Studies in vitro on the involvement of O-sulphate esters in the formation of O-methylated 3,4-dihydroxybenzoic acid by rat liver.

The involvement of O-sulphate esters in the directed O-methylation was investigated in vitro with a dialysed "high-speed' supernatant from rat liver as the enzyme preparation and the catechol compound 3,4-dihydroxybenzoic acid as the substrate. The enzyme reactions involved were studied separately with the O-methylated and O-sulphated derivatives. The rate of hydrolysis by arylsulphatase was 14.5 nmol/min per mg of protein for 3-methoxy-4-sulphonyloxybenzoic acid and 10.1 nmol/min per mg of protein for 4-methoxy-3-sulphonyloxybenzoic acid. The sulphotransferase activity towards the guaiacols 4-hydroxy-3-methoxybenzoic acid and 3-hydroxy-4-methoxybenzoic acid was 570pmol of 4-O-sulphated and 350pmol of 3-O-sulphated product formed/min per mg of protein. The 3-O- and 4-O-sulphate esters of 3,4-dihydroxybenzoic acid could not serve as substrates for the catechol O-methyltransferase reaction. When either ester was incubated in the presence of S-adenosyl-L-methionine, but without the arylsulphatase inhibitor KH2PO4, 3,4-dihydroxybenzoic acid was formed, which was subsequently O-methylated in a meta/para ratio of 4.6. It is concluded that O-methylation can precede O-sulphation but that O-sulphation prevents further metabolism by O-methylation. Also O-sulphate esters do not have a directing effect on O-methylation. From the study of the simultaneous action of sulphotransferase and catechol O-methyltransferase on 3,4-dihydroxybenzoic acid we conclude that O-sulphation and O-methylation proceed independently of each other under the assay conditions used, both directed preferentially to the 3-hydroxy group.     

Comparison of catechol-Q-methyltransferase from rat brain, erythrocytes and liver.

Catechol-O-methyltransferase activity from supernatant of brain, red blood cells and liver of the rat were characterized by the following items: 1) substrate specificity with respect to five catechol substrates, 2) meta/para ratio of O-methylation, 3) apparent Michaelis constants, 4) pH dependence of reaction rate. Under identical conditions, a progressive decrease in the rate of O-methylation by rat liver supernatant can be noticed in the sequence of 3.4-dihydroxy-acetophenone > 3.4-dihydroxybenzoic acid > 3.4-dihydroxyphenylacetic acid. Using 3.4-dihydroxybenzaldehyde as substrate, the meta/para ratio of O-methylation and the apparent Michaelis constants are similar for catechol-O-methyltransferase from brain and red blood cells and differ from the values obtained with liver supernatant. In phosphate buffer, the pH curves are almost identical for catechol-O-methyltransferase from brain, red blood cells and liver, in glycine/NaOH buffer, however, the liver enzyme shows a broad peak. From our experiments it is obvious that the biochemical characteristics selected are similar for soluble catechol-O-methyltransferase from brain and erythrocytes which differ from that obtained with liver enzyme.     

Extensive conjugation of dopamine (3,4-dihydroxyphenethylamine) metabolites in cultured human skin fibroblasts and rat hepatoma cells.

[G-3H]Dopamine (3,4-dihydroxyphenethylamine) metabolism in human skin fibroblasts and rat hepatoma cells in culture was determined by high-pressure liquid-chromatographic analysis of both cell extract and uptake medium. Conjugated metabolites were selectively hydrolysed by incubation with arylsulphatase or beta-glucuronidase before analysis. The principal metabolites of dopamine in fibroblast cells are 3-methoxytyramine 4-O-sulphate and 3-methoxytyramine. No significant differences, either in the amounts of these metabolites or in the amount of dopamine metabolism, were observed in fibroblasts from both normal and homocystinuric individuals. In rat hepatoma cells, the major metabolite of dopamine was 3-methoxytyramine 4- or 3-O-glucuronide; lower concentrations of dopamine 4- or 3-O-glucuronide, 4-hydroxy-3-methoxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid and two unidentified glucuronide conjugates were also observed. Significant differences in the relative concentrations of these metabolites in cell and uptake medium were observed in both cell systems.     

Diacylglycerol hydrolysis in rat liver lysosomes

The matrix of rat liver lysosomes exhibits high hydrolytic activity towards 1,2-diacylglycerol with an optimum at pH 4.0. The lipolytic reaction follows Michaelis-Menten kinetics (apparent Vmax 470 nmol hydrolysed/min per mg protein; apparent Km 71 microM 1,2-dioleoylglycerol). Formation of 1- and 2-monooleoylglycerols indicates an initial attack at both the primary and secondary ester bonds. The lysosomal matrix also catalyses (re)acylation reactions, i.e. the formation of 1,2-diacylglycerol from 2-monoacylglycerol and free fatty acid. However, (re)acylation proceeds at a far lower rate than deacylation of diacylglycerol. Lysosomal diacylglycerol hydrolysis is sensitive towards non-ionic detergents, cationic amphiphilic drugs and the lipase inhibitor RHC 80267.     

Determination of catechol-O-methyltransferase activity in brain tissue by high-performance liquid chromatography with on-line radiochemical detection

A sensitive assay for catechol-O-methyltransferase (COMT) activity by high-performance liquid chromatography with on-line radiochemical detection was described. The method was based on the measurement of 3H-labeled 3-O- and 4-O-methylated products of the substrate, 3,4-dihydroxybenzoic acid, using S-adenosyl-L-[methyl-3H]methionine as the methyl donor, or the measurement of 14C-labeled 3-O- and 4-O-methylated products of the substrate, [7-14C]dopamine. The reaction products were determined from the incubation mixture after removal of protein by injecting an aliquot into the liquid chromatograph. The detection limit with counting efficiency of 40% was 0.45 pmol 3H-labeled product, and 0.04 pmol 14C-labeled product with 61% counting efficiency. The method is suitable for assaying membrane-bound and soluble COMT activities in the brain tissue and for calculation of meta/para product ratios.     

Enzymic formation of esters of methyl sterol precursors of cholesterol

For investigation of the reactions of cholesterol biosynthesis, a number of workers use the 10,000 g supernatant fraction (or similar preparations) obtained from cell-free homogenates of rat liver. We have found that esters of methyl sterol biosynthetic intermediates are formed by this crude source of enzymes. Esters of C(30)-, C(29)-, C(28)-, and C(27)-sterol intermediates have been isolated by silicic acid chromatography of an acetone extract of incubation mixtures. Competition between ester formation and demethylation of the C(28)-sterol intermediate has been demonstrated. With 4alpha-methyl-5alpha-cholest-7-en-3beta-ol as substrate, maximal velocities of ester formation (0.36 nmole/30 min per mg of protein) were almost equivalent to maximal velocities of demethylation (0.45 nmole/30 min per mg of protein). Ester formation may be eliminated by carrying out incubations with microsomal preparations; ester formation may be restored completely upon addition (to the microsomes) of either coenzyme A and ATP or the supernatant fraction resulting from centrifugation at 105,000 g. Ester formation has been examined similarly with broken-cell preparations of rat skin. With $$Word$$ as substrate, the rate of ester formation was more than six times the rate of methyl sterol demethylation. The very significant competition between esterification and demethylation of methyl sterol intermediates of skin suggests that sterol intermediates accumulate in rat skin because of the rapid formation of esters that may not be further metabolized.     

Studies on the sulphation of 3,4-dihydroxyphenylethylamine (dopamine) and related compounds by rat tissues

The formation of sulpho-conjugates of 3,4-dihydroxyphenylethylamine (dopamine) and related compounds was examined in preparations of rat tissues. Liver high-speed-supernatant preparations readily transferred sulphate from adenosine 3'-phosphate 5'-sulphato-phosphate to dopamine under standard conditions. The main product was identified as the 3-O-sulphate. The preparation also sulphated the 3- and 4-methoxy derivatives but to a lesser extent (44% and 95% respectively) relative to dopamine. Brain preparations possessed only half the activity of liver but formed both the 3- and 4-O-sulphates in the molar ratio of 1.7:1. l-3,4-Dihydroxyphenylalanine (l-dopa) in both tissue preparations did not yield any significant amount of sulpho-conjugate when the dopa decarboxylase present was inhibited. The sulphotransferase activity of preparations was doubled in the presence of dithiothreitol and it was concluded that l-tyrosine methyl ester sulphotransferase was the enzyme involved. A method for the preparation of authentic dopamine 3-O-sulphate and 4-O-sulphate was developed.     

Charge effects on phospholipid monolayers in relation to cell motility

A new sensitive method for the assay of retinyl ester hydrolase in vitro was developed and applied to liver homogenates of 18 young pigs with depleted-to-adequate liver vitamin A reserves. Radioactive substrate was not required, because the formation of retinol could be adequately quantitated by reversed-phase high-performance liquid chromatography. Optimal hydrolase activity was observed with 500 microM retinyl palmitate, 100 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 2 mg/ml Triton X-100 at pH 8.0. The relative rates of hydrolysis of six different retinyl esters by liver homogenate were: retinyl linolenate (100%), myristate (99%), palmitate (47%), oleate (38%), linoleate (31%), and stearate (29%). The enzyme was found primarily in the membrane-containing fractions of liver (59 +/- 3%, S.E.) and kidney (76 +/- 3%), with considerably lower overall activity in kidney (57-375 nmol/h per g of tissue) than in liver (394-1040 nmol/h per g). Retinyl ester hydrolase activity in these pigs was independent of serum retinol values, which ranged from 3 to 24 micrograms/dl, and of liver vitamin A concentrations from 0 to 32 micrograms/g. Pig liver retinyl ester hydrolase differs from the rat liver enzyme in its substrate specificity, bile acid stimulation, and interanimal variability.     

Assay of catechol O-methyltransferase by determination of the m-and p-O-methylated products using high-performance liquid chromatography.

A new assay of catechol O-methyltransferase (EC 2.1.1.6) is described by determination of the m- and p-O-methylated products of 3,4-dihydroxybenzoic acid. The method involves DEAE-Sephadex A-25 chromatography and reversed-phase high-performance liquid chromatography on a LiChrosorb 5 RP 18 column. The liquid chromatographic solvent system consisted of 0.05 m acetic acid in methanol:water (1:4, $\text{v}\text{v}$), pH 3,2. meta/para ratios of O-methylation are easily obtained by this method. Dimethylation has not been observed with a partially purified enzyme preparation from rat liver.     

The kinetic parameters of the uptake of very-low-density lipoprotein remnant cholesteryl esters by perfused rat livers

The aim of this study was to determine the kinetic parameters of the hepatic uptake of VLDL remnant cholesteryl esters. Rat livers were perfused in situ with a broad range of remnant [3H]cholesteryl ester concentrations of known specific radioactivity. Following exactly 3 min of perfusion, hepatic lipids were extracted and labelled cholesteryl esters were separated by thin-layer chromatography and counted. The rate of cholesteryl ester uptake was a saturable process and the apparent kinetic parameters were determined from the Lineweaver-Burk plot of the data. Km and Vmax were calculated to be 72 microM and 35 nmol cholesteryl ester/min per g liver, respectively. For the purpose of comparison, we have expressed our kinetic parameters in terms of number of particles (Vmax = 0.022 nmol particles/min per g liver and Km = 45 nM) and compared our values with those obtained with chylomicron remnants by another group of investigators (Sherrill, B.C., Innerarity, T.L. and Mahley, R.W. (1980) J. Biol. Chem. 255, 1804-1807). We found that the maximal capacity for the removal of VLDL particles was similar to what was observed with rat chylomicron remnants. In contrast, the Km for the uptake process of VLDL remnant particles was approximately four times higher than that of rat chylomicron remnant particles. Our results are consistent with the hypothesis that hepatic removal of both chylomicron and VLDL remnants is mediated by the same receptor, but suggest that the affinity of VLDL remnants for the hepatic removal process is substantially lower, possibly due to structural differences between the two remnant particles.     

A continuous fluorescence displacement assay for the measurement of phospholipase A2 and other lipases that release long-chain fatty acids.

1. A new continuous fluorescence assay for phospholipase A2 is described which involves the displacement of the highly fluorescent fatty-acid probe 11-(dansylamino)undecanoic acid from rat liver fatty-acid-binding protein by long-chain fatty acids released as a result of phospholipase A2-catalysed hydrolysis of phospholipids. The initial rate of decrease in fluorescence is linearly related to enzyme activity. 2. The assay will detect enzyme activity down to about 10 pmol/min per ml and gives a linear response up to about 10 nmol/min per ml. 3. The assay will work with all phospholipids that have been tested including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol. Substrates carrying a net negative charge showed the highest rates of hydrolysis. 4. The assay will work, in principle, with an enzyme catalysing the release of long-chain fatty acids from a fatty-acylated substrate. This has been confirmed with pancreatic lipase and cholesterol esterase.     

Properties of liver retinyl ester hydrolase in young pigs

A new sensitive method for the assay of retinyl ester hydrolase in vitro was developed and applied to liver homogenates of 18 young pigs with depleted-to-adequate liver vitamin A reserves. Radioactive substrate was not required, because the formation of retinol could be adequately quantitated by reversed-phase high-performance liquid chromatography. Optimal hydrolase activity was observed with 500 μM retinyl palmitate, 100 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 2 mg/ml Triton X-100 at pH 8.0. The relative rates of hydrolysis of six different retinyl esters by liver homogenate were: retinyl linolenate (100%), myristate (99%), palmitate (47%), oleate (38%), linoleate (31%), and streate (29%). The enzyme was found primarily in the membrane-containing fractions of liver (59±3%, S.E.) and kidney (76±3%), with considerably lower overall activity in kidney (57–375 nmol/h per g of tissue) than in liver (394–1040 nmol/h per g). Retinyl ester hydrolase activity in these pigs was independent of serum retinol values, which ranged from 3 to 24 μg/dl, and of liver vitamin A concentrations from 0 to 32 μg/g. Pig liver retinyl ester hydrolase from the rat liver enzyme in its substrate specificity, bile acid stimulation, and interanimal variability.     

Purification and some properties of arylsulphatases A and B from rabbit kidney cortex.

Arylsulphatases A and B (EC 3.1.6.1) of rabbit kidney cortex were purified 5250- and 7720-fold respectively by a multiple-column-chromatography method. The specific activity toward 4-nitrocatechol sulphate was 42mumol/min per mg for arylsulphatase A and 62 mumol/min per mg for arylsulphatase B. Each enzyme migrated as a single band on polyacrylamide-gel electrophoresis, and the enzyme activity corresponded to the band of protein on the gel. The rate of hydrolysis of ascorbic acid 2-sulphate by arylsulphatase A was three times that for cerebroside 3-sulphate. Arylsulphatase B hydrolysed UDP-N--acetylgalactosamine 4-sulphate and glucosamine 4,6-disulphate, but not galactosamine 6-sulphate.     

Functional relationship between initial oxidation of 7-ethoxycoumarin and subsequent conjugation of 7-hydroxycoumarin in isolated perfused rat livers

Functional relationship between the initial mixed function oxidation of 7-ethoxycoumarin (EC) to 7-hydroxycoumarin (HC) and the subsequent conjugation of this metabolite to sulfate ester and glucuronide has been studied using isolated perfused rat livers. When increasing concentrations of EC (from 25 to 200 microM) were infused, perfused liver can oxidize only up to about 60 nmol of the infused EC to HC per min/g liver tissue. Most of this HC metabolite was released as sulfate ester, but there was a dose dependent shift to a more significant glucuronidation at the expense of the sulfate form. The dose dependent shift observed upon infusions with increasing dose of EC was not extensive so that the major portion of metabolite released was always the sulfate ester. However, the shift observed with HC was extensive and the major portion released was the glucuronide conjugate. Upon infusions with increasing concentrations of HC, the maximal rates of sulfation and glucuronidation were found to be 60 nmol and 120 nmol of HC conjugated per min/g liver tissue, respectively. Furthermore, the ranges in the rates of conjugation for the infused HC were divided into a sulfate ester 'zone' (less than 20 nmol), a dose-dependent shift 'zone' (between 20 and 180 nmol) with the 'cross-over' occurring at 80 nmol/min/g liver, and reaching the maximal conjugation 'capacity' rate (180 nmol), above which the unconjugated free form of HC was released. Under conditions when EC was infused into normal rat livers, the calculated maximal oxidation rate was only 60 nmol of HC produced/min/g liver. Consequently, under such a condition, the oxidation rate may never reach the 'cross-over' rate and this explains the lack of extensive dose-dependent shift and further indicates that there remained a large reserve conjugation capacity (120 nmol/min/g).     

The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5''-nucleotidase.

An alkaline 5'-nucleotidase with properties similar to those of membrane-bound 5'-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.     

Detoxification of DNA hydroperoxide by glutathione transferases and the purification and characterization of glutathione transferases of the rat liver nucleus.

DNA peroxidized by exposure to ionizing radiation in the presence of oxygen is a substrate for the Se-independent GSH peroxidase activity of several GSH transferases, GSH transferases 5-5, 3-3 and 4-4 being the most active in the rat liver soluble supernatant fraction (500, 35 and 20 nmol/min per mg of protein respectively) and GSH transferases mu and pi the most active, so far found, in the human liver soluble supernatant fraction (80 and 10 nmol/min per mg respectively). Although the GSH transferase content of the rat nucleus was found to be much lower than that of the soluble supernatant, nuclear GSH transferases are likely to be more important in the detoxification of DNA hydroperoxide produced in vivo. Two nuclear fractions were studied, one extracted with 0.075 M-saline/0.025 M-EDTA, pH 8.0, and the other extracted from the residue with 8.5 M-urea. The saline/EDTA fraction contained subunits 1, 2, 3, 4 and a novel subunit, similar but not identical to 5, provisionally referred to as 5*, in the proportions 40:25:5:5:25 respectively. The 8.5 M-urea-extracted fraction contained principally subunit 5* together with a small amount of subunit 6 in the proportion 95:5 respectively. GSH transferase 5*-5* purified from the 8.5 M-urea extract has the highest activity towards DNA hydroperoxide of any GSH transferase so far studied (1.5 mumol/min per mg). A Se-dependent GSH peroxidase fraction from rat liver was also active towards DNA hydroperoxide; however, since this enzyme accounts for only 14% of the GSH peroxidase activity detectable in the nucleus, GSH transferases may be the more important source of this activity. The possible role of GSH transferases, in particular GSH transferase 5*-5*, in DNA repair is discussed.     

Catechol-O-methyltransferase and aromatic L-amino acid decarboxylase activities in human gastrointestinal tissues

Human gastrointestinal samples from the corpus, antrum, bulbus, jejunum and ileum were assayed for soluble and membrane-bound catechol-O-methyltransferase (COMT) and aromatic L-amino acid decarboxylase (AADC) activity in vitro. The mean soluble COMT activities with 3,4-dihydroxybenzoic acid (DBA) and 3,4-dihydroxyphenylalanine (L-DOPA) as substrate were 70-242 and 70-174 pmol/min mg, respectively. The membrane-bound COMT activities ranged from 33 to 60 pmol/min mg in the different parts of the intestine. The AADC activities, measured with L-DOPA as the substrate, increased from 114 pmol/min mg in the corpus to 3488 pmol/min mg in the jejunum. The affinity of the soluble COMT was approximately 20 times higher for DBA (Km 15-19 microM) than for L-DOPA (Km 300-600 microM). The Km-values for L-DOPA of AADC and COMT were of the same order of magnitude. The specific COMT inhibitors, nitecapone and OR-611, effectively inhibited in vitro the human intestinal COMT activity. Nanomolar concentrations caused 50% inhibition with both DBA and L-DOPA as substrate.     

Topological arrangement in microsomal membranes of hepatic haem oxygenase induced by cobalt chloride.

Arysulphatase A was purified from rabbit testis. The purification was accomplished by a four-step procedure involving (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, SP(sulphopropyl)-Sephadex and affinity chromatography on concanavalin A-Sepharose. The specific activity of purified preparation was 135 mumol/min per mg of protein, which represented an increase of 900-fold above that of the crude homogenate. The purified enzyme (20-50 micrograms) was found to move electrophoretically as a single band on polyacrylamide gel at pH 7.2 and 8.4. The homogeneous enzyme was shown to be a glycoprotein with 0.8% (w/w) of N-acetylneuraminic acid and 20% neutral sugar. The treatment of purified enzyme with bacterial neuraminidase had no effect on enzyme activity or kinetic properties, but it changed the elution prolife of rabbit testis arylsulphatase A through DEAE-Sephadex. The purified enzyme was strongly inhibited by Cu2+, Fe3+ and Ag+. It hydrolysed several sulphate esters including cerebroside 3-sulphate, ascorbic acid 2-sulphate and steroid sulphates. Pure arysulphatase was effective in dispersing the cumulus cells of rabbit ova.     

Characterization and analysis of early enzymes for petrobactin biosynthesis in Bacillus anthracis

Recently, iron acquisition and, more specifically, enzymes involved in siderophore biosynthesis have become attractive targets for discovery of new antibiotics. Accordingly, targeted inhibition of the biosynthesis of petrobactin, a virulence-associated siderophore encoded by the asb locus in Bacillus anthracis, may hold promise as a potential therapy against anthrax. This study describes the biochemical characterization of AsbC, the first reported 3,4-dihydroxybenzoic acid-AMP ligase, and a key component in the biosynthesis of DHB-spermidine (DHB-SP), the first isolable intermediate in petrobactin biosynthesis. AsbC catalyzes adenylation to the corresponding AMP ester of the unusual precursor 3,4-dihydroxybenzoate, in addition to benzoate substrates bearing hydrogen bond-donating substituents at the para and meta positions on the phenyl ring. In a second reaction, AsbC catalyzes transfer of the activated starter unit to AsbD, an aryl carrier protein similar to acyl and peptidyl carrier proteins that function in fatty acid, polyketide, and nonribosomal peptide biosynthesis. A third protein, AsbE, is shown to be responsible for condensation of 3,4-dihydroxybenzoyl-AsbD with spermidine, providing the DHB-spermidine arms that are linked to citrate for assembly of petrobactin. On the basis of the selective substrate profile of AsbC, a nonhydrolyzable analogue of 3,4-DHB-AMP was synthesized and shown to effectively inhibit AsbC function in vitro.     

Haem transport to the liver by haemopexin. Receptor-mediated uptake with recycling of the protein

Rat [(59)Fe]haem-(125)I-labelled haemopexin complexes (700pmol/rat) associate rapidly and exclusively with the liver after intravenous injection into anaesthetized rats. The two isotopes exhibit different patterns of accumulation. Liver (125)I-labelled haemopexin is maximum 10min after injection (20+/-4.9pmol/g of liver) and then declines by 2h to the low values (about 3pmol/g of liver) seen after injection of the apoprotein. In contrast, [(59)Fe]haem accumulates in the liver for at least 2h. Haemopexin undergoes no extensive proteolysis during 2h of haem transport as shown by precipitation with acid (98%) and specific antiserum (92%) and by electrophoresis. Moreover, only 1-2% of the dose is located in extrahepatic tissues, and there is no significant urinary excretion of either (125)I or (59)Fe. Hepatic uptake at 10min is saturable, reaching 200pmol of haemopexin/g of liver and 350pmol of haem/g of liver at a dose of 9nmol/rat, whereas uptake of the apoprotein is 3-5% of the dose. This suggests that the interaction of haem-haemopexin with the liver is a specific receptor-mediated process. The complex probably interacts via the protein moiety, since the haem analogues mesohaem and deuterohaem do not affect association of the protein with the liver but the species of haemopexin does. Increasing amounts of protein are associated with the liver 5min after injection in the order: human>rabbit>rat, and haem uptake is consistently increased. For both rat and rabbit haemopexin saturation is reached at the same concentration of protein, i.e. 180-200pmol/g of liver, indicating that the different protein species bind to a common receptor. We propose that haemopexin transports haem to the liver by a specific receptor-mediated process and then returns to the circulation.