Effect of obstructive jaundice on the fate of a nephrophilic organic anion in the rat

Renal transtubular transport of phenolsulfophthalein (PSP), a nephrophilic organic anion that circulates bound to albumin, was studied in normal and bile-duct-ligated rats. Intravenously injected PSP disappeared from the circulation more rapidly in bile-duct-ligated jaundiced rats than in intact animals. However, urinary excretion of PSP was significantly lower in the former than in the latter. Kinetic analysis revealed that binding of PSP to plasma protein(s) was significantly lower with jaundiced rats than with intact animals. Addition of albumin to plasma samples from bile-duct-ligated rats markedly increased PSP binding. The decreased PSP binding returned to normal levels after treating the jaundiced plasma with bilirubin oxidase, an enzyme that degrades amphiphilic bilirubin to water soluble metabolites. These results suggest that bilirubin might be the major metabolite that occupied the PSP binding site(s) on albumin in jaundiced rats. When PSP was injected bound to equimolar amount of albumin, the rate of PSP disappearance from the circulation decreased and urinary excretion of the ligand increased markedly; urinary excretion of PSP was significantly larger in bile-duct-ligated rats than in intact animals. These results suggest that the renal transport capacity for amphiphilic organic anions, such as PSP, might be increased compensatively in bile-duct-ligated animals, and that the apparent decrease in renal secretory transport for PSP might result from, at least in part, random distribution of the ligand to extrarenal tissues due to decrease in the binding activity of albumin.     

Differences in the protein binding of several drugs and bilirubin in serum and heparinized plasma of rats.

The purpose of this investigation was to compare the protein binding of salicylic acid, phenytoin, warfarin and bilirubin in serum and heparinized plasma of rats. Protein binding was determined by equilibrium dialysis (drugs) or by a reaction rate method (bilirubin), using serum and plasma obtained from the same animals. The three drugs were significantly less protein bound in heparinized plasma than in serum; this difference was particularly pronounced in the case of warfarin. Addition of heparin to serum also resulted in a decrease in the protein binding of the drugs but to a lesser extent than in plasma. The protein binding of bilirubin was more extensive in plasma than in serum, irrespective of the anticoagulant used (heparin, sodium citrate, or disodium ethylenediamine-tetraacetate). It may be desirable to perform all binding studies with serum rather than plasma.     

Enantioselective binding of mephobarbital to plasma proteins

The enantioselective protein binding of mephobarbital (MPB) was investigated in human plasma and human serum albumin solutions by equilibrium dialysis. A small but statistically significant difference was observed in the in vitro plasma protein binding of the enantiomers; (S)-MPB was approximately 59% bound and (R)-MPB approximately 67% bound. The binding to albumin [(S)-MPB: approximately 29% bound, and (R)-MPB: approximately 41% bound] was less than to plasma proteins but showed somewhat greater enantioselectivity, suggesting that albumin binding is a major source of the enantioselectivity in plasma. The effects of MPB concentration, of varying enantiomeric concentration ratio, and of phenobarbital on the enantioselective binding of MPB were studied. The effect of age was also investigated by measuring the binding in plasma from 8 young (18-25 yr) and 8 elderly (greater than 60 yr) male subjects who took single doses of MPB. The results were in close agreement with the in vitro binding data, and the binding of both enantiomers was marginally but significantly lower in the young compared with the elderly subjects. These differences in binding were consistent with previously observed pharmacokinetic differences between the two subject groups.     

Binding of norgestrel to human plasma proteins.

Binding of [14, 15-3H](+/-)-norgestrel to human plasma proteins has been investigated. Norgestrel showed greater affinity to plasma than to human serum albumin indicating specific norgestrel binding protein(s) in the plasma. alpha1-acid glycoprotein showed high affinity for norgestrel when compared with human serum albumin. The binding protein was eluted at pH 5.8 by step by step elution on a DEAE-cellulose column. Norgestrel binding to plasma proteins was not affected at 60 degrees C. The optimal binding occurred between pH 7 and 8. Ligand specificity of the binding protein revealed that progesterone was able to compete for the norgestrel binding sites, whereas corticosterone, testosterone, oestradiol, and norethindrone acetate did not show much competition. The molecular weight of the binding protein was found to be approximately 43 000. Sucrose density gradient analysis indicated that norgestrel bound to a macromolecular component of sedimentation coefficient 2.9 S. The association constant (Kass) and dissociation constant (Kdiss) of norgestrel-binding plasma protein was found to be 1.4-10(6) M-1 and 0.7-10(-6) M respectively. The number of binding sites was 0.5-10(-9) mol/mg protein. Norgestrel-binding protein in the plasma appeared to be a protein different from human serum albumin, corticosteroid-binding globulin and sex-steroid-binding protein. This binding protein showed some similarities to alpha1-acid glycoprotein.     

The plasma binding protein for vitamin D is a site of discrimination against vitamin D-2 compounds by the chick

The binding of 25-hydroxy-[26,27-3H]vitamin D-3 and 25-hydroxy-[26,27-3H]vitamin D-2 to the vitamin D binding protein in the plasma of both rats and chicks has been studied. In the case of rats, sucrose density gradient analysis, competitive displacement, and Scatchard analysis demonstrate that 25-hydroxyvitamin D-3 and 25-hydroxyvitamin D-2 are bound equally well to the vitamin D binding protein. In contrast, 25-hydroxyvitamin D-2 is poorly bound, while 25-hydroxyvitamin D-3 is tightly bound to the vitamin D binding protein in chick plasma. On the other hand, the chick intestinal receptor binds 1,25-dihydroxyvitamin D-2 and 1,25-dihydroxyvitamin D-3 equally well with a KD of 7.10(-11) M for both compounds. These results strongly suggest that the failure of the plasma transport protein in chicks to bind the vitamin D-2 compounds may be responsible for their relative ineffectiveness in these animals.     

Role of plasma albumin in renal elimination of a mercapturic acid. Analyses in normal and mutant analbuminemic rats

Biosynthesis of N-acetylcysteine S-conjugates of xenobiotics (mercapturic acids) occurs via interorgan metabolism and the renal transtubular transport system plays an important role in elimination of the final metabolites from the organism. To assess the behavior of a mercapturic acid in the circulation, plasma clearance of radioactive S-benzyl-N-acetylcysteine and its interaction with plasma proteins were studied in normal and mutant analbuminemic rats (NAR). Intravenously injected S-benzyl-N-acetylcysteine rapidly disappeared from the circulation both in NAR and normal animals. However, its plasma clearance was significantly higher in NAR (45.7 ml kg-1 min-1) than in normal rats (25.2 ml kg-1 min-1). Ultrafiltration analysis revealed that 18.4% and 80.1% of the mercapturate bound to plasma protein(s) from NAR and normal rats, respectively, at 50 microM ligand concentration. The mercapturic acid bound to plasma albumin with an association constant of 2.24 X 10(5) M-1 and the number of binding sites was 1.18/mol albumin. The binding was competitively inhibited by probenecid and L-tryptophan. Concomitant administration of this mercapturic acid with equimolar amounts of albumin resulted in a marked decrease in the plasma clearance (26.2 ml kg-1 min-1) and an increase in the urinary secretion of this ligand in NAR. 30 min after injection of the mercapturic acid (10 mumol/kg body weight), 27.3% and 60.4% of the injected dose was recovered from urine and kidneys of NAR and normal rats respectively. About 41% of the dose was recovered in NAR urine when the ligand was injected bound to an equimolar amount of albumin. These results suggested that albumin is important for the renal accumulation and urinary elimination of the circulating mercapturic acid.     

Transhepatic transport of taurocholic acid in normal and mutant analbuminemic rats

To elucidate a possible function of plasma albumin in vectorial transport of various cholephilic organic anions, such as bile acids, plasma clearance and transhepatic transport of radioactive taurocholate were studied in vivo in normal and mutant analbuminemic rats. Intravenous administration of taurocholate was followed by its rapid disappearance from the circulation in both animal groups. However, plasma clearance of taurocholate was significantly larger in analbuminemic (68.3 ml/min per kg of body weight) than in normal rats (29.8 ml/min per kg of body weight) at a dose of 8 mumol/kg of body weight. The increased plasma clearance in analbuminemic rats was accompanied by a more prompt biliary secretion of the ligand than occurred in normal animals; 79 and 42% of the injected dose was recovered in analbuminemic and normal rat bile, respectively, within 10 min after administration. Ultrafiltration analysis revealed that the binding of taurocholate to serum protein(s) was significantly lower in analbuminemic rats as compared with that in normal rat serum; 24 and 76% of taurocholate bound to protein fractions of analbuminemic and normal rat serum, respectively, at 0.5-mM ligand concentration. Binding of taurocholate to cytosolic proteins of normal and analbuminemic liver were similar; 23 and 28% of taurocholate bound to protein fractions from analbuminemic and normal rat, respectively, at 10 mg protein/ml and 20-microM ligand concentration. These results indicate that plasma albumin does not play a role in directing circulating taurocholate to the liver and that transhepatic transport of the bile acid increases with the increase in concentration of unbound ligand in the circulation.     

Serum protein binding characteristics of cyproterone acetate, gestodene, levonorgestrel and norethisterone in rat, rabbit, dog, monkey and man

Protein binding characteristics including percentage of total binding, total binding capacity (pmol/mg protein), degree of specific binding, competition with dihydrotestosterone (DHT) and estradiol (E2) binding sites and dissociation constants (Kd) of low and high affinity binding sites were investigated for the progestins cyproterone acetate (CPA), gestodene (G), norethisterone (NET) and levonorgestrel (LN) in serum or plasma pools from man and four laboratory animal species (rat, rabbit, dog and monkey). Serum pools from animals were constructed from samples obtained either prior to or 1 day after pretreatment with ethinyl estradiol (EE2) (5 micrograms/kg/day for 7 days). Human plasma pools differed by SHBG levels (normal/induced). All serum pools were characterized by protein content and distribution. Equilibrium dialysis or dextran-coated charcoal (DCC) methods were used to separate bound and free steroids labelled with tritium. All progestins were highly (greater than 80%) bound to proteins in all undiluted samples. Total binding capacity was highest in rat and lowest in monkey. Human plasma showed a capacity of 1.5-2.1 microgram steroid/ml. In man, monkey and rabbit LN and G were specifically bound to the same degree as DHT, whereas NET binding was 50% lower. Specific binding of CPA to dog serum was 2-3 times higher than for other steroids. Two (high and low affinity) binding sites were found for LN, G and NET in man, monkey and rabbit and in dog for LN. Kd values for high affinity binding ranged from 3.5 (G in man) to 23 (NET in man) x 10(-9)M. Kd values of low affinity binding varied from 0.5 (CPA in dog) to 4 (NET in man) x 10(-6)M. E2 and DHT competition experiments confirmed the concept of SHBG as a carrier protein of 19-nor-progestins and DHT and its occurrence in man, monkey and rabbit. A sex hormone binding protein (SBP) in the dog seems to be responsible for the relatively high specific binding of CPA. SHBG is inducible by means of EE2 in man and monkey, but not in rabbit. EE2 may induce SBP in the dog. Comparison of in vitro Kds (high affinity binding) and in vivo metabolic clearance rates showed the same rankings for LN, G and NET in man, monkey and rabbit.     

Removal of apoprotein-B-containing lipoproteins by plasmapheresis using immobilized phosphorylcholine-binding protein affinity adsorbent

Rat serum phosphorylcholine binding protein was earlier shown to bind lipoproteins containing apoproteins B and E from human very low and low density lipoproteins. The present studies were undertaken to show the effectiveness of rat serum phosphorylcholine-binding protein immobilized on Sepharose affinity column to remove apoprotein-B-containing lipoproteins from normal and hypercholesterolemic rabbit plasma, when used in a plasmapheresis system. The maximum in vitro binding of very low and low density lipoproteins from hypercholesterolemic rabbit plasma to the affinity adsorbent was Ca2+ dependent, and the cholesterol bound to the column at the optimum calcium concentration (2.5 mM) was 21% of the total plasma cholesterol applied. The in vivo binding of total cholesterol from normal and hypercholesterolemic rabbit plasma during plasmapheresis ranged from 0.22 to 7.7%. Total mass of cholesterol bound ranged from 3.86 and 27.52 mg at plasma cholesterol concentrations 13.8 and 282 mg/dL, respectively. Most (greater than 95%) of the bound cholesterol was associated with very low and low density lipoproteins. These studies show the ability of immobilized rat serum phosphorylcholine-binding protein to lower the atherogenic apoprotein-B-containing lipoproteins from plasma of hypercholesterolemic rabbits.     

Growth hormone-dependent insulin-like growth factor (IGF) binding protein from human plasma differs from other human IGF binding proteins

A growth hormone-dependent binding protein for insulin-like growth factors (IGF-I and IGF-II) has been isolated from human plasma. Analyzed on SDS gels, the preparation contained a major protein band of 53 kDa, and a minor band of 47 kDa. After transfer to nitrocellulose, both species bound iodinated IGF-I, and could be detected using an antibody raised against the purified preparation. In contrast, an IGF binding protein purified from human amniotic fluid bound IGF-I but was not detectable immunologically. The amino acid comparison of the plasma binding protein preparation was different from that reported for amniotic fluid and HEP G2 hepatoma proteins, and the unique amino-terminal sequence, Gly-Ala-Ser-Ser-Ala-Gly-Leu-Gly-Pro-Val-, was different from that of the amniotic fluid and hepatoma proteins. This study indicates that the growth hormone-dependent IGF binding protein of human plasma is structurally and immunologically distinct from other IGF binding proteins.     

Cadmium-binding to transferrin in the plasma of the common carp Cyprinus carpio

In this study, it was investigated by autoradiography with radioactive cadmium after Western blotting of two-dimensional electrophoresis gels, to which proteins cadmium is mainly bound in plasma of common carp Cyprinus carpio. The obtained results demonstrate that in carp plasma, cadmium is primarily bound to two high molecular weight proteins. Relative small amounts are bound to a protein with M(r) approximately 60000. The other metal-binding protein, with M(r) approximately 70000 and pI approximately 6.7 was identified as transferrin. The conditional equilibrium constants for the binding of cadmium ions to the two metal-binding sites of this protein were calculated as logK(1)=5.40+/-0.12 and logK(2)=4.66+/-0.21, which are comparable to those of human transferrin under the same experimental conditions. Transport of cadmium in plasma of carp was found to be different from that of brown trout Salmo trutta and man, where cadmium is mainly bound to albumin and transferrin. The prominent binding of cadmium to transferrin can be explained by the absence or at least the very low concentrations in which albumin is present in carp plasma.     

Protein binding and stability of norepinephrine in human blood plasma. Involvement of prealbumin, alpha 1-acid glycoprotein and albumin

The binding of norepinephrine (NE) to plasma proteins of fresh human blood obtained from healthy volunteers was studied by ultrafiltration at different NE concentrations and incubation times at 37 degrees C. At 1.7 nM L-[3H]-NE binding was approximately 25%. The binding was rapid and was not influenced by the incubation time. [3H]-NE could be dissociated from its binding sites by acid precipitation and, after HPLC, showed to be unchanged NE. No difference in NE binding was found between plasma collected in EGTA-GSH or heparin solution. There was no degradation of NE when incubated in plasma at 37 degrees C for 10 h, even without the addition of antioxidants. Therefore, in the present study, binding represented interaction of unchanged NE with plasma proteins. The whole plasma binding was saturable over the range of 0.66 nM to 0.59 mM of NE. Scatchard plot of specific binding revealed high-affinity sites with a Kd of 5.4 nM and a Bmax of 3.9 fmoles.mg-1 protein, and low-affinity sites with a Kd of 2.7 microM and a Bmax of 3.3 pmoles.mg-1 protein. Electrophoretic characterization of NE-binding proteins showed that about 60% of bound NE was associated to albumin, and 20% to prealbumin. NE binding to pure human plasma proteins was also studied using ultrafiltration. Scatchard analyses revealed a single class of very high-affinity binding sites for prealbumin (Kd 4.9 nM), a single class of binding sites for alpha 1-acid glycoprotein (Kd 54 microM) and two classes of binding sites for albumin with high (Kd 1.7 microM) and low (Kd 0.8 mM) affinities respectively. The main results obtained in this study - a) reversibility of NE binding, b) stability of free and bound NE in plasma, c) involvement of the prealbumin as a specific binding protein - point out to a specific transport for NE in human blood plasma.     

The binding of silica to proteins from plasma and lungs of rat: in vitro

Silica dissolving out from the slate dust was found to bind with plasma protein and purified bovine serum albumin. At 24 h of incubation at 37 degrees C binding affinity of silica (microgram of silica bound/mg of protein) with plasma protein and bovine serum albumin was found to be 0.59 and 0.44, respectively. By molecular exclusion chromatography using Sephadex G-200, silica binding protein of plasma was determined to be of mol. wt. around 67000. Similar proteins having silica binding capacity (mol. wt. 70000 and 85000) were also found in rat lung but these proteins unlike their plasma counterpart were glycoprotein in nature. Polyacrylamide gel electrophoresis of plasma and protein rich lung fraction show that proteins upon binding with silica undergo mobility changes. Significance of the existence of silica binding protein in plasma and lung of rat in relation to silica toxicity is discussed.     

Cholecystokinin induces the interaction of its receptor with a guanine nucleotide binding protein

To evaluate the relation between the pancreatic cholecystokinin (CCK) receptor and guanine nucleotide-binding protein(s) we studied the effects of nucleotides on 125I-CCK binding to pancreatic acinar plasma membranes, 125I-CCK binding to solubilized 125I-CCK receptors, and the stability of the solubilized 125I-CCK-receptor complex. In plasma membranes, guanine nucleotides both inhibited CCK binding and increased the dissociation of CCK from its receptor. The potency of the nucleotides studied was GTP gamma S = GMP-PNP greater than GTP much greater than ATP. When membranes were solubilized with digitonin, subsequent binding of CCK was insensitive to guanine nucleotides including GTP, GMP-PNP and GTP gamma S. However, if CCK binding occurred before solubilization of the membranes, guanine nucleotides increased dissociation at concentrations and with a specificity similar to that observed for effects on intact pancreatic membranes. It is concluded that guanine nucleotides act via a protein which is separable from the receptor to induce dissociation of bound CCK. Moreover, CCK binding induces an association in the plasma membrane of the CCK receptor with this guanine nucleotide binding protein.     

Oestriol binding to plasma proteins

Simple diffusion experiments indicated that oestriol was retained by human pregnancy plasma more effectively than by albumin solutions of a corresponding concentration. Oestriol bound (Ka = 6 X 10(6) l/mol at 4 degrees C) to a glycoprotein which had been isolated from plasma by adsorption to Concanavalin A. The free energy of binding at 37 degrees C was -38 kJ/mol. Competition experiments indicated that the oestriol binding glycoprotein had properties expected of sex hormone binding globulin. The distribution of oestriol among the protein fractions of human pregnancy plasma--glycoprotein bound 7.8%, albumin bound 78.6%, unbound 13.6%--suggests that this glycoprotein plays little part in the transport of oestriol.     

Variations in riboflavin binding by human plasma: identification of immunoglobulins as the major proteins responsible

Riboflavin binding by plasma proteins from healthy human subjects was examined by equilibrium dialysis using a physiological concentration of [2-14C]riboflavin (0.04 microM). Binding ranged from 0.080 to 0.917 pmole of riboflavin/mg of protein (with a mean +/- SD of 0.274 +/- 0.206), which corresponded to 4.14 to 49.4 pmole/ml of plasma (15.5 +/- 11.0) (N = 34). Males and females yielded similar results. Upon fractionation of plasma by gel filtration, the major riboflavin-binding components eluted with albumin and gamma-globulins. Albumin was purified and found to bind riboflavin only very weakly (Kd = 3.8 to 10.4 mM), although FMN and photochemical degradation products (e.g., lumiflavine and lumichrome) were more tightly bound. Binding in the gamma-globulin fraction was attributed to IgG and IGA because the binding protein(s) and immunoglobulins copurified using various methods were removed by treatment of plasma with protein A-agarose, and were coincident upon immunoelectrophoresis followed by autoradiography to detect [2-14C]riboflavin. Differences among the plasma samples correlated with the binding recovered with the immunoglobulins. Binding was not directly related to the total IgG or IgA levels of subjects. Hence, it appears that the binding is due to a subfraction of these proteins. These findings suggest that riboflavin-binding immunoglobulins are a major cause of variations in riboflavin binding in human circulation, and may therefore affect the utilization of this micronutrient.     

Characterization of glucagon receptors in Golgi fractions of rat liver: evidence for receptors that are uncoupled from adenylyl cyclase

Glucagon receptors have been identified and characterized in intermediate (Gi) and heavy (Gh) Golgi fractions from rat liver. At saturation, plasma membranes bound 3500 fmol of hormone/mg of membrane protein, while Gi and Gh bound 24 and 60 fmol of 125I-glucagon/mg of protein, respectively. Half-maximal saturation of binding to plasma membranes, Gi, and Gh occurred at approximately 4, 10, and 20 nM 125I-glucagon, respectively. Trichloroacetic acid precipitation of intact, but not degraded, glucagon was used to correct binding isotherms for hormone degradation. After such correction, half-maximal saturation of binding to plasma membranes, Gi, and Gh was observed in the presence of approximately 2, 7, and 14 nM hormone, respectively. After 90 min of dissociation in the absence of guanosine 5'-triphosphate (GTP), 86% of 125I-glucagon remained bound to plasma membranes, whereas only 42% remained bound to Golgi membranes. GTP significantly increased the fraction of 125I-glucagon released from plasma membranes but only slightly augmented the dissociation of hormone from Golgi fractions. 125I-Glucagon/receptor complexes solubilized from plasma membranes fractionated by gel filtration as high molecular weight (Kav = 0.16), GTP-sensitive complexes and lower molecular weight (Kav = 0.46), GTP-insensitive complexes. 125I-Glucagon complexes solubilized from Golgi membranes fractionated almost exclusively as the lower molecular weight species. The lower affinity of Golgi than plasma membrane receptors for hormone, the ability of glucagon to stimulate plasma membrane, but not Golgi membrane, adenylyl cyclase, and the near absence of high molecular weight, GTP-sensitive complexes in solubilized Golgi membranes demonstrate that plasma membrane contamination of Golgi fractions cannot account for the 125I-glucagon binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroid-protein interactions in bovine plasma.

The binding of testosterone, corticosterone and 17alpha-hydroxyprogesterone by bull plasma protein was studied by equilibrium dialysis. Testosterone in bovine plasma was bound by a CBG-like protein and by a specific protein (testosterone-binding protein or TBP) of limited capacity and high affinity. The TBP was specific for C18 and C19 steroids with a 17 beta-hydroxy group. Precision of the steroid-protein binding measurements was tested and was satisfactory. The testosterone-binding capacity in bull plasma samples did not seem to be related to testosterone levels in peripheral plasma. Significant differences between bulls and cows with regard to the binding capacity were observed.     

Characterization of low density lipoprotein binding to human adipocytes and adipocyte membranes

125I-labeled low density lipoprotein (LDL) binding to purified plasma membranes prepared from freshly isolated human adipocytes was saturable, specific, and displaceable by unlabeled ligand. The maximum specific binding capacity measured at saturating concentrations of 125I-LDL was 1.95 +/- 1.17 micrograms of LDL bound/mg of membrane protein (mean +/- S.D., n = 16). In contrast to cultured fibroblasts, specific binding of LDL to adipocyte membranes was calcium-independent, was not affected by EDTA or NaCl, and was not destroyed by pronase. Plasma membranes purified directly from homogenized adipose tissue also showed calcium-independent LDL specific binding (0.58 +/- 0.33 micrograms of LDL bound/mg of membrane protein, mean +/- S.D. n = 11). Specific binding, internalization, and degradation of 125I-methylated LDL was demonstrated in isolated adipocytes and competition experiments showed that native and methylated LDL interacted with adipocytes through some common recognition mechanism(s). Compared to native LDL, specific binding of methylated LDL to adipocyte membranes was significantly reduced (43%), indicating that interaction of LDL with adipocyte was dependent in part on the lysine residues of apolipoprotein B. LDL binding to adipocyte plasma membranes was also competitively inhibited by human high density lipoprotein subfractions HDL2 and HDL3. Thus, LDL metabolism in mature adipocytes appears to be regulated by mechanisms distinctly different from a variety of cultured mesenchymal cells. In addition, the ability of adipocytes to bind, internalize, and degrade significant amounts of methylated LDL supports the view that adipose tissue is involved in the metabolism of modified lipoproteins in vivo.     

Ontogeny of insulin-receptor interaction: Correlation with circulatory insulin levels

Interaction of [¹²⁵I]-insulin with intact hepatocytes and its correlation with circulatory insulin level was examined. The hepatocytes from new-born rats bound lowest amount of [¹²⁵I]-insulin (1.39±0.41 pM/mg cell protein) when circulatory insulin level was high (8±1.5 ΜU/ml). Hepatocytes from 7 day and 21 day old animals demonstrated a more or less similar relationship, Cells from 31 day old animals exhibited maximum insulin binding, activity (5.13±0.18.pM/mg cell protein) against a low serum insulin level (4.25±0.25 ΜU/ ml). Scatchard analysis of insulin binding shows that the affinity is higher in the hepatocytes from new-born animals than in the hepatocytes of 31 day old animals. Higher binding observed in the latter case may be due to a greater number of binding sites. Hepatocytes from one year old rats bound very little insulin (2.50±0.36 pM/mg cell protein) against a high circulatory insulin level (9.25±0.85 ΜU/ml). In view of these results, it appears that the down-regulation hypothesis holds true during ontogeny too.