intrachain disulfide bonds, arranged linearly, involve half-cystine residues at positions 101 and 164 and at positions 203 and 259. They form two loops of 62 and 55 residues, respectively, separated by 38 residues. Computer analysis of the sequence suggests the existence of internal homology between the amino-terminal portion (residues 1--90) and the region of the first disulfide loop (residues 91--180). There is a significant homology between the second disulfide loop region of the chain (residues 182-271) and immunoglobulin (Ig) constant domains and beta 2-microglobulin [Orr, H. T., Lancet, D., Robb, R. J., López de Castro, J. A., & Strominger, J. L. (1979A) Nature (London) (in press)]. However, no such homology to Ig is apparent in the amino-terminal or in the first disulfide loop regions.     

Galactose-binding lectin from the seeds of champedak (Artocarpus integer): sequences of its subunits and interactions with human serum O-glycosylated glycoproteins

Our group has previously reported the isolation, partial characterisation, and application of a Galbeta1-3GalNAc- and IgA1-reactive lectin from the seeds of champedak (Artocarpus integer). In the present study, we have subjected the purified lectin to reverse-phase high performance liquid chromatography and sequenced its subunits. Determination of the N-terminal sequence of the first 47 residues of the large subunit demonstrated at least 95% homology to the N-terminal sequence of the alpha chains of a few other galactose-binding Artocarpus lectins. The two smaller subunits of the lectin, each comprised of 21 amino acid residues, demonstrated minor sequence variability. Their sequences were generally comparable to the beta chains of the other galactose-binding Artocarpus lectins. When used to probe human serum glycopeptides that were separated by two-dimensional gel electrophoresis, the lectin demonstrated strong apparent interactions with glycopeptides of IgA1, hemopexin, alpha2-HS glycoprotein, alpha1-antichymotrypsin, and a few unknown glycoproteins. Immobilisation of the lectin to Sepharose generated an affinity column that may be used to isolate the O-glycosylated serum glycoproteins.     

Sequence of the precursor to rat ornithine aminotransferase deduced from a cDNA clone

The nucleotide sequence of ornithine aminotransferase mRNA from rat liver, including the entire coding and 3' untranslated regions, was determined from two overlapping cDNA clones. The mRNA encodes a precursor polypeptide of 439 amino acid residues with a molecular weight of 48,332. The deduced amino acid composition of the proposed mature enzyme sequence (residues 35 through 439) was in good agreement with that reported for the purified protein. The amino-terminal segment of the precursor corresponding to residues 1 through 34 has an overall positive charge, containing 6 basic residues and only a single acidic residue, and is postulated to be the mitochondrial leader sequence. The first 22 amino acid residues of the proposed leader sequences share 54% homology with the leader peptide of rat ornithine transcarbamylase precursor and more limited homology to the leader peptides of other nuclear-encoded mitochondrial matrix proteins. Homology was also observed between residues 286 through 362 ornithine aminotransferase precursor and a region containing the pyridoxyl phosphate binding domain of mitochondrial aspartate aminotransferase.     

Structural variants of human T200 glycoprotein (leukocyte-common antigen).

Structural variation in the primary structure of human T200 glycoprotein has been detected. Three cDNA variants have been characterized each of which encode T200 molecules that differ in size as a result of sequence differences in their amino-terminal regions. The largest form of the molecule is distinguished from the smallest by an insert of 161 amino acids, after the first eight amino-terminal residues. The other variant has an insert at the same location of 47 amino acids identical to residues 75-121 in the larger insert. Both extra domains are rich in serine and threonine residues and are likely to display multiple O-linked oligosaccharides. These structural variants which probably arise by cell-type-specific alternative splicing provide a molecular basis for the previously observed structural and antigenic heterogeneity of T200 glycoprotein. In addition to the variable amino-terminal region, the external domain of human T200 glycoprotein consists of a second cysteine-rich region of about 400 amino acids, a single transmembrane-spanning region and a large cytoplasmic domain of 707 amino acids shared by all of the structural variants and highly conserved between species. The gene encoding human T200 is located on the long arm of chromosome 1.     

Trypsin inhibitor from the seeds of Acacia confusa.

A trypsin inhibitor (ACTI) was isolated and purified from the seeds of Acacia confusa by gel filtration, and trypsin-Sepharose 4B column affinity chromatography. The molecular weight of ACTI was found to be 21,000 +/- 1,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid composition analysis. ACTI contained four half-cystine and no methionine residues, and was rich in aspartic acid, glutamic acid, glycine, leucine, and lysine residues. The native trypsin inhibitor was composed of two polypeptide chains, and it inhibited trypsin and alpha-chymotrypsin stoichiometrically at the molar ratio of 1:1 and 2:1, respectively. The amino-terminal sequence analysis of the A. confusa trypsin inhibitor A and B chains revealed a more extensive homology with Acacia elata and silk tree trypsin inhibitors, and a less extensive homology with Kunitz soybean trypsin inhibitor.     

Complete amino acid sequence of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes

The complete amino acid sequence of porcine hepatic microsomal NADPH-cytochrome P-450 reductase has been determined by microsequence analysis on several sets of proteolytic fragments. Sequence studies were performed initially on a 20-kilodalton (kDa) fragment and then on 80-kDa fragment. The amino-terminal end of the mature protein was blocked with an acetyl group, followed by 676 amino acid residues. It has been revealed that the COOH-terminal 20-kDa fragment has been derived from original enzyme by cleavage at the Asn-Gly (residues 502-503) linkage by an unknown mechanism. An NADPH-protected cysteine residue is located at residue 565, near a region exhibiting high sequence homology with ferredoxin-NADP+ reductase. The FMN and FAD binding regions are possibly located in the amino-terminal region and the middle part of the protein molecule, respectively, as suggested by Porter and Kasper [Porter, T. D., & Kasper, C. B. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 973-977]. When this sequence is compared with that of rat enzyme, 60 amino acid residues are substituted, probably due to species differences. However, total sequence homology between these enzymes is 90%. Hydropathy plot analysis reveals that two regions from residues 27-43 and from residues 523-544 exhibit a high degree of hydrophobicity, suggesting membrane binding or interaction with cytochrome P-450.