Purification of insulin-like growth factor I receptor from human placental membranes

Insulin-like growth factor (IGF) I receptor was purified from Triton X-100-solubilized human placental membranes by wheat germ agglutinin-Sepharose chromatography followed by immunoaffinity chromatography using alpha IR-3, a monoclonal antibody directed against the IGF-I receptor. Purification of 3200-fold and 2800-fold was achieved from wheat germ agglutinin-Sepharose eluates with regard to IGF-I binding and kinase activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed two major protein bands corresponding to the alpha and beta subunits of the receptor, which accounted for at least 90% of the protein content. The purified receptor bound 10-20 micrograms of IGF-I/mg of protein and was more than 95% free of contamination by insulin receptor. It sedimented in glycerol gradients as a single species with a sedimentation coefficient of 13.7 S and gave three protein bands with Mr = approximately 300,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, indicating that alpha 2 beta 2 is an intact form of the IGF-I receptor. The purified receptor, when incubated with [gamma-32P] ATP, became phosphorylated at tyrosine residues of its beta subunit. This was stimulated 3-fold by IGF-I. It also had IGF-I-stimulated tyrosine kinase activity (5264 pmol of 32P incorporated/min/mg of protein) toward a synthetic peptide corresponding to the autophosphorylation site of pp60src. These data strongly suggest that it is a tyrosine-specific protein kinase.     

Purification of human placental trophoblast interferon by two-dimensional high performance liquid chromatography

Human placental trophoblast challenged with Sendai virus induced IFNs mainly of the beta-type (75%) and relatively low levels of the alpha-type (25%). A two-step high performance liquid chromatographic procedure ("two-dimensional HPLC") has been developed for the complete purification of the placental trophoblast interferon beta (tro-IFN-beta) from serum-containing culture supernatant. The method involved a combination of high performance liquid affinity chromatography (HPLAC) on Cibacron Blue 3GA immobilized on an activated pressure stable macroporous synthetic polymer, 2-hydroxyethyl methacrylate vinyl sulphone (HEMA-BIO 1000 VS), as the first dimension and reversed-phase high performance liquid chromatography (RP-HPLC) on Separon SGX C-18 as the second. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot experiments showed that the tro-IFN-beta was present as a 24 kDa protein. Densitometric scanning analysis of Coomassie-stained gel revealed the purity of the final preparation to be greater than 99%. The purified tro-IFN-beta had a specific activity of 1.03 x 10(8) IU/mg of protein and the overall recovery was 81% of the total IFN-beta activity in the crude preparation and 61% of the total IFN activity.     

Purification of lysosomal membrane-bound glucocerebrosidase from human cultured fibroblasts using high-performance liquid chromatography

Glucocerebrosidase from human skin fibroblasts was purified more than 2300-fold to apparent homogeneity with an overall yield of 39% using taurocholate extraction, ammonium sulfate fractionation, and high-performance hydrophobic interaction and gel permeation column chromatography. This relatively high yield is attributed to two modifications from previously published procedures: (i) the elimination of a butanol delipidation step that resulted in substantial loss of enzyme activity; and (ii) the use of 2% (w/v) sodium taurocholate instead of 1-2% sodium cholate that resulted in more than 90% solubilization of total membrane-bound enzyme activity. Confluent monolayers of human cultured skin fibroblasts (approximately 3.6 x 10(8) cells) were harvested from 10 roller bottles. Glucocerebrosidase in the cell pellet was solubilized with 2% (w/v) sodium taurocholate, fractionated in 14% ammonium sulfate, and applied to a high-performance hydrophobic interaction phenyl-5PW column. After an ammonium sulfate descending linear gradient step, glucocerebrosidase was eluted from the column at 4% cholate concentration using a 0-5% linear cholate gradient. There was a 36-fold purification and 80% recovery. In the subsequent step, concentrated glucocerebrosidase fractions from the phenyl column were injected into two Bio-Sil TSK-250 gel permeation columns joined in series. Glucocerebrosidase peak activity was eluted at 263 ml corresponding to Mr 76,000. There was an 18-fold purification and 78% recovery. The enzyme preparation was then recycled through the phenyl-5PW column in order to remove a remaining contaminant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of synthetic oligodeoxyribonucleotides by ion-exchange high-performance liquid chromatography

Synthetic oligodeoxyribonucleotides rangin from 11 to 37 nucleotides in length and with varying base compositions, prepared by both the phosphotriester and phosphite procedures, have been purified by ion-exchange high-performance liquid chromatography on Whatman Partisil 10/SAX columns using phosphate buffer gradients. The effects of different buffer systems on elution times and resolution have been evaluated. Oligomer composition and length had a marked effect on the resolution achieved. In general the use of formamide buffers gave the best results, particularly in the case of 2′-deoxyguanosine-rich sequences. These methods have also been successfully applied to the purification of mixtures of synthetic oligodeoxynucleotides.     

Purification of myosin light chains by high-performance liquid chromatography

Three procedures for the purification of myosin light chains-1, -2, and -3 from avian fast white muscle fibers using high-performance liquid chromatography are described. Two involve the reverse-phase mode, the other, anion exchange. The procedures allow preparation of microgram to milligram amounts of the proteins and are suitable for the study of myosin light chains in small muscles and biopsy muscle samples. The elution order of light chain-1 and light chain-3, two proteins with extensive sequence homology, is discussed in terms of the unusual amino acid sequence and structure of the N-terminal peptide of light chain-1.     

Purification of growth hormones by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (HPLC) on a column of Radial-Pak C₁₈ cartridge was utilized for the purification of a variety of growth hormone (GH) proteins from mammalian, avian, amphibian and fish pituitary glands. Recovery of GH from pituitary glands of up to 0.43% of total protein was obtained with a high degree of homogeneity as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The HPLC-purified GHs show reactions of identity or near identity by immuno-diffusion studies on agar gel. This method offers a convenient and rapid purification of vertebrate GH on an analytical or preparative scale.     

Purification of the insulin receptor from human placental membranes

Insulin receptors were purified from human placental microsomal membranes by solubilisation with Triton X-100 followed by Sepharose 6B chromatography, phosphate gradient elution from hydroxyapatite and affinity chromatography on concanavalin A-Sepharose. 2000-fold purification was achieved with 63% overall recovery. The purified receptor gave a single band on 3.75% polyacrylamide (0.1% Triton X-100) gel electrophoresis. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis there was a major band at 75,000 and a minor band at 80,000 daltons. The purified receptor rechromatographed on Sepharose 6B with an apparent molecular weight of 300,000.     

Purification of cholesterol-esterifying enzymes from rat liver cytosol by high-performance liquid chromatography

Three enzymes esterifying cholesterol with long-chain fatty acids were purified approximately 31 000-fold to apparent homogeneity from the cytosol of normal rat liver. The enzymatic activity was tested by incubation of active fractions with tritiated cholesterol and separation of newly formed esters from non-reacted cholesterol by a passage through silica gel cartridges with subsequent assay for radioactivity by liquid scintillation. For the purification of enzymes, active proteins were precipitated by (NH₄)₂SO₄ to 35% saturation. The bulk of inactive proteins was removed by size-exclusion chromatography on TSK G3000 SW. The active fraction was subsequently separated on Separon HEMA BIO 1000 DEAE in gradients of 0–500 mM KCl into three enzymatic activities differing in their retention and these proteins were finally purified by affinity HPLC on columns of cholesterol immobilized on HEMA BIO 1000 E-H. Final purified enzymes showed the same single band in polyacrylamide gel electrophoresis corresponding to 16.5 kDa. Combination of individual enzymes did not increase the overall yield of cholesteryl esters but the reaction-rate was significantly accelerated. These proteins are apparently subunits of a larger complex (M_r 65 000) that can be demonstrated by electrophoresis in the absence of 2-mercaptoethanol. Results presented in this paper indicate that because of good and rapid separation of active proteins, HPLC may be a method of choice for enzyme purifications.     

Glycosylation of annexin I and annexin II.

Human placental annexin I and annexin II were shown to be glycosylated by one-dimensional affinity blotting with the lectin concanavalin A, which recognizes D-mannose and D-glucose residues. Further evidence that annexin I and annexin II are glycosylated was provided by the finding that these proteins incorporated D-[2,6-3H]mannose and D-[6-3H]glucose when they were biosynthesized by the human squamous carcinoma cell line SqCC/Y1. Annexin I and annexin II could be rapidly purified from a human placental membrane extract by concanavalin A-Sepharose, which indicated that these proteins contain two biantennary mannosyl residues.     

Purification of dye-labeled oligonucleotides by ion-pair reversed-phase high-performance liquid chromatography

Singly- and dually-labeled synthetic oligonucleotides were purified by ion-pair reversed-phase high-performance liquid chromatography using a 50x4.6-mm column packed with porous, 2.5 micrometer C(18) sorbent. We studied the mechanism of dye-labeled oligonucleotide retention in order to improve the quality of purification. By-products of oligonucleotide synthesis were characterized by liquid chromatography with mass spectrometry detection (LC-MS). We purified oligonucleotides labeled with 6-carboxyfluorescein (6FAM), hexachlorofluorescein (HEX), tetrachlorofluorescein (TET), carboxytetramethylrhodamine (TAMRA) and indodicarboxycyanine (Cy3) dyes, as well as dually-labeled TaqMan probes. Purification of a 0.1-micromole oligonucleotide synthesis in a single injection was demonstrated.     

Purification of human brain metallothionein by organic and reversed-phase high-performance liquid chromatography under acidic conditions

A simplified high-performance liquid chromatographic method for the detection of metallothioneins, notably metallothionein-III, has been developed. In order to purify metallothionein, differential acetone precipitation at 50% (v/v) and at 80% (v/v) was employed on a 20% normal human brain homogenate. The reconstituted pellet was injected into a C₁₈ microbore reversed-phase HPLC column, equilibrated with 0.1% trifluoroacetic acid, and developed at a flow-rate of 800 μl/min with a linear gradient from 0% to 60% acetonitrile in 0.094% trifluoroacetic acid for 60 min. Western blots indicated that metallothioneins-I and II eluted at 16% acetonitrile and metallothionein-III eluted at 37% acetonitrile.     

Purification of liver and hepatoma membrane proteins by high-performance liquid chromatography

This paper documents the recovery of selected proteins from hepatic plasma membranes. Initial purification, achieved by a series of stepwise extractions, facilitates the subsequent purification by HPLC. Examples are provided to illustrate the recovery of specific proteins from two Morris hepatoma lines and the liver.     

Purification of fatty acid methyl esters by high-performance liquid chromatography

The determination by gas chromatography (GC) of fatty acid methyl esters (FAMEs) prepared from complex biological samples is subject to interference from cholesterol. During sample injection on the GC system of FAMEs prepared from tissues that contain cholesterol, we observed a major contaminant that co-eluted with docosahexaenoic acid (DHA, 22:6n-3). To address this problem, FAMEs were purified on an amino-phase high-performance liquid chromatography (HPLC) column using a hexane–isopropanol gradient. The HPLC retention times for both the FAME fraction and cholesterol were stable and reproducible when the amino column was used for sample purification. The purified extracts were analyzed by GC without artifacts or impurity peaks after 50 analytical runs. The method described here will be useful for measurement of 22:6n-3 and other fatty acids important for studies of nutrition or pathology.     

Purification of major variable-surface glycoproteins from African trypanosomes by reverse-phase high-performance liquid chromatography

Reverse-phase high-performance liquid chromatography (RP-HPLC) was used in a one-step procedure to purify and analyze several different major variable-surface glycoproteins (VSGs) from lysates of African trypanosomes. RP-HPLC was used to fractionate lysates of trypanosomes and the VSG localized to the major peak of the elution profile using a rabbit antiserum to the cross-reacting determinant of the VSG. Polyacrylamide gel electrophoresis of HPLC fractions showed that the purity of isolated VSGs was equivalent to or better than that attained using conventional purification procedures. The elution positions of purified VSGs from a variety of cloned trypanosomes were identical, indicating the presence of a common hydrophobic feature on the surface of these highly polymorphic antigens. Preliminary experiments have shown that purification of VSG from trypanosome lysates may be scaled up to preparative levels. The results show that RP-HPLC is a useful procedure for rapid preparation of highly purified trypanosome VSGs and that analysis of their various molecular forms will be facilitated by the application of HPLC methods.     

Purification of the major GTP-binding proteins from human placental membranes

Using minor modifications of procedures developed to purify GTP-binding proteins (G-proteins) from rabbit liver, we have purified the major G-proteins present in human placental membranes. One, referred to as Gi, is the major substrate for pertussis toxin-catalyzed ADP-ribosylation and has an alpha-subunit of 41,000 daltons, and beta-subunit of 36,000 and 35,000 daltons, and a gamma-subunit of 10,000 daltons. The other protein, referred to as Gp, was identified by its ability to bind guanine nucleotides specifically with high affinity. This activity was resolved from Gs and Gi by the second step of purification (AcA-34 chromatography) and was further purified through heptylamine-Sepharose and hydroxylapatite. The guanine nucleotide-binding site, which can be resolved by high performance liquid chromatography procedures and identified by a photolyzable GTP analogue, is associated with a 21,000-dalton protein (Gp alpha) that copurifies with beta gamma-subunits indistinguishable from the beta gamma-subunits associated with Gs and Gi. This protein represents a potentially novel member of the structurally and functionally homologous family of G-proteins that are transducing elements in the receptor-mediated regulation of a variety of cellular processes.     

Assay of human fibrinopeptides by high-performance liquid chromatography

Fibrinopeptides A, AP, and B, desarginine fibrinopeptide B, and a previously unknown peptide corresponding to B beta 3-14 were resolved within 10 min by an HPLC technique using an isocratic solvent system (22% acetonitrile in 0.1% trifluoroacetic acid) and a 0.46 X 10-cm Spherisorb ODS-2 (3-micron) octadecylsilane column. Fibrinopeptides A and AY eluted in the same peptide peak. The method was used to evaluate a carboxypeptidase which converts fibrinopeptide B into its desarginine form. Fifty percent inhibition of this activity occurred at 1.7 mM epsilon-aminocaproic acid (EACA). At saturating substrate concentrations the rates of total fibrinopeptides A and B release were unaffected by 125 mM EACA, a concentration at which the carboxypeptidase activity is completely inhibited.     

Purification of reduced nicotinamide adenine dinucleotide by ion-exchange and high-performance liquid chromatography

Combining ion-exchange (AG MP-1) and reversed-phase (C-18) partition chromatography accomplishes a higher degree of purification of NADH than either method can provide alone. Final elution in 95% ethanol, dehydration with anhydrous sodium sulfate, and storage in dry 1,2-propanediol over molecular sieves prevents hydrolysis of the purified dinucleotide.     

Purification and assay of bovine parathyroid hormone by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (HPLC) has been used to purify a crude extract of bovine parathyroid glands, in a single run on an analytical column, to give a high yield of homogeneous material with full bioactivity in in vivo bioassay. Bovine parathyroid hormone (bPTH) prepared and purified by conventional procedures has been rapidly and quantitatively separated from its oxidation and other degradation products, from hormone fragments and from non-hormonal contaminants. Recovery of bPTH, monitored by region-specific immunoassays, in vivo bioassay and re-chromatography on HPLC was > 93%. The detection limit of the HPLC system, using endogenous tryptophan fluorescence, was 20 ng bPTH.     

Purification of radiolabeled and native polypeptides by gel permeation high-performance liquid chromatography

Some results and observations concerning the use of protein columns are presented. The combined use of four protein columns having different fractionation ranges together with a volatile triethylamine formate buffer allowed the sieving of various polypeptides according to their molecular weights over a range of 500 to 150,000. The addition of 4 or 6 m guanidine-HCl permitted the reduction of aggregation with no sacrifice in resolution or linearity. With that denaturant, rapid separation, and molecular weight determination in the range 500–90,000 is easily accomplished. Moreover, sample recoveries as determined with radiolabeled proteins always exceeded 70% while radioimmunoassay techniques can be directly applied to the column eluate. Applications to quick identification of natural fragments of a serine protease, tonin, analysis of maturation products of pro-opiomelanocortin in an in vitro pulse experiment and finally quantitation by radioimmunoassays of pituitary peptides and elution of their ¹²⁵I-labeled derivatives are described.