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PubMed1973

Hydrophobic chromatography: use for purification of glycogen synthetase.

A homologous series of omega-aminoalkylagaroses [Sepharose-NH(CH(2))(n)NH(2)] that varied in the length of their hydrocarbon side chains was synthesized. This family of agaroses was used for a new type of chromatography, in which retention of proteins is achieved mainly through lipophilic interactions between the hydrocarbon side chains on the agarose and accessible hydrophobic pockets in the protein. When an extract of rabbit muscle was subjected to chromatography on these modified agaroses, the columns with short "arms" (n = 2 and n = 3) excluded glycogen synthetase (EC 2.4.1.11), but the enzyme was retained on delta-aminobutyl-agarose (n = 4), from which it could be eluted with a linear NaCl gradient. Higher members of this series (e.g., n = 6) bind the synthetase so tightly that it can be eluted only in a denatured form. A column of delta-aminobutyl-agarose, which retained the synthetase, excluded glycogen phosphorylase (EC 2.4.1.1), which in this column series and under the same conditions requires side chains 5-(or 6)-carbon-atoms long for retention. Therefore, it is possible to isolate glycogen synthetase by passage of muscle extract through delta-aminobutyl-agarose, then to extract phosphorylase by subjecting the excluded proteins to chromatography on omega-aminohexyl-agarose (n = 6). On a preparative scale, the synthetase (I form) was purified 25- to 50-fold in one step. This paper describes some basic features and potential uses of hydrophobic chromatography. The relevance of the results presented here to the design and use of affinity chromatography columns is discussed.

Animals↗

PubMed1994

Purification to homogeneity of an active opioid receptor from rat brain by affinity chromatography.

Active opioid binding proteins were solubilized from rat brain membranes in high yield with sodium deoxycholate in the presence of NaCl. Purification of opioid binding proteins was accomplished by opioid antagonist affinity chromatography. Chromatography using the delta-opioid antagonist N,N-diallyl-Tyr-D-Leu-Gly-Tyr-Leu attached to omega-aminododecyl-agarose (Affi-G) (procedure A) yielded a partially purified protein that binds selectively the delta-opioid agonist [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr ([3H]DSLET), with a Kd of 19 +/- 3 nM and a Bmax of 5.1 +/- 0.4 nmol/mg of protein. Subsequently, Lens culinaris agglutinin-Sepharose 4B chromatography of the Affi-G eluate resulted in isolation of an electrophoretically homogeneous protein of 58 kDa that binds selectively [3H]DSLET with a Kd of 21 +/- 3 nM and a Bmax of 16.5 +/- 1.0 nmol/mg of protein. Chromatography using the nonselective antagonist 6-aminonaloxone coupled to 6-aminohexanoic acid-Sepharose 4B (Affi-NAL) (procedure B) resulted in isolation of a protein that binds selectively [3H]DSLET with a Kd of 32 +/- 2 nM and a Bmax of 12.4 +/- 0.5 nmol/mg of protein, and NaDodSO4/PAGE revealed a major band of apparent molecular mass 58 kDa. Polyclonal antibodies (Anti-R IgG) raised against the Affi-NAL protein inhibit the specific [3H]DSLET binding to the Affi-NAL eluate and to the solubilized membranes. Moreover, the Anti-R IgG inhibits the specific binding of radiolabeled Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol (DAMGO; mu-agonist), DSLET (delta-agonist), and naloxone to homogenates of rat brain membranes with equal potency. Furthermore, immunoaffinity chromatography of solubilized membranes resulted in the retention of a major protein of apparent molecular mass 58 kDa. In addition, immunoblotting of solubilized membranes and purified proteins from the Affi-G and Affi-NAL matrices revealed that the Anti-R IgG interacts with a protein of 58 kDa.

Analgesics↗

PubMed2004

Purification of hemoglobin by ion exchange chromatography in flow-through mode with PEG as an escort.

Development of hemoglobin-based blood substitutes requires production of highly purified hemoglobin. Process of hemoglobin purification by ion exchange chromatography in flow-through mode was researched and optimized. Three kinds of media including, QMA Spherosil LS (Biosepra, France) and Q Sepharose Big Beads (Amersham Bioscience, Sweden), and an anion exchange membrane column, Mustang Q (PALL, USA) were investigated and compared. Adding polyethylene glycol (PEG) as an escort in ion exchange chromatography improved the purity and recovery, and the recovery in the chromatography was increased from 75 to 95%. The mechanism of PEG effects on chromatography was discussed. The optimal chromatography step, in combination with hypotonic dilution hemolyzing and membrane separation, formed an integrated hemoglobin purification process. The total recovery in the process was 87.6%. The activity of hemoglobin was well preserved: P50 23.2 mmHg, and Hill coefficient 2.31. The product appeared as a single band in SDS-PAGE, and GF-HPLC showed only one peak. The purity of the prepared hemoglobin was more than 99.9%. The optimized process is time saving and suitable for large-scale preparation of hemoglobin to provide materials for further preparation of blood substitutes.

Animals↗

PubMed1985

Modification of the N-linked oligosaccharides in cell surface glycoproteins during chick embryo development. A using lectin affinity and a high resolution chromatography study.

Important differences in asparagine-linked glycopeptides were observed in vitro cultured fibroblasts derived from chick embryo at different stages of development. Cells from 8-day and 16-day embryos were labeled metabolically with [3H]mannose. Cell surface glycopeptides obtained after mild trypsin treatment were extensively digested with pronase and then chromatographed on concanavalin-A-Sepharose and other immobilized lectins. The most important changes concerned the complex type chains. The ratio between triantennary plus tetraantennary and biantennary chains increased about 2.5-fold from the 8th to the 16th day of development. In the same way, complex chains with bisecting N-acetylglucosamine increased from 8-day to 16-day cells as shown by Phaseolus-vulgaris-erythroagglutinin--agarose chromatography. In 16-day cells, the majority of triantennary chains (60%) with alpha-linked mannose substituted at C2 and C6 positions and biantennary chains (50%) were shown to contain fucosyl (alpha 1----6)N-acetylglucosaminyl structure in the core region by their ability to bind to a lentil lectin affinity column. Similarly, in 8-day cells, triantennary chains (50%) were more fucosylated than biantennary chains (35%). Thus, complex structures exhibited an increased fucosylation of their invariable core from the 8th to the 16th day of development, except for fucosylated triantennary chains which were retained on Phaseolus vulgaris Leucoagglutin and on lentil lectin. These latter structures were present at the surface of 8-day cells and absent at the surface of 16-day cells. After chromatography on Bio-Gel P6 and treatment with endo-beta-N-acetylglucosaminidase H, the [3H]-mannose-labeled glycopeptides were separated by high resolution chromatography into glycopeptides with complex chains and glycopeptides with high-mannose chains. Analysis of the high-mannose oligosaccharides released after endo-beta-N-acetylglucosaminidase H treatment by chromatography on Bio-Gel P4 indicated that the same type of high-mannose chains were present at the surface of 8-day and 16-day cells. Quantification of mannose, galactose and sialic acid residues using gas liquid chromatography was consistent with a decrease of the relative amount of oligomannose chains and an increase of the relative amount of complex type chains in 16-day cells compared to 8-day cells. Thus N-linked oligosaccharides derived from cell surface glycoproteins undergo changes during embryo development resulting in greater complexity of carbohydrate chains.

Animals↗

PubMed1981

Characterization of alkylgalactolipids from calf brain by high performance liquid chromatography.

Minor nonpolar galactolipids were isolated from the total lipids of calf brain stem by column chromatography and were separated by preparative thin-layer chromatography into four groups. The material recovered from the bottom band of the thin-layer chromatography consisted of monogalactosyl diglyceride and its 1-0-alkyl isomer, alkylgalactolipid, present in a molar ratio of 11:9. After perbenzoylation, they were separated by preparative thin-layer chromatography and characterized. The fatty acid compositions of these lipids were similar to each other and to those of the ester-linked fatty acids of cerebroside esters. The major alkyl group of alkylgalactolipid was palmityl, and the other, minor componenents were oleyl, myristyl, and stearyl ethers. Perbenzoylated derivatives of these lipids were further separated by reverse-phase high performance liquid chromatography. The chromatograms from these two lipids were similar; however, most of the peaks were still mixtures of homologs containing different fatty acids or an alkyl group.

Animals↗

PubMed2005

The secretion of ibuprofen metabolites interferes with the capillary chromatography of urinary homovanillic acid and 4-hydroxy-3-methoxymandelic acid in neuroblastoma diagnosis.

Neuroblastoma is the most common extracranial solid tumor in children. Abnormal secretion of catecholamines in tissues and body fluids allows for the differential diagnosis of neuroblastoma from other neoplasms and its distinction from non-neoplastic inflammatory diseases. This is achieved by assaying homovanillic acid and 4-hydroxy-3-methoxymandelic acid, the catabolites of catecholamine metabolism. In the course of an evaluation of children with suspected neuroblastoma, homovanillic acid and 4-hydroxy-3-methoxymandelic acid were analyzed in urine samples by capillary gas chromatography with flame ionization detection after extraction and derivatization of these compounds as trimethylsilyl derivatives. In three urine samples a significant increase in biogenic amines was observed, but these results were not confirmed by thin-layer chromatography. Patient history revealed that these children had been treated with ibuprofen, an analgesic and anti-inflammatory drug. To verify how ibuprofen or its metabolites may have interfered with capillary gas chromatography with flame ionization detection, we analyzed the same samples by capillary gas chromatography-mass spectrometry. In urine samples from patients on the drug, the presence of a peak identified as the trimethylsilyl ester of hydroxyibuprofen, which had the same retention time as 4-hydroxy-3-methoxymandelic acid, was found to interfere with the capillary gas chromatography with flame ionization detection analysis of the metabolite. This interference must be taken into account during the laboratory diagnosis of neuroblastoma.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1986

Transformation of androgen receptors from mouse submandibular glands by gel chromatography.

[3H]Methyltrienolone-bound and unbound androgen receptors from mouse submandibular glands bound to DNA-cellulose to a similar extent after gel chromatography. The receptors sedimented at 6 S in a low-salt gradient whereas if receptors were transformed with KCl (0.4 mol/l) they sedimented at 4 S. On DEAE-anion exchange chromatography both were eluted at a concentration of 0.07 mol KCl/l. These results suggest that two states of transformed androgen receptors exist. A 6 S transformed receptor may well derive by partial dissociation of an 8 S non-transformed receptor and this is caused by gel chromatography. Molybdate ions completely prevent both the transformation induced by gel chromatography and the secondary transformation during DEAE-anion exchange chromatography.

Animals↗

PubMed1984

Preparation of purified erythropoietin by high performance liquid chromatography.

Highly purified erythropoietin was not available in quantities needed to carry out planned investigations and, therefore, the use of high performance liquid chromatography was explored. This technique permits the separation of proteins with high efficiency and resolution. Three types of chromatography were used. Size exclusion or gel permeation, reversed phase, and ion exchange columns were utilized with different solvent systems. The chromatographic fractions were assayed either by an exhypoxic polycythemic mouse assay or by the fetal liver cell assay. In addition, selected fractions were tested for their capability to stimulate CFU-E and BFU-E colony formation in methyl cellulose. The results of the techniques of size exclusion and ion exchange chromatography were found to be rapid and reproducible. Although reversed phase chromatography gave excellent resolution, the results were somewhat variable. Using different chromatographic combinations, erythropoietin with a specific bioactivity in the range of 50,000 u/mg protein was isolated. Although the erythropoietin gene has now been cloned and the hormone purified by utilizing monoclonal antibodies, high performance liquid chromatography may be useful in the removal of unwanted contaminants, as a tool in chemical characterization, and as a possible method for the hormone's identification and measurement in clinical laboratories.

Anemia, Aplastic↗

PubMed1983

Purification of the major component of the pregnancy-specific beta 1-glycoprotein (SP1) by affinity chromatography, and application to a highly sensitive radioimmunoassay.

The major component of the pregnancy-specific beta 1-glycoprotein (SP1) was purified from pregnancy sera by ammonium sulphate precipitation (1.95 mol/l), anion-exchange chromatography at pH 7.0, positive affinity chromatography, negative affinity chromatography, anion-exchange chromatography at pH 5.0 and, finally, concentration by ultrafiltration or lyophilization. Essentially, the first anion-exchange chromatography separated the major component from three minor components, termed SP1 (gamma), SP1 (alpha 2(2)) and SP1 (alpha 2(1)) on the basis of their electrophoretic mobilities. The yield was 3-6% and the purity about 98-99%. A highly sensitive radioimmunoassay to determine SP1 was developed (detection limit, about 0.5 microgram/l). By using a commercial antiserum against SP1, the slopes of the dose-response curves for purified SP1, a reference preparation from the International Agency for Cancer Research, a commercial SP1 standard, and two of three minor SP1 components were all similar to that of a pool of pregnancy sera, whereas the third minor component, SP1 (gamma), showed a marked difference in slope.

Chemical Phenomena↗

PubMed2003

Purification of soluble human epidermal growth factor (hEGF) from recombinant Escherichia coli culture broth by using expanded-bed adsorption chromatography.

Human epidermal growth factor (hEGF) secreted by recombinant Escherichia coli was purified from culture broth by expanded-bed adsorption (EBA) chromatography, strong anion-exchange chromatography and finally preparative reversed-phase HPLC (RP-HPLC). The EBA chromatography step simultaneously captured the hEGF by cationic exchanger and removed the cellular biomass from the diluted culture broth. This step was carried out at high throughput, and resulted in a high yield (>90%) and a purification factor of approx. 20-fold to >80% purity. Its process performance was well maintained during a 16-fold scale-up. After the successive purification steps of anion-exchange chromatography and RP-HPLC, the overall yield was approx. 84% and the purity was satisfactory (>99.5%). It was concluded that the purification process was very efficient and scaleable, warranting its implementation in large-scale manufacturing.

Adsorption↗

PubMed2002

Dynamics of protein uptake within the adsorbent particle during packed bed chromatography.

A new experimental set-up for on-line visualization of the intra-particle uptake kinetics during packed bed chromatography has been designed and tested. Confocal laser scanning microscopy was used to analyze the dynamics of protein adsorption to porous stationary phases. In combination with this, a flow cell was developed that could be packed with chromatography media and operated as a fully functional mini-scale chromatography column. Adsorption profiles of single- and two-component mixtures containing BSA and IgG 2a during packed bed cation-exchange chromatography were investigated. The two proteins appear to exhibit different transport characteristics. For BSA a classical "shrinking core" behavior could be detected. The profiles obtained during IgG 2a adsorption point toward a different transport mode, which deviates from the classical pore-diffusion picture. For the two-component system, a superposition of the single-component profiles combined with a classical displacement of the weaker bound protein species was found. The results indicate that depending on the adsorbed protein the uptake can vary tremendously, even for adsorption to the same chromatographic support. It is clearly shown that the new microcolumn allows in situ quantitative investigations of protein adsorption dynamics within a single particle, which adds a new tool to the available methods for characterizing and optimizing protein adsorption chromatography.

Adsorption↗

PubMed2005

A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography.

Membrane separation and chromatographic technologies are regarded as an attractive alternative to conventional academic small-scale ultracentrifugation procedures used for retrovirus purification. However, despite the increasing demands for purified retroviral vector preparations, new chromatography adsorbents with high specificity for the virus have not been reported. Heparin affinity chromatography is presented here as a novel convenient tool for retrovirus purification. The ability of bioactive retroviral particles to specifically bind to heparin ligands immobilized on a chromatographic gel is shown. A purification factor of 63 with a recovery of 61% of functional retroparticles was achieved using this single step. Tentacle heparin affinity supports captured retroviral particles more efficiently than conventional heparin affinity chromatography supports with which a lower recovery was obtained (18%). Intact, infective retroviral particles were recovered by elution with low salt concentrations (350 mM NaCl). Mild conditions for retrovirus elution from chromatographic columns are required to preserve virus infectivity. VSV-G pseudotyped retroviruses have shown to be very sensitive to high ionic strength, losing 50% of their activity and showing membrane damage after a short exposure to 1M NaCl. We also report a complete scaleable downstream processing scheme for the purification of MoMLV-derived vectors that involves sequential microfiltration and ultra/diafiltration steps for virus clarification and concentration respectively, followed by fractionation by heparin affinity chromatography and final polishing by size-exclusion chromatography. Overall, by using this strategy, a 38% yield of infective particles can be achieved with a final purification factor of 2,000.

Chromatography, Affinity↗

PubMed1981

Combined high performance liquid chromatography mass spectrometry.

Combined gas chromatography mass spectrometry, because of its ability to provide both high sensitivity and specificity, has become a vital technique for the identification and quantitation of natural and synthetic chemicals in a wide variety of areas. Unfortunately, a very large number of organic compounds are not directly amenable to gas chromatography since they are thermally labile and/or of low volatility. Chemical modification of compounds of interest can assist in some cases, but it is not universally applicable. High performance liquid chromatography has been increasingly utilized for studies of compounds of these types since heat is not usually involved in the analysis. Combined liquid chromatography mass spectrometry would lend a new dimension to these studies, enabling the separating ability of the liquid chromatograph to be combined with the sensitivity and specificity of the mass spectrometer. Approaches to liquid chromatography mass spectrometry are discussed. Using examples from our liquid chromatographic mass spectrometric studies of natural compounds, drugs and pesticides, the current abilities of such systems to identify and quantify natural and synthetic chemicals present in a complex matrix of other chemicals are described.

Carbohydrates↗

PubMed1990

Application of micellar electrokinetic chromatography to pharmaceutical analysis.

Electrokinetic chromatography is a new type of analytical separation method which belongs to the group of high performance capillary electrophoretic techniques but whose separation principle is based on that of chromatography. The solute distributes itself between a carrier and the surrounding medium. The carrier, which corresponds to the stationary phase in conventional chromatography, can be transported by electrophoresis with a different velocity from the surrounding medium. The separation is achieved by the differential solute distribution and the differential migration of the carrier. The charged molecules or charged molecular aggregates are employed as the carrier. Various kinds of carriers are available for electrokinetic chromatography along with different partition mechanisms. Among them, micellar electrokinetic chromatography, which employs an ionic micelle as a carrier, has become the most popular method because of its unique and attractive characteristics as well as the separating capability of electrically neutral or nonionic solutes in comparison with capillary zone electrophoresis. The present paper describes the principle, separation characteristics and its application to the analysis of pharmaceuticals.

Chromatography↗

PubMedDate not supplied

Characterization of specific interactions of coenzymes, regulatory nucleotides and cibacron blue with nucleotide binding domains of enzymes by analytical affinity chromatography.

The dissociation constant for the complex of rhodanese and Cibacron Blue, determined by analytical affinity chromatography using rhodanese immobilized on controlled-pore glass (CPG) beads (200 nm pore diameter) and aminohexyl-Cibacron Blue, was 44 microM which agreed well with the kinetic inhibition constant, suggesting that the dye binds at or near the active site of this enzyme. Formation of a binary complex of the dye and lactate dehydrogenase (LDH) was also characterized by direct chromatography of LDH on CPG/immobilized Cibacron Blue (KD = 0.29 microM). The binary complex formed between LDH and NADH was characterized by analytical affinity chromatography using both CPG/immobilized LDH and immobilized Cibacron Blue. Since the dye competes with NADH in binding to the active site of LDH, competitive elution chromatography using the immobilized dye allows determination of the dissociation constant of the soluble LDH.NADH complex. Agreement between the dissociation constants determined by direct chromatography of NADH on immobilized LDH (KD = 1.4 microM) and that determined for the soluble complex (KD = 2.4 microM) indicates that immobilization of LDH did not affect the interaction. Formation of various binary, ternary and quaternary complexes of bovine liver glutamate dehydrogenase (GDH) with glutamate, NADPH, NADH, and ADP was also investigated using immobilized GDH. This approach allows characterization of the enzyme/ligand interactions without the complicating effect of enzyme self-association. The affinity for NADPH is considerably greater in the ternary complex (including glutamate) as compared to the binary complex (0.38 microM vs 22 microM); however, occupancy of the regulatory site by ADP greatly reduces the affinity in both complexes (6.4 microM and 43 microM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Nondestructive detection of gangliosides with lipophilic fluorochromes and their employment for preparative high-performance thin-layer chromatography.

A simple and effective procedure for the isolation and purification of gangliosides by preparative thin-layer chromatography is described. The method is based on nondestructive visualization of gangliosides on silica gel-precoated thin-layer chromatography plates by staining with uncharged lipophilic fluorochromes. Fluorescent dyes were added in low concentrations into the mobile phase (0.002%, w/v) without any interference of the ganglioside separation. After uv localization, the fluorescent zones were scraped off the plate and the silica gel was extracted with chloroform/methanol/water (30/60/8). In the following step fluorochromes were removed from gangliosides containing crude extracts by anion-exchange chromatography on DEAE-Sepharose. After desalting, impurities were removed by Iatrobeads chromatography. The method described offers an easy to handle and successful preparative thin-layer chromatography strategy to obtain pure gangliosides in microgram and miligram quantities.

Animals↗

PubMed1998

High-resolution separation and quantification of neutral lipid and phospholipid species in mammalian cells and sera by multi-one-dimensional thin-layer chromatography.

An improvement of current methods is needed for simple, rapid, and precise quantification of cellular lipids, including rare species of biologically active cellular lipids, such as phosphatidic acid (PA) and diradylglycerol (DG). In addition, further analysis of hydrolyzed acyl chains from these species by methods such as gas chromatography requires complete separations. Methods have been developed for the quantification of neutral lipids and several phospholipids extracted from mammalian cells and sera. Lipid masses were determined for the major classes of the neutral, nonpolar lipids, and of the phospholipids. The lipid classes were separated by a multistep thin-layer chromatography (TLC) procedure in different solvent systems, a method which we have designated as multi-one-dimensional thin-layer chromatography (MOD-TLC). Resolved lipid bands were visualized by the lipophilic dye primulin (direct yellow 59) and scanned by an automated laser-fluorescence detector. The mass of each band was determined by comparing band intensities of unknown samples to dilution curves of authentic standards. With modifications in solvent mixtures and length of separation times, the majority of biological lipids could be resolved and quantified with MOD-TLC methods. Since the detection method is nondestructive, purified lipids could then be recovered by scraping the visualized bands and extracting the lipids from the silica. The structural identities of the recovered lipids were confirmed by fast-atom bombardment and electrospray mass spectrometry. Extracted lipids were also hydrolyzed to release acyl chains and acyl chain species were determined in comparison to authentic standards by gas chromatography. PA and DG levels in ECV.304 cells were found to be 4. 6 and 3.3%, respectively, of PC levels, with a PA/DG ratio of 1.4, which is in accord with published experience using other methods and different cell types. PA in human serum was detected at 0.6% of PC, indicating the sensitivity of the technique. In contrast to two-dimensional thin-layer chromatography, which allows for good resolution of some lipid species, but cannot be used to analyze more than a single experimental point per plate, MOD-TLC allows for direct comparative analysis of multiple samples on a single TLC plate, while still providing good resolution for the quantification of most major classes of lipid species.

Anthocyanins↗

PubMed2002

Rapid identification of Wilson's disease carriers by denaturing high-performance liquid chromatography.

BACKGROUND: Wilson's disease is an autosomal recessive disorder characterized by decreased biliary copper excretion and reduced copper incorporation into ceruloplasmin. The disease gene ATP7B maps to chromosome 13q14.3, contains 21 exons, and encodes a copper-transporting P-type ATPase. ATP7B mutations are scattered over the entire gene, and scanning methods to detect mutation carriers are in demand. We have tested the usefulness of denaturing high-performance liquid chromatography for mutation detection in Wilson's disease. METHODS: Genomic DNA from five Sardinian Wilson's disease families (32 individuals, 8 patients) was subjected to polymerase chain reactions for ATP7B exons 2-21 and the 5' untranslated region. PCR products were analyzed by chromatography and by direct sequencing. RESULTS: Three disease-causing mutations and seven sequence variants were detected by chromatography. Five patients were homozygotes for -441/-427del, and three were compound heterozygotes for V1146M plus 1512-13insT (N505X) and for -441/-427del plus V1146M, respectively. Eighteen unaffected individuals were mutation carriers. Sequence variants comprised V290V, A406S, L456V, R832K, A1140V, the novel K952R, and T991T. The novel intronic IVS18+6c>t change escaped detection by chromatography. CONCLUSIONS: Denaturing high-performance liquid chromatography is a dependable tool for ATP7B screening that is superior to traditional haplotyping. This method allows for fast, sensitive, and specific mutation detection and identification of carriers in Wilson's disease families.

Adenosine Triphosphatases↗