Isolation of phosphoglycerate kinases by affinity chromatography

A variety of Sepharose derivatives containing DL-O-phosphorylserine or adenosine nucleotides with different points of attachment, has been synthesized and tested for affinity to phosphoglycerate kinase. The most effective gels contained periodate-oxidized ATP or ADP bound via the ribose by hydrazone formation to adipoyl-dihydrazo-Sepharose. The effect of pH, magnesium and buffer ions on the binding capacity of the ATP derivative of Sepharose has been examined. Optimal elution of phosphoglycerate kinase was investigated using different combinations of adenosine nucleotides, 3-phosphogylcerate and magnesium ions. A method is presented giving conditions for the purification of phosphoglycerate kinase from different sources (spinach, human erythrocytes, human, rabbit and trout muscle). It includes extract preparation, affinity chromatography and gel filtration. The method is greatly superior to known isolation procedures by virtue of its technical simplicity, excellent yield (85-100%) and reproducability. The capacity of the ATP-ribosyl-adipoyl-dihydrazo-Sepharose was 5 mg phosphoglycerate kinase per 1 g of matrix. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate indicated that the final products are homogeneous. The phosphoglycerate kinases from different sources appear to have the same affinity for this ATP derivative of Sepharose, the same molecular weight and the same specific activity.     

Further properties and possible mechanism of action of adenosine 5′-triphosphate–d-glucose 6-phosphotransferase from rat liver

1. Magnesium ions are the most effective bivalent ions in the glucokinase reaction. 2. The molecular weight of rat hepatic glucokinase is 48000-49000 as assessed by gel filtration on Sephadex G-100. 3. Anomalous kinetic behaviour at low glucose concentrations appears to be due to the formation during the purification procedure of fragments possessing modified catalytic properties, but is unlikely to be of physiological significance. 4. Extension of previous studies (Parry & Walker, 1966) suggests that glucokinase catalyses a reaction of the random Bi Bi type similar to that of yeast hexokinase. 5. The inhibitory effects of various thiol reagents suggest that a thiol group may be involved at or near the binding site of the acceptor molecule.     

Optimisation of conditions for the affinity chromatography of human enterokinase on immobilised p-aminobenzamidine. Improvement of the preparative procedure by inclusion of negative affinity chromatography with glycylglycyl-aniline.

The affinity chromatography of human enterokinase using p-aminobenzamidine as the ligand [Grant, D.A.W. & Hermon-Taylor, J. (1976) Biochem. J. 155, 243-254] has been reassessed and the optimal conditions for the synthesis and operation of the derivatised gel defined. Satisfactory adsorbants were only produced using high concentrations of both CNBr and spacer-arm in the initial coupling slurry. Under these conditions it seemed likely that the majority of the ligand in a sterically favourable position to bind enterokinase was on the external surface of the bead. Trypsin binding to the adsorbant was not so critically dependent on the synthetic conditions and correlated closely with the degree of substitution. Dilution of the adsorbant with unlabelled Sepharose 4B indicated that there was more than one binding site per enterokinase molecule. The highest affinity was presumably for the active site, with adsorption supported by secondary interactions with spacer-arm or gel matrix not necessarily on the same bead. Maximal resolution was obtained by prolonged washing of the gel after loading; two populations of intestinal aminopeptidase were identified. Substitution of aniline for p-aminobenzamidine abolished specific enterokinase adsorption and improved the purification procedure by further removal of onon-specifically adsorbed contaminants.     

Studies on the use of sepharose-N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucopyranose for the large-scale purification of hepatic glucokinase.

The synthesis of N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucose is described and it was shown to be a competitive inhibitor (Ki, 0.75 mM) with respect to glucose of rat hepatic glucokinase (EC 2.7.1.2). After attachment to CNBr-activated Sepharose 4B, this derivative was able to remove glucokinase quantitatively from crude liver extracts and release it when the columns were developed with glucose, glucosamine, N-acetyl-glucosamine or KC1. Repeated exposure of the columns to liver extracts led to rapid loss in their effectiveness as affinity matrices because proteins other than glucokinase are bound to the columns. The nature of such protein binding and methods for the rejuvenation of "used" columns are discussed along with the effect of the mode of preparation of the Sepharose-ligand conjugate and the concentration of bound ligand on the purification of glucokinase. Glucose 6-phosphate dehydrogenase is cited as an example of both non-specific protein binding to the affinity column and of the importance of the control of ligand concentration in removing such non-specifically bound proteins. Some guidelines emerged that should be generally applicable to other systems, particularly those which involve affinity chromatography of enzymes that are present in tissue extracts in very low amounts and possess only a relatively low association constant for the immobilized ligand.     

Hexokinase isoenzymes of RIN-m5F insulinoma cells. Expression of glucokinase gene in insulin-producing cells.

We have analysed the pattern of expression of the hexokinase isoenzyme group in RIN-m5F insulinoma cells. Three hexokinase forms were resolved by DEAE-cellulose chromatography. The most abundant isoenzyme co-eluted with hexokinase type II from rat adipose tissue and displayed a Km for glucose of 0.15 mM, similar to the adipose-tissue enzyme. Hexokinase type II was in large part associated with a particulate subcellular fraction in RIN-m5F cells. The two other hexokinases separated by ion-exchange chromatography were an enzyme similar to hexokinase type I from brain and glucokinase (or hexokinase type IV). The latter isoenzyme was identified as the liver-type glucokinase by the following properties: co-elution with hepatic glucokinase from DEAE-cellulose and DEAE-Sephadex; sigmoid saturation kinetics with glucose with half-maximal velocity at 5.6 mM and Hill coefficient (h) of 1.54; suppression of enzyme activity by antibodies raised against rat liver glucokinase; apparent Mr of 56,500 and pI of 5.6, as shown by immunoblotting after one- and two-dimensional gel electrophoresis; peptide map identical with that of hepatic glucokinase after proteolysis with chymotrypsin and papain. These data indicate that the gene coding for hepatic glucokinase is expressed in RIN-m5F cells, a finding consistent with indirect evidence for the presence of glucokinase in the beta-cell of the islet of Langerhans. On the other hand, the overall pattern of hexokinases is distinctly different in RIN-m5F cells and islets of Langerhans, since hexokinase type II appears to be lacking in islets. Alteration in hexokinase expression after tumoral transformation has been reported in other systems.     

The purification in high yield and characterization of rat hepatic glucokinase.

A new improved procedure for the purification of rat hepatic glucokinase (ATP-d-glucose 6-phosphotransferase, EC 2.7.1.2) is given. A key step is affinity chromatography on Sepharose-N-(6-aminohexanoyl)-2-amino-2-deoxy-d-glucopyranose. A homogeneous enzyme, specific activity 150 units/mg of protein, is obtained in about 40% yield. The molecular weight of the pure enzyme was determined by several procedures. In particular, sedimentation-equilibrium studies under a variety of conditions indicate a molecular weight of 48000 and no evidence for dimerization; reports in the literature of other values are discussed in the light of this evidence on the pure enzyme. The amino acid composition suggests that hepatic glucokinase is closely related to rat brain hexokinase and also the wheat "light" hexokinases.     

Is rat skeletal muscle hexokinase an allosteric enzyme?

A recent report by M. Gregoriou, I. P. Trayer, and A. Cornish-Bowden (1986, Eur. J. Biochem. 161, 171-176) showed that the mechanism for rat skeletal muscle hexokinase contains two allosteric sites: one for ATP and one for glucose 6-phosphate. In this report, we show that the allosteric mechanism is at variance with a large amount of kinetic data for the skeletal muscle hexokinase reaction in the literature. In addition, the allosteric mechanism conflicts with isotope exchange at chemical equilibrium data reported by M. Gregoriou, I. P. Trayer, and A. Cornish-Bowden (1983, Eur. J. Biochem. 134, 283-288).     

Preparation of adenosine nucleotide derivatives suitable for affinity chromatography

Methods of synthesizing a series of chemically-defined AMP, ADP, ATP, adenylyl imidodiphosphate and pyrophosphate derivatives suitable for affinity chromatography are extensively described. Each derivative has a single primary amino group at the end of a hexamethylene ;spacer' chain for attachment to CNBr-activated agarose. The synthesis of the derivative where the ;spacer' arm is attached directly to the 8 position of the adenine ring to produce 8-(6-aminohexyl)amino-AMP involves the direct bromination of AMP in the 8 position followed by displacement of the halogen by 1,6-diaminohexane. This monophosphate derivative can then be converted into the corresponding di- or triphosphate forms by direct phosphate condensation with carbonyl di-imidazole. A second series of adenosine phosphate derivatives with the phosphate moieties unsubstituted has been similarly prepared from N(6)-(6-aminohexyl)-AMP (Guilford et al., 1972). A third type of ligand has been synthesized by condensing the phosphoryl imidazolide of AMP with 6-aminohex-1-yl phosphate. This compound, P(1)-(6-aminohex-1-yl) P(2)-(5'-adenosyl) pyrophosphate, has an unsubstituted adenine ring. The synthesis of a fourth type of ligand, 6-aminohex-1-yl pyrophosphate, was done by heating 6-aminohexan-1-ol with crystalline pyrophosphoric acid under reduced pressure. The structures of the synthesized compounds were confirmed by chemical, electrophoretic and chromatographic methods and by u.v. spectrometry. The general applicability of the synthetic methods used is discussed in relation to the preparation of other affinity adsorbents. Examples are given where these derivatives have been successful in reversibly binding dehydrogenases, kinases and myosin and its proteolytic subfragments. The partial purification of rat liver glucokinase on an ADP derivative is shown.     

Preparation of a stable folate-sepharose complex for affinity chromatography.

A stable folic acid affinity gel has been developed for the purification of nanograms of protein that bind folic acid or its derivatives. The affinity gel was prepared by first coupling folic acid covalently to bovine serum albumin, followed by covalent coupling of the albumin to p-benzoquinone-activated Sepharose. After the albumin-folic acid complex was formed, it was treated with charcoal to remove ionically bound folate which would otherwise elute from the gel and decrease the recovery of the binding protein. The p-benzoquinone activation resulted in a more stable binding of the albumin to the Sepharose.     

Inositol 1,4,5-trisphosphate affinity chromatography

Inositol 1,4,5-trisphosphate (IP3) affinity columns were made by coupling IP3 analogs to a supporting matrix. Sepharose 4B. IP3 5-phosphatase activity. IP3 3-kinase activity and IP3 binding activity from rat brain were absorbed on the IP3 columns. and were eluted by increasing KC1 concentration. This purification procedure increased the specific activities of these parameters 5-200-fold. Thus Sepharose 4B immobilized IP3 analogs can specifically interact with IP3-binding proteins, demonstrating that IP3 affinity columns are a good method for purifying such proteins. Furthermore, our results suggest that IP3 analogs can be linked to other molecules to make useful derivatives without loss of their biological activities.     

New affinity adsorbents containing deoxycytidine, deoxyadenosine, or deoxyguanosine and their interactions with deoxynucleoside-metabolizing enzymes

3'-(4-Aminophenyl phosphate) derivatives of deoxycytidine (dCyd), deoxyadenosine (dAdo), and deoxyguanosine ( dGuo ) were synthesized. The inhibitory effects of these compounds on mammalian and bacterial deoxynucleoside kinases and several other deoxynucleoside-metabolizing enzymes were examined. The same derivatives were coupled to carboxyl-terminal Sepharose CL-6B (3-8 mumol of ligand/mL of gel), and each of the resulting affinity adsorbents was tested with various partially purified enzymes. Reasonable correlation between the inhibitory effect of a soluble deoxynucleoside 3'-phosphate diester and affinity of the corresponding Sepharose adsorbent for the enzyme was observed. Among the three dCyd kinases examined, only the bovine mitochondrial enzyme was adsorbed onto the dCyd-Sepharose column and eluted biospecifically by 1 mM dCyd (1400-fold purification). Its Ki toward the dCyd derivative was relatively low (1.1 mM), whereas no measurable inhibition was seen with mammalian cytosol or bacterial enzymes that did not stick to the column. The Ki of the dAdo derivative toward three dAdo kinases was more than 5 mM in each case, and none of these were retained by dAdo-Sepharose. Among the other dAdo-metabolizing enzymes examined, nucleoside phosphotransferase from barley (Ki = 1.2 mM) was adsorbed to dAdo-Sepharose at pH 5.0 and was biospecifically eluted with dAdo or AMP after suppressing ionic binding by adjusting the pH to 6.0 (480-fold purification to homogeneity). Mammalian mitochondrial dGuo kinase (beef liver) showed the lowest Ki (0.16 mM) among the enzymes tested and was biospecifically purified with dGuo -Sepharose (2800-fold purification).(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of the lymphocytosis promoting factor-haemagglutinin of Bordetella pertussis by affinity chromatography.

The lymphocytosis promoting factor-haemagglutinin of Bordetella pertussis was isolated from solutions obtained after cell disintegration by a novel affinity chromatographic method using an adsorbent composed of human haptoglobin covalently attached to a Sepharose 4B matrix. The haemagglutinin was bound to the adsorbent at pH 6.5 and eluted by a stepwise change to a pH 10 buffer. A 300--600-fold purification of the haemagglutinin was achieved by this one-step process. The chemical and biological properties of the haemagglutinin isolated by affinity chromatography were found to be similar to those of the protein isolated by other workers from culture supernatants. The affinity chromatographic method was found to be specific for the purification of the lymphocytosis promoting factor-haemagglutinin and no purification of the fimbrial-haemagglutinin of Bordetella pertussis was achieved by the method.     

Synthesis of a naphthylvinylpyridine derivative and its use for affinity chromatography of choline acetyltransferase

N-(10-carboxy)decamethylene-4(1-naphthylvinyl)pyridinium chloride, a derivative of the choline acetyltransferase (CAT) inhibitor naphthylvinylpyridine (NVP) was synthesized and used as a ligand for affinity chromatography of choline acetyltransferase. The preparation of this inhibitor included the quaternization of naphthylvinylpyridine with 11-Br-undecanoic acid methyl ester to obtain N-(10-carbomethoxy)decamethylene-4-(1-naphthylvinyl)pyridinium bromide, followed by hydrolysis to free the carboxylic group. This inhibitor (C11-NVP+) had a potency comparable to that of N-methyl-4(1-naphthylvinyl) pyridinium iodide (C1-NVP+) which is the most potent derivative of NVP but which lacks a functional group for conjugation to Sepharose. The C11-NVP+ was then bound through the carboxylic group to aminoalkyl Sepharose by a carbodiimide promoted condensation reaction. Interaction of CAT with the inhibitor retarded its elution from a column of Sepharose-C11-NVP+ and permitted the purification of the enzyme to electrophoretic homogeneity starting from a preparation in which CAT represented about 20% of the total proteins. Conventional procedures of protein purification had previously been unsuccessful in isolating the enzyme in pure form. Inhibition studies showed that CAT could exhibit either a "high" or a "low" sensitivity to inhibition by naphthylvinylpyridine and its derivatives (I50 with C1-NVP+ = 0.57 microM or 5.2 microM). A direct relationship existed between the sensitivity of CAT to these inhibitors and the retention of the enzyme by the affinity column.     

Purification and physical characterization of glutathione S-transferase K. Differential use of S-hexylglutathione and glutathione affinity matrices to isolate a novel glutathione S-transferase from rat liver.

A novel hepatic enzyme, glutathione S-transferase K, is described that, unlike previously characterized transferases, possesses little affinity for S-hexylglutathione-Sepharose 6B but can be isolated because it binds to a glutathione affinity matrix. A purification scheme for this new enzyme was devised, with the use of DEAE-cellulose, S-hexylglutathione-Sepharose 6B, glutathione-Sepharose 6B and hydroxyapatite chromatography. The final hydroxyapatite step results in the elution of three chromatographically interconvertible forms, K1, K2 and K3. The purified protein has an isoelectric point of 6.1 and comprises subunits that are designated Yk (Mr 25,000); during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it migrates marginally faster than the Ya subunit but slower than the pulmonary Yf monomer (Mr 24,500). Transferase K displays catalytic, immunochemical and physical properties that are distinct from those of other liver transferases. Tryptic peptide maps suggest that transferase K is a homodimer, or comprises closely homologous subunits. The tryptic fingerprints also demonstrate that, although transferase K is structurally separate from previously described hepatic forms, a limited sequence homology exists between the Yk, Ya and Yc polypeptides. These structural data are in accord with the immunochemical results presented in the accompanying paper [Hayes & Mantle (1986) Biochem. J. 233, 779-788].     

Dihydrotestosterone derivatives: relative binding affinity versus affinity purification.

Mono esters of a homologous series of diacids of dihydrotestosterone were synthesized and converted to the corresponding n-butyl amides. The relative binding affinities of these amides to androgen receptor were compared with the degree of purification of rat prostate androgen receptor by affinity columns prepared by linking the steroidal acid to amino Sepharose. There was good correlation between binding of the amide model to androgen receptor and the extent of purification by the affinity resin.     

Preparation and properties of an affinity support for purification of cyclic AMP receptor protein from Escherichia coli.

Reaction of cyclic AMP with 1,1'-carbonyldiimidazole produces an intermediate which reacts with primary amines to provide a stable 2'-O-carbamyl derivative. This chemistry has been used to tether cyclic AMP to a Sepharose gel. The resulting affinity support has been used to effect a simple, nondenaturing purification of cyclic AMP receptor protein from crude cell extracts.     

Phosphorylation of D-glucosamine by rat liver glucokinase.

D-Glucosamine was found to be phosphorylated by a rat liver extract in the presence of a high concentration of glucose, which was formerly believed to be a strong competitive inhibitor of this reaction. Results suggested that glucosamine may be phosphorylated by high Km hexokinase, i.e. glucokinase [EC 2.7.1.2]. The enzyme involved was separated from specific N-acetyl-D-glucosamine kinase [EC 2.7.1.59]. The phosphorylation was not inhibited by a physiological level of glucose or glucose 6-phosphate, which strongly inhibited low Km hexokinase. The apparent Km of glucokinase for glucosamine was estimated as 8 mM, which is ten times that of low Km hexokinase.     

Purification of cobalt-activated acylase by affinity chromatography

alpha-Hydroxyisocaproyltyrosine (HyIc-Tyr-OH), a potent competitive inhibitor of the cobalt-activated acylase form 2, was synthesized. Its derivative, alpha-aminopentyl-HyIc-Tyr-OEt was coupled to cyanogen bromide-activated Sepharose 4B and was used for about 100-fold purification of the acylase from human liver by affinity chromatography. The preparation obtained did not show aminoacylase, aspartyl acylase or alanylarylamidase activities. The same chromatographic method was also applied to isolate form 2 of the serum acylase from patients with viral hepatitis and guinea pig placenta.     

Purification of acetyllactosamine-specific tomato lectin by erythroglycan-sepharose affinity chromatography

Tomato lectin is specific for oligomers of poly-N-acetyllactosamine containing 3 repeating Gal(beta 1-4)GlcNAc (beta 1-3)-disaccharides. As such it is highly useful for purifying oligosaccharides or glycopeptides with poly-N-acetyllactosamine character. We have found the lectin very useful as an affinity reagent for isolating glycoproteins or glycoprotein domains having poly-N-acetyllactosamine glycosylation. Conventional preparation of tomato lectin by ovomucoid-Sepharose affinity chromatography was found to be unsatisfactory due to instability of column and bleeding of ovomucoid into eluents requiring the necessity for additional purification steps following affinity chromatography. We prepared a column of human erythrocyte band 3 carbohydrate glycopeptide (erythroglycan) attached to Sepharose as an affinity matrix. The purification of tomato lectin to homogeneity in one step on this column matrix is described in this report.     

Easy assembly of ligands for glycosidase affinity chromatography.

An improved, high-yield synthesis of the corresponding N-carboxypentyl derivatives of three iminoalditol glycosidase inhibitors has been developed for affinity chromatography enzyme purification. Reductive amination of 1-deoxynojirimycin (or its D-manno or D-galacto analogues) with methyl 5-formylvalerate and NaBH3CN at neutral pH afforted an aminoester which upon hydrolysis with aqueous 5% HCl gave the desired aminoacid in 97% overall yield. These amino acids could then be covalently attached using water-soluble carbodi-imide to 6-aminohexyl Sepharose 4B.