Biomedical subjects

H Vorum

Publications and source records attributed to H Vorum.

At least 55 records · Page 3Linked to original sources

PubMed1995

Evidence that epithelial glycoprotein 330/megalin mediates uptake of polybasic drugs.

Glycoprotein 330 (gp330) is an endocytic receptor expressed in the renal proximal tubules and some other absorptive epithelia, e.g., in the inner ear. The present study shows that the antifibrinolytic polypeptide, aprotinin, and the nephro- and ototoxic antibiotics, aminoglycosides, and polymyxin B compete for binding of 125I-urokinase-plasminogen activator inhibitor type-1 complexes to purified rabbit gp330. Half maximal inhibition was measured at 4 microM for aprotinin, 50 microM for gentamicin, and 0.5 microM for polymyxin B. Drug binding to gp330 was validated by equilibrium dialysis of [3H] gentamicin-gp330 incubations and binding/uptake studies in rat proximal tubules and gp330-expressing L2 carcinoma cells. Analyses of mutant aprotinins expressed in Saccharomyces cerevisiae revealed that basic residues are essential for the binding to gp330 and renal uptake. The polybasic drugs also antagonized ligand binding to the human alpha 2-macroglobulin receptor. However, the rapid glomerular filtration of the drugs suggests kidney gp330 to be the quantitatively most important target. In conclusion, a novel role of gp330 as a drug receptor is demonstrated. The new insight into the mechanism of epithelial uptake of polybasic drugs might provide a basis for future design of drugs with reduced toxicity.

Animals↗

PubMed1995

Calcium ion binding to clinically relevant chemical modifications of human serum albumin.

Calcium binding to glycated, penicilloylated, acetylated, and normal defatted human serum albumin as well as to mercapt- and nonmercaptalbumin was studied by equilibrium dialysis of radioactive Ca2+. Binding was quantified by five Scatchard constants [ni = 1, (i = 1-4) and n5 = 10]. Glycation resulted in increased k1- and k2-values and unchanged k3-k5-values, whereas penicilloylation increased all five association constants. The increments were greater the more pronounced the modification, and the enhancements caused by penicilloylation were, for the same degree of modification, greater than those produced by glycation. In contrast, acetylation by acetylsalicylate did not affect calcium binding. Likewise, binding to mercapt- and nonmercaptalbumin was the same, a finding showing that the thiol group of cysteine 34 is not important for calcium binding. D-Glucose and penicillin G are known to react with lysine residues of albumin, and the enhancement of binding resulting from glycation or penicilloylation is probably brought about by unspecific electrostatic effects, possibly supplemented by conformational changes of the protein molecule. The relative importance of the three domains of human serum albumin for calcium binding is discussed.

Acetylation↗

PubMed1994

Measurement of palmitate availability in serum samples: method and utility.

Palmitic acid shows a very low and unknown solubility at neutral pH. Binding equilibria of palmitate to human serum albumin accordingly cannot be investigated by measuring free and bound ligand concentrations as in conventional binding studies. It is feasible, on the other hand, to describe the binding equilibria in relative terms, by measuring the concentration, p, of reserve albumin, previously defined as the concentration of a purified standard albumin preparation which in buffered solution binds a trace amount of palmitate as tight as it is bound in the sample. Palmitate availability is calculated as C/p, when C is the concentration of bound palmitate. The general binding equation is modified to contain the availability beside relative binding constants, Li = Ki/K1,St, where K1,St is the first stoichiometric binding constant for palmitate to the standard albumin preparation. Availabilities and relative binding constants can replace free concentrations and usual binding constants in considerations of biochemical transport and enzymatic mechanisms. A method is described for measuring the concentration of reserve albumin for binding of palmitate, based upon determination of dialytic exchange rates for palmitate among identical equilibrium samples. A technique for reproducibly adding radiolabelled palmitate to the samples is given.

Adult↗

PubMed1994

High-affinity binding of two molecules of warfarin and phenprocoumon to human serum albumin.

Binding equilibria of warfarin, 3-(alpha-acetonylbenzyl)-4-hydroxycoumarin, and phenprocoumon, 3-(alpha-ethylbenzyl)-4-hydroxycoumarin, to defatted human serum albumin (Kabi Vitrum) were studied by equilibrium dialysis in a 33 mM sodium phosphate buffer (pH 7.4) at 37 degrees C. The binding data were analysed in terms of several acceptable sets of binding constants using a computerized curve fitting procedure. The findings were consistent with binding of two warfarin or two phenprocoumon molecules with high affinity and additional molecules bound with lower affinity. The binding of warfarin or phenprocoumon was explained by a model with two independent and equal high-affinity binding sites besides several independent weak sites in the albumin molecule (p < 0.01, by F-test). The findings were not consistent with binding of warfarin or phenprocoumon to a single high-affinity site besides several weak sites. A model of sequential binding of several ligand molecules to one locus is proposed.

Binding Sites↗

PubMed1994

The human keratinocyte two-dimensional protein database (update 1994): towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.

The master two-dimensional (2-D) gel database of human keratinocytes currently lists 3087 cellular proteins (2168 isoelectric focusing, IEF; and 919 none-quilibrium pH gradient electrophoresis, NEPHGE), many of which correspond to posttranslational modifications, 890 polypeptides have been identified (protein name, organelle components, etc.) using one or a combination of procedures that include (i) comigration with known human proteins, (ii) 2-D gel immunoblotting using specific antibodies (iii) microsequencing of Coomassie Brilliant Blue stained proteins, (iv) mass spectrometry and (v) vaccinia virus expression of full length cDNAs. These are listed both in alphabetical order and with increasing SSP number, together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Furthermore, we list 239 microsequenced proteins recorded in the database. We also report a database of proteins recovered from the medium of noncultured, unfractionated keratinocytes. This database lists 398 polypeptides (309 IEF; 89 NEPHGE) of which 76 have been identified. The aim of the comprehensive databases is to gather, through a systematic study of keratinocytes, qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and, ultimately, to pinpoint signaling pathways and components affected in various skin diseases, cancer included.

Amino Acid Sequence↗

PubMed1993

New procedure for analysing drug binding data, exemplified by warfarin binding to human serum albumin.

Determination of binding constants for multiple binding of a ligand usually results in highly variable figures. We have found that the variations depend mainly upon cooperativity of ligand binding, and that cooperativity is generally absent on binding to human serum albumin. When this is taken into account it becomes possible to obtain binding constants with only slight variation. A computerized curve fitting procedure for analysing binding data has been established consisting of the following steps. (1) Fitting of Scatchard's equation to observed binding equilibrium data to obtain a best-fit set of Scatchard binding constants. (2) Repetition of the fitting procedure, not to obtain a best fit but to generate 30 acceptable sets of Scatchard binding constants. (3) Fitting of Adair's equation to the observed points to obtain a best fit. If the sum of weighted and squared deviations is significantly smaller than the fitting of Scatchard's equation, cooperativity should be considered. If not, cooperativity cannot be demonstrated and the binding constants obtained by fitting Scatchard's equation can be accepted, with the variations found. (4) Final transformation of all Scatchard constants to Adair's. To illustrate the method warfarin data obtained by equilibrium dialysis was used.

Binding Sites↗

PubMed1993

Variation in the binding affinity of warfarin and phenprocoumon to human serum albumin in relation to surgery.

The binding equilibria of warfarin and phenprocoumon with defatted human serum albumin were studied by equilibrium dialysis in 33 mM sodium phosphate buffer, pH 7.4, 37 degrees C. The binding isotherms for both ligands were consistent with binding to two similar and independent sites in the albumin molecule. The binding affinity of warfarin was markedly increased on adding palmitic acid up to palmitate 4 mol per mol albumin and then it decreased. The binding affinity of phenprocoumon varied similarly but to a lesser degree. Serum samples were obtained from 14 patients undergoing knee joint surgery, six consecutive samples from each patient. The binding affinity of warfarin and phenprocoumon added in low concentrations to the serum samples was consistently less than to purified albumin. The binding affinity for warfarin increased slightly with increasing fatty acid concentrations during surgery, but the increase was much less than expected from the in vitro studies. The binding of phenprocoumon in the serum samples was not influenced by changing fatty acid concentration. The binding affinity for both drugs decreased markedly during the three days following surgery.

Aged↗

PubMed1993

Valproate and palmitate binding to serum albumin in valproate-treated patients. Relation to obesity.

Binding of valproate and palmitate to serum albumin was studied in 29 valproate-treated epileptic patients. The results were compared with similar observations in a reference group of 43 non valproate-treated individuals. The binding affinity for palmitate was decreased (P < 0.0001) resulting in increased availability of long-chain fatty acids (P = 0.008) due to competitive valproate binding in the valproate-treated patients. The findings support a hypothesis on the pathogenesis of obesity as a complication of valproate treatment of epilepsy.

Adult↗

PubMed1993

Quantitative analyses of the interaction between calcium ions and human serum albumin.

We examined the suitability of nine organic buffers for studying calcium binding to albumin by equilibrium dialysis. Results obtained with defatted human serum albumin showed that 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 2-([tris(hydroxymethyl)methyl] amino)ethanesulfonic acid were superior. Scatchard analysis of the experimental data from an extensive study performed in HEPES at pH 7.4 and 20 degrees C revealed (putting n(i) = 1, i = 1-4) k1 = 367 L/mol, k2 = 314 L/mol, k3 = 291 L/mol, and k4 = 179 L/mol. The very weak binding was characterized as n5 = 10 and k5 = 40 L/mol. The results were also analyzed in terms of stoichiometric association constants. The constants K1 and K2 were calculated to be 1513 and 647 L/mol, respectively, whereas the other constants were considered undeterminable. pH studies showed that in the interval 6.8-7.4, binding was not influenced by changes in acidity. Increasing pH to above the physiological value resulted in increased binding. At pH 8.0, k1 was increased almost fourfold, whereas k2 and k3 were approximately doubled. These findings indicate that the neutral to basic transition is important for the calcium-binding properties of albumin. The transition is a reversible, gradual conformational change of the protein at pH 6-9.

Buffers↗

PubMed1992

Solubility of long-chain fatty acids in phosphate buffer at pH 7.4.

The solubility of the saturated fatty acids lauric, myristic, palmitic, and stearic acid and the unsaturated oleic acid at 37 degrees C in phosphate buffer (pH 7.4) was estimated by using two independent methods. The one was a conventional solubility technique measuring the concentration of dissolved fatty acid in buffer by using radioactive compounds. The other was a dialysis exchange technique monitoring possible aggregation of solvated fatty acid anions by measuring the rate of diffusion of labelled compound across a dialysis membrane under conditions of chemical equilibrium. It was found that the results were strongly dependent on the radiochemical purity of the fatty acids. Using highly purified samples of radioactively labelled fatty acids, the solubility of monomeric laurate was shown to be greater than 500 microM, whereas the solubility of monomeric myristate was found to be 20-30 microM. Palmitate, stearate, and oleate solutions, on the other hand, showed a tendency to aggregation even at concentrations below 1 microM. Special attention was given to palmitate, as a reference compound for long-chain fatty acids, and the solubility of monomeric palmitate was estimated to be lower than 10(-10) M.

Autoradiography↗

PubMed1992

Fatty acid and drug binding to a low-affinity component of human serum albumin, purified by affinity chromatography.

Binding equilibria for decanoate to a defatted, commercially available human serum albumin preparation were investigated by dialysis exchange rate determinations. The binding isotherm could not be fitted by the general binding equation. It was necessary to assume that the preparation was a mixture of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture, whereas warfarin was not bound at all to the low-affinity component.

Chromatography, Affinity↗

PubMed1991

Binding of long-chain fatty acids to serum albumin in healthy humans. Relationship to obesity.

Equilibria of the binding of palmitate to serum albumin in adults are studied by dialysis-exchange-rate determinations. The results are used for a description of binding equilibria of fatty acids in general, as follows. 1. The reserve albumin concentration, p, for binding of palmitate is used as an approximate measure of p*, the reserve albumin concentration for binding of mixed fatty acids present in serum. 2. The total availability of fatty acids is defined as C*/p*, where C* is the total concentration of non-esterified fatty acid. 3. The fatty-acid-binding property of albumin is described by L* = p*/P = alpha C*/P, where P is the albumin concentration. The numerical value of alpha is -0.05. The above parameters are measured in sera from four healthy volunteers, in whom large variations of serum fatty acid concentration occurred. A group of 64 healthy students showed considerable variation of L* from one individual to another. It is found that L* decrease significantly with increasing body mass index (body mass divided by the square of the body length). In 42 patients with diabetes type I, L* was independent of body mass index. These findings are consistent with a previously formulated hypothesis of mechanism of obesity.

Adult↗

PubMed1991

Na(+)- and H(+)-gradient-dependent transport of alpha-aminoisobutyrate by luminal membrane vesicles from rabbit proximal tubule.

1. The characteristics of renal transport of alpha-aminoisobutyrate (AIB) by luminal membrane vesicles isolated from either the proximal convoluted part (pars convoluta) or the proximal straight part (pars recta) of rabbit proximal tubule were investigated. 2. Transport of AIB in vesicles from pars convoluta was mediated by both Na(+)-dependent and Na(+)-independent systems, which in the presence of an inwardly directed H+ gradient can drive the uphill transport of AIB into these vesicles. 3. By contrast, in luminal membrane vesicles from pars recta, transient accumulation of AIB was only dependent on Na+. Lowering pH without a H+ gradient (pHi = pH0 = 5.5) completely abolished the Na(+)-dependent transient accumulation of AIB in these vesicle preparations. 4. Attempts to determine the stoichiometry of both the Na(+)-AIB and H(+)-AIB transporters located in these two segments of proximal tubule suggested that one Na+ and one H+ ion may be involved in the transport of AIB. 5. Sodium-dependent uptake of AIB in vesicles from pars convoluta was competitively inhibited by L-serine and L-phenylalanine, whereas the presence of L-proline, L-alanine and glycine had no significant effect. By contrast, the H(+)-gradient-dependent uptake of AIB was drastically reduced (30% of the control value) by L-proline, L-alanine and glycine, while L-serine and L-phenylalanine had no significant effect. 6. On the other hand, pars recta vesicles exhibited a different transport specificity. L-Phenylalanine, L-serine, L-alanine and glycine, but not L-proline competitively inhibited the uptake of AIB, providing evidence for the existence of a common transport system for AIB, L-phenylalanine, L-serine, L-alanine and glycine in this segment of rabbit proximal tubule.

Amiloride↗

PubMed1990

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.

Diabetes Mellitus, Type 1↗

PubMed1990

Two-dimensional electrophoresis of the fatty acid binding protein from human heart: evidence for a thiol group which can form an intermolecular disulfide bond.

A 100,000 g supernatant from human heart muscle, containing cytosolic proteins with some contaminating plasma proteins, was analyzed for fatty acid binding protein (FABP) by two-dimensional electrophoresis (2-DE) using isoelectric focusing under nondenaturing conditions in the first dimension. FABP purified from human heart muscle was found to comigrate with a major spot in 2-DE gels of the supernatant. This spot was comparable with those of the myoglobins in staining intensity. When purified FABP was charged with [3H]palmitate and subjected to nondenaturing 2-DE, radioactivity always comigrated with this protein. Under denaturing and reducing conditions in the second dimension, FABP was found to have a pI of 5.3 and an apparent molecular weight of 15,000. Isoforms of FABP, reported here for the first time to occur in human heart muscle, were observed as minor spots focusing at pH 5.1 and 5.7. When electrophoresis in the second dimension was carried out under denaturing but nonreducing conditions, an additional protein appeared at pH 5.3 with an apparent molecular weight of about 30,000. This protein was identified as a dimer of FABP and evidence for the involvement of an intermolecular disulfide bond in this dimerization is presented.

Carrier Proteins↗

PubMed1990

Valproate and palmitate binding to human serum albumin: an hypothesis on obesity.

Binding equilibria of valproate (2-n-propyl-pentanoic acid anion) with defatted human serum albumin were studied by equilibrium dialysis in a 66 mM sodium phosphate buffer, pH 7.4, 37 degrees. Three hundred and fifty-six observed points for bound versus free valproate concentration were obtained and analyzed in terms of stepwise binding. It was found that the best fit resulted from a model in which 67% of the albumin was capable of binding valproate, whereas 33% did not bind. Thirty acceptable variants of the curve fitting were generated in order to assess the variation of the binding constants. The binding albumin component combines with three molecules of valproate with high affinity and with at least seven additional molecules that are loosely bound. Saturation of the protein cannot be reached. At very high concentrations of free valproate (above 10 mM) irreversible changes in the albumin take place, resulting in poor reproducibility in the amount of bound valproate. In the presence of palmitate, 0.5, 1, and 1.5 mol/mol of albumin, binding of valproate is decreased by a competitive mechanism. It is hypothesized that obesity, developing as a complication of valproate treatment of epilepsy, results from increased availability of long-chain fatty acids due to competitive valproate binding.

Humans↗

PubMed1989

Serum albumin binding of palmitate and stearate. Multiple binding theory for insoluble ligands.

In usual studies of ligand binding to a carrier, free and bound ligand concentrations are measured in equilibrium mixtures with varying carrier and ligand concentrations. The observed data are then analyzed by a binding equation such as Scatchard's or the general binding equation. With palmitic, stearic and oleic acids as ligands we found that the aqueous solubility is too low to allow this procedure. We have consequently transformed the general binding equation so that it does not contain parameters related to aqueous solutions of the ligand. While the classical binding equations describe affinities of transfer of a ligand from an aqueous solution to the carrier, the new equation is valid for transfer of a ligand from one bound state to another, i.e. for relative binding description. The relative binding constants, L1, L2, L3 ... Li, in the new equation thus define the transfer affinity for the ligand from a 1:1 complex with a standard carrier to an i:1 complex of the ligand with the carrier investigated. Binding of palmitate and stearate to human serum albumin was studied by determination of dialytic exchange rates between identical fatty acid/albumin solutions. The results were analyzed by the new equation without reference to ligands in aqueous solution.

Chlorides↗

PubMed1988

Energetics of renal Na+ and H+/L-alanine co-transport systems.

The stoichiometric properties of Na+- and H+-dependent L-alanine transporters recently identified in luminal-membrane vesicles prepared from proximal convoluted tubules (pars convoluta) and proximal straight tubules (pars recta) of rabbit kidney were studied. We provide indirect evidence suggesting that one Na+ and one H+ ion are co-transported with the L-alanine molecule via Na+-dependent and H+-dependent transport systems located in vesicles from pars convoluta. Furthermore, our experimental data suggest that both the high-affinity and the low-affinity Na+-dependent L-alanine transport systems of pars recta vesicles operate with a 1:1 stoichiometry.

Alanine↗