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.     

Biochemical characterization of cholesteryl ester transfer protein inhibitors

Cholesteryl ester transfer protein (CETP) has been identified as a novel target for increasing HDL cholesterol levels. In this report, we describe the biochemical characterization of anacetrapib, a potent inhibitor of CETP. To better understand the mechanism by which anacetrapib inhibits CETP activity, its biochemical properties were compared with CETP inhibitors from distinct structural classes, including torcetrapib and dalcetrapib. Anacetrapib and torcetrapib inhibited CETP-mediated cholesteryl ester and triglyceride transfer with similar potencies, whereas dalcetrapib was a significantly less potent inhibitor. Inhibition of CETP by both anacetrapib and torcetrapib was not time dependent, whereas the potency of dalcetrapib significantly increased with extended preincubation. Anacetrapib, torcetrapib, and dalcetrapib compete with one another for binding CETP; however anacetrapib binds reversibly and dalcetrapib covalently to CETP. In addition, dalcetrapib was found to covalently label both human and mouse plasma proteins. Each CETP inhibitor induced tight binding of CETP to HDL, indicating that these inhibitors promote the formation of a complex between CETP and HDL, resulting in inhibition of CETP activity.     

Limited pepsin digestion of bovine plasma albumin

Limited pepsin digestion of bovine plasma albumin at pH 3.7 and 25 °C in the presence of octanoic acid gave two fragments, A and B, each in about 16% yield. In the absence of octanoic acid fragment A was rapidly degraded further into smaller fragments. Sodium dodecylsulfate gel electrophoreses and amino acid analyses of fragments A and B indicated their molecular weights to be about 29,000 and 34,000, respectively. Comparative studies of the cyanogen bromide peptides of fragments A and B with those of intact albumin established that fragments A and B represent, respectively, the carboxyl and the amino terminal portions of the albumin molecule.In Tris-HCl buffer (pH 7.95) at 25 °C, fragment A has one primary binding site for octanoic acid with a binding constant about one-eighth of that of albumin. This binding constant is doubled in the presence of an equimolar amount of fragment B, although fragment B itself shows very weak activity, less than one three-hundreth of that of albumin. l- and d-tryptophans competitively bind at the same primary octanoate binding site of fragment A, just as is the case with albumin.These findings together with those of other studies suggest that the albumin molecule might consist of several compact regions and that the interactions of these regions within the molecule vary with the solvent environment and upon binding of organic ligands.     

Low plasma albumin levels are associated with increased plasma protein glycation and HbA1c in diabetes

Albumin is one of the most abundant plasma proteins and is heavily glycated in diabetes. In this study, we have addressed whether variation in the albumin levels influence glycation of plasma proteins and HbA1c. The study was performed in three systems: (1) streptozotocin (STZ)-induced diabetic mice plasma, (2) diabetic clinical plasma, and (3) in vitro glycated plasma. Diabetic mice and clinical plasma samples were categorized as diabetic high albumin plasma (DHAP) and diabetic low albumin plasma (DLAP) on the basis of their albumin levels. For the in vitro experiment, two albumin levels, high albumin plasma (HAP) and low albumin plasma (LAP), were created by differential depletion of plasma albumin. Protein glycation was studied by using a combination of two-dimensional electrophoresis (2DE), Western blotting, and LC-MS(E). In both mice and clinical experiments, an increased plasma protein glycation was observed in DLAP than in DHAP. Additionally, plasma albumin levels were negatively correlated with HbA1c. The in vitro experiment with differential depletion of albumin mechanistically showed that the low albumin levels are associated with increased plasma protein glycation and that albumin competes for glycation with other plasma proteins.     

Stereoselective binding of zopiclone to human plasma proteins

The binding of racemic zopiclone (ZOP) and of its two enantiomers to plasma proteins, albumin and α1‐acid glycoprotein were compared. Our work shows that the binding of ZOP to human plasma proteins is stereoselective. The total plasma protein binding percentages were 79.3 ± 5.5%, 83.8 ± 5.2%, and 75.1 ± 2.1%, for racemic zopiclone, (−)zopiclone and (+)zopiclone, respectively. These results were confirmed by the analysis of samples obtained from healthy volunteers after the oral administration of ZOP. The anticoagulant used for sampling was also shown to have an influence on the percentage binding and on its stereoselectivity. Considering albumin and α1‐acid glycoprotein separately, stereoselectivity was also observed. Chirality 11:129–132, 1999. © 1999 Wiley‐Liss, Inc.     

Behavior of [185W]thiotungstates injected into sheep and the influence of copper: their fate and the effect of the compounds upon plasma copper

[185W]trithio- and tetrathiotungstates (0.5 mg W) were injected intravenously into sheep. The compounds circulated in plasma bound reversibly to plasma proteins, particularly to albumin. After the first few minutes, levels declined exponentially with a T 1/2 of 12-14 hr. The initial movement of [185W]trithiotungstate from the plasma compartment was delayed transiently by the immediate injection of copper (2-6 mg); the longer-term metabolism was unaffected. The final fate of the compounds appeared to be hydrolysis and excretion in urine as [185W]tungstate. 185W from [185W]trithiotungstate appeared more rapidly than from [185W]tetrathiotungstate, but in both the rate was unaffected by copper injections. Since the appearance in urine did not correspond to the disappearance from plasma, it was suggested that the hydrolysis occurred in extravascular tissues and that the liver might be the site. A control experiment showed that [185W]tungstate in plasma was very rapidly cleared (and appeared in urine). At higher W levels (25-50 mg W per sheep per day), systematic copper metabolism was perturbed since plasma copper levels rose. The experiments demonstrated that in sheep the behavior and the effects of thiotungstates and thiomolybdates are sufficiently similar for 185W to be used as a more convenient alternative to 99Mo for longer-term studies on the interaction of the compounds with copper metabolism in animals.     

Interaction of prostaglandins with blood plasma proteins. I. Binding of prostaglandin E 2 to human plasma proteins and its effect on the physiological activity of prostaglandin E 2 in vitro and in vivo

The binding of (PGE2) prostaglandin E2 to human plasma proteins was investigated by DEAE-Sephadex column chromatography and acrylamide gel electrophoresis, and quantitatively assessed by equilibrium dialysis. PGE2 added to human plasma in vitro was found to become mainly bound to plasma albumin. This binding was also demonstrated by adding PGE2 to human serum albumin solutions. The binding of PGE2 to human serum albumin inhibits the contraction-producing effect of PGE2 on the isolated gerbil colon in vitro. The depressor effect of PGE2 on the rat blood pressure was used to assess the in vivo effect of PGE2 albumin interaction. The blood pressure lowering activities of free and albumin-bound PGE2 were found to be the same when administered either intravenously or intraarterially. The significance of these observations with regard to estimation of PG concentration in whole blood or plasma, and their possible effects on PG metabolism is discussed.     

Binding of [3H]ctyochalasin B and [3H]colchicine to isolated liver plasma membranes.

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([3H]cytochalasin B and [3H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [3H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB3H4. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insensitive to changes of pH or ionic strength. At 10(-6) M [3H]cytochalasin B, glucose of p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 A) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10(-5) M [3H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [3H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes. [3H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [3H]cytochalasin B binding and cytochalasin B stimulated 3H colchicine binding. [3H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Binding of bismuth to serum proteins: implication for targets of Bi(III) in blood plasma

Bismuth complexes have been widely used in clinical treatment as antiulcer drugs. However, different adverse effects have been observed and the diagnosis is generally confirmed by the detection of bismuth in blood or blood plasma. In this study, binding of bismuth to human serum albumin was studied by fluorescence spectroscopy with the binding constant logK(a) to be 11.2. Competitive binding of bismuth to human albumin and transferrin was carried out at pH 7.4 by FPLC and ICP-MS. It was found that over 70% of bismuth binds to transferrin even in the presence of a large excess of albumin (albumin/transferrin=13:1) at pH 7.4, 10 mM bicarbonate. The distribution of bismuth between the two proteins was almost unchanged when Cys(34) of albumin was blocked. However, all bismuth binds to albumin when iron-saturated transferrin was used. Almost all of the bismuth was distributed over the fractions containing transferrin (70%) and albumin (<30%) in serum. The percentage of bismuth associated with transferrin was further increased by 15% with elevated transferrin in serum. Binding of bismuth to transferrin is much stronger than human albumin. Transferrin is probably the major target of bismuth in blood plasma, and it may play a role in the pharmacology of bismuth.     

Changes of plasma dopamine-beta-hydroxylase activity and other plasma constituents during the cold pressor test

The effect of the cold pressor test on plasma DBH activity in ten healthy human subjects was investigated. Parallel changes of other plasma constituents were ascertained as well. Plasma DBH activity rose by over ten per cent in six of the sen subjects and declined by 14 per cent or more in two subjects; the correlations of altertions in DBH activity with changes of high molecular weight plasma constituents were high (r=0.565 to 0.902); correlations with blood urea nitrogen and plasma glucose were low (r=0.002 to 0.248). The results suggest that factors other than neuronal DBH release may be important in alterations of plasma DBH activity following $\text{acute}$ stresses produced by the cold pressor test in man.     

Apo A-I and apo A-II inhibit hepatic triglyceride lipase from human postheparin plasma

Like α-amanitin, tRNA binds reversibly to yeast RNA polymerases A or B, in a 1:1 stoechiometry and inhibits enzyme B preferentially. Kinetic studies showed that the binding sites for the two inhibitors are completely independent. The dissociation constant of the enzyme-inhibitor complexes was determined. Only in the case of tRNA, it varied with the nature and concentration of the template. The stimulation factor P₃₇ does not interfere with the binding of both inhibitors.     

Multiple fatty acid binding to albumin in human blood plasma

Binding equilibria of long-chain fatty acids to human serum albumin, in serum or plasma, were studied by a dialysis exchange rate technique. Palmitate was added to citrated plasma in vitro and it was observed that between six and ten palmitate molecules were bound to albumin with nearly equal affinity. Observations in vivo gave similar results in the following series: (a) in two volunteers with increased fatty acid concentrations after fasting, exercise, and a cold shower: (b) in three male volunteers in whom high concentrations of non-esterified fatty acids, up to 4.6 mM, were induced by intravenous administration of a preparation of lecithin/glycocholate mixed micelles, and (c) in 81 patients with diabetes mellitus, type I. The binding pattern of palmitate in serum or plasma is essentially different from that observed with palmitate added to buffered solutions of pure albumin when two molecules are tightly bound and about four additional molecules with lower affinity. The differences may partly be explained by the presence of chloride ions in blood plasma, reducing the affinity for binding of the first two fatty acid molecules, and partly by facilitated binding of several molecules of mixed fatty acids, as found in plasma.     

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.     

Effects of forskolin analogs, phosphodiesterase inhibitors and 8-bromo cyclic AMP on plasma exudations induced with bradykinin and prostaglandin E1 in rat skin

The effects of forskolin analogs, phosphodiesterase inhibitors and 8-bromo cyclic AMP on plasma exudations induced with bradykinin and prostaglandin E1 in rat skin were investigated using [125I]bovine serum albumin (125I-BSA). Forskolin, forskolin 7-ethyl carbonate and 7-desacetylforskolin, which are potent activators of adenylate cyclase, greatly potentiated the bradykinin-induced plasma exudation and inhibited the prostaglandin E1-induced response. On the other hand, 14,15-dihydroforskolin and 1,9-dideoxyforskolin, which are weak or inactive as activators of adenylate cyclase, did not have any significant effect on bradykinin and prostaglandin E1-induced plasma exudations. The phosphodiesterase inhibitors, ZK 62711, dipyridamole, HL 725, and 3-isobutyl-1-methylxanthine potentiated the bradykinin-induced plasma exudation and inhibited the prostaglandin E1-induced response. Papaverine had biphasic effects on the bradykinin-response and slight inhibitory effects on the prostaglandin E1-response. 8-Bromo cyclic AMP in the doses of 0.01 to 1 microgram potentiated the bradykinin-induced plasma exudation, but had no effect at doses of 10 and 100 micrograms. 8-Bromo cyclic AMP at all doses significantly inhibited the prostaglandin E1-induced response. The results suggest that the effects of forskolin and its analogs on plasma exudations induced with bradykinin and prostaglandin E1 in rat skin derive from activation of cyclic AMP-generating systems.     

Inhibition of platelet [3H]-imipramine binding by human plasma protein fractions

Inhibition of high-affinity [3H]-imipramine binding to platelet membranes by human plasma fractions and isolated plasma proteins was investigated. Several plasma proteins were found to contribute to the observed apparent inhibition and this contribution was assessed in terms of inhibitor units. Alpha 1 acid glycoprotein, high density and low density lipoprotein, IgG and alpha 1-antitrypsin were identified as effective non-specific inhibitors. Alpha-1-acid glycoprotein was confirmed to be the most potent plasma protein inhibitor. Cohn fractions were evaluated for the presence of the postulated endocoid of [3H]-imipramine binding site.     

Sources of the proteins of rat bile

The protein composition of rat bile has been studied systematically using two-dimensional agarose-polyacrylamide gel electrophoresis, with or without prior absorption by immobilised antisera, and by crossed immunoelectrophoresis. Sixteen bile proteins were distinguished. Of these, thirteen are immunologically identical to proteins present in rat serum and only one is identical to a protein present in rat liver plasma membrane but not in rat serum. Of the remaining two proteins, one is bile lipoprotein and the other has many of the properties of immunoglobulin A secretory component.The serum-related proteins in rat bile fall into two distinct groups. In the first group are immunoglobulin A and an α₂-globulin. These proteins are major constituents of bile but only minor constituents of serum. In the second group are albumin and some other major serum proteins which are found in bile at concentrations less than 1% of their concentrations in serum. The relative proportions of these proteins in bile appear to differ from their proportions in serum. It therefore appears that, although the majority of bile proteins are derived from serum, there cannot be direct leakage of serum into bile. Examination of the proteins contained within liver lysosomes indicates that, although discharge of lysosomal contents at the bile canalicular face of the hepatocyte may contribute to the bile proteins, an additional mechanism, with a considerable degree of selectivity, must also be involved in the transport of proteins from serum to bile.     

Binding of [3H]cytochalasin B and [3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([³H]cytochalasin B and [³H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [³H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB³H₄. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10^?6 M [³H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10^?5 M [³H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [³H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes.[³H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [³H]cytochalasin B binding and cytochalasin B stimulated ³H colchicine binding. [³H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Binding of physostigmine to rat and human plasma and crystalline serum albumins

The binding of 3H-physostigmine (3H-Ph) to human and rat plasma proteins and crystalline serum albumin was studied by ultrafiltration technique. This study showed that the percentage of 3H-Ph bound to rat plasma slightly decreased from 49% to 41% whereas human plasma showed an increase in binding from 29% to 43% over a 50-fold increase in drug concentration. Human plasma samples which were collected in a bag coated with citrate phosphate dextrose adenine-1 solution bound 50% less 3H-Ph than samples collected with EDTA indicating a drug-drug interaction between 3H-Ph and anticoagulants. No significant change in binding was observed if the samples were frozen prior to use. Scatchard plots for binding of 3H-Ph resulted in a positive slope for human plasma and a negative slope for rat plasma; whereas curvilinear Scatchard plots with negative slopes were obtained for binding to human and rat crystalline serum albumin.     

The interaction of living boar sperm and sperm plasma membrane vesicles with the porcine zona pellucida

Enzymological, morphological, and immunological methods were used to characterize further the interaction of noncapacitated boar spermatozoa with the porcine zona pellucida. Transmission electron microscopy showed that sperm usually bind to the zona over the head region of the cell. Only the plasma membrane is involved in this binding. Bound sperm will undergo the acrosome reaction when treated with calcium and the ionophore A23187. The ability of intact sperm to bind to porcine eggs in vitro and the ability of sperm plasma membrane vesicles to absorb univalent antibody to the sperm binding site for the zona were used to determine the effects of various physical, chemical, and enzymological treatments on the sperm binding sites. These sites were resistant to a number of enzymes including proteases and polysaccharidases, but were inactivated by heat and trichloroacetic acid. Binding sites on the zona were inactivated by extracts from small quantities of sperm. Binding was also blocked by Fab antibody to whole zonae absorbed to other swine tissue and by similarly absorbed Fab antibody to sperm plasma membranes. These data provide further support for the presence of zona recognition sites on the plasma membrane of noncapacitated boar sperm. The binding sites on the sperm plasma membrane do not appear to be peripheral membrane proteins nor major constituents of a surface glycocalyx.     

Limited pepsin digestion of human plasma albumin.

Limited pepsin digestion of human plasma albumin at pH 3.5 and 0 degrees in the presence of octanoate caused cleavage at residue 307 of the albumin molecule to yield two fragments. Thw two fragments corresponding to the NH2- and the COOH-terminal halves of the molecule were isolated in yields of about 15%. The COOH-terminal fragment is a mixture in which about 85% of the molecules had an additional cleavage at residue 422 of the albumin molecule. The COOH-terminal fragment with the additional cleavage at residue 422 contains two peptides which are linked by a disulfide bridge at residues 391 and 437 of the albumin molecule. Both the NH2- and the COOH-terminal fragment of human albumin showed no detectable binding of octanoate anions, that is, less than 1/170 of the binding constant of the primary site of human albumin. These findings differ from earlier observations on limited pepsin digestion of bovine plasma albumin where the corresponding COOH-terminal fragment had the octanoate-binding activity, about 1/8 of the primary binding constant of bovine albumin, while the NH2-terminal fragment did not. The COOH-terminal fragment of bovine albumin did not have cleavage at residue 422 as in the corresponding fragment of human albumin. However, it is not clear that the loss of octanoate-binding activity of fragment C of human albumin is a direct consequence of the cleavage at residue 422.