Further studies on bovine serum amylase. 3. Affinity chromatography

The major bovine serum isoamylases controlled by the AmI locus have been examined by gel filtration. On Sephadex G-200 the isoamylases can be resolved into two classes. The AmI A and AmI B have apparent molecular weights of 307 000 daltons whilst the AmI C isozyme has an apparent molecular weight of 44 400 daltons. The separation of the isozymes into two classes according to their elution behaviour on Sephadex G-200 has been shown to be an affinity separation. All three AmI isozymes are eluted from a non-dextran media (BioGel A1.5m) with apparent molecular weights of 417 000 daltons. The affinity separation on Sephadex G-200 has been shown to be inhibited by the addition of 1% (w/v) maltose to the elution buffer. In the presence of 1 % (w/v) maltose all three AmI isozymes are coeluted from Sephadex G-200 with apparent molecular weights of 321000 daltons. The maltase and amylase activities of the AmI isozymes were eluted coincidentally under all the conditions studied.     

Further studies on bovine serum amylase. 2. Affinity electrophoresis

The polymorphism of bovine serum amylase, which is controlled by the Ami locus, has previously only been demonstrated by starch gel electrophoresis. The addition of maltose to starch gels has been demonstrated to inhibit any subsequent separation of the Ami isozymes by starch gel electrophoresis. When electrophoresis was conducted in a support medium in the absence of starch no polymorphic variation was detected amongst samples from animals of different Ami phenotypes. The addition of starch to agarose gels has been shown to facilitate the subsequent detection of the Ami polymorphism by agarose/starch gel electrophoresis. The electrophoretic resolution of the Ami isozymes has been demonstrated to depend upon differences in affinity for starch rather than differences in net charge. The starch gel electrophoretic separation of the Ami isozymes is. therefore, another example of affinity electrophoresis. All the Ami amylases have been shown to share a common isoelectric point of pH 3.5.     

Further studies on bovine serum amylase - the effect of gel buffer pH

This paper describes the effect of gel buffer pH on the resolution of bovine serum amylase (Amylase I) isozymes in starch gel and the consequences for the understanding of the genetics of this locus. The two main findings are: (1) the existence of a satellite isozyme E to isozyme C which at pH 7.3 has the same mobility as the B isozyme but which at pH 8.0 migrates slower than B, and (2) the finding of three alleles AmI A, AmI B and AmI C in British cattle populations previously reported as having only AmI B and AmI C .     

Further studies on bovine serum amylase--the effect of gel buffer pH.

This paper describes the effect of gel buffer pH on the resolution of bovine serum amylase (Amylase I) isozymes in starch gel and the consequences for the understanding of the genetics of this locus. The two main findings are: (1) the existence of a satellite isozyme E to isozyme C which at pH 7.3 has the same mobility as the B isozyme but which at pH 8.0 migrates slower than B, and (2) the finding of three alleles Aml A, Aml B and Aml C in British cattle populations previously reported as having only Aml B and Aml C.     

Further studies on single fibres of bovine muscles.

Young & Davey (1981) (Biochem. J. 195, 317-327) identified numbers of polymorphs of myofibrillar proteins by sodium dodecyl sulphate/polyacrylamide gel electrophoresis of single muscle fibres isolated from three bovine muscles. Fibres were classed according to the distribution of polymorphs. The study has now been extended to eight diverse bovine muscles. The previous distinction made between fast and slow fibres is valid without exception in the extended study. Within these classes, variations in myofibrillar expression are examined within and between fibres, muscles and animals. Two slow muscles are contrasted; masseter is homogeneous in fibre type, whereas diaphragm is subtly heterogeneous, possibly arising from greater physiological demands. Of the myofibrillar polymorphs, attention is concentrated on two variants of fast-muscle myosin heavy chain. Both are present in all fast and mixed muscles examined, except sternomandibularis, and each is respectively associated with certain unidentified proteins. Within a muscle the fast-muscle myosin light-chain expression is the same irrespective of the heavy-chain variant. Histochemical techniques demonstrated that the variants are respectively associated with types IIA and IIB as defined by other investigators.     

Control of proteoglycan biosynthesis. Further studies on the effect of serum on cultured bovine articular cartilage.

Proteoglycan synthesis in explant cultures of adult bovine articular cartilage is stimulated in a dose-dependent manner when the tissue is cultured in the presence of foetal-calf serum. The stimulation of proteoglycan synthesis is paralleled by a similar increase in DNA synthesis; however, when DNA synthesis is inhibited by hydroxyurea the stimulation of proteoglycan synthesis by serum remains essentially the same. The apparent half-life of the pool of proteoglycan core protein precursor was measured in freshly isolated tissue as well as in tissue cultured for 7 days in the presence and in the absence of foetal-calf serum; under all conditions the half-life was the same, suggesting that this value is independent of the net rate of proteoglycan synthesis. In the presence of actinomycin D, an inhibitor of RNA synthesis, there was a difference in the apparent half-life of the available pool of mRNA coding for proteoglycan core protein: 8.5 h for tissue maintained in the presence of serum and 3.8 h for tissue cultured in the absence of serum. It is suggested that proteoglycan synthesis is stimulated by serum factors at the level of DNA-dependent RNA synthesis. Concomitant with an increase in the rate of proteoglycan synthesis induced by the presence of serum in the culture medium, an increase in the concentrations of several glycosyltransferases involved in chondroitin sulphate synthesis was also observed.     

Studies on bovine serum amylase; evidence for two loci

Two genetically controlled polymorphic systems of amylase enzymes are described in sera from Friesian, Jersey, Guernsey, Hereford, Ayrshire and Charolais cattle. One of these Ami has been described previously and is controlled by two codominant alleles in British cattle, AmI ^B and AmI ^C. Evidence presented here suggests that it could be a gamma.amylase. The other enzyme which is described here for the first time has many of the properties of an alpha amylase. It is also polymorphic and controlled by two codominant alleles AmII ^A and AmII ^B. AmII ^A is only present in low frequency. The inheritance of both enzyme systems has been studied in 419 families.     

Affinity chromatography: purification of bovine trypsin and thrombin

Bovine trypsin has been purified by affinity chromatography on agarose beads containing covalently bound p-aminophenylguanidine, p-aminobenzamidine, or m-aminobenzamidine. Bovine thrombin was purified on a m-aminobenzamidine-agarose column containing a high concentration of the inhibitor. The values of the inhibition constant, K_i, for these inhibitors were determined for both enzymes and found to be 5–10 times poorer for thrombin than for trypsin. Only those benzamidines with low K_i values and coupled in high concentration to the agarose matrix were satisfactory for thrombin purification. Affinity-purified trypsin and thrombin were both greater than 90% active as measured by active site titration.     

Raman studies of bovine serum albumin.

The Raman Spectra of bovine serum albumin have been obtained in the solute state, in alkaline and acidic solutions, and in the gel. The reversible denaturations of bovine serum albumin solutions by heat, acid's, and alkali were studied and a new mechanism for heat denaturation has been proposed based on a continuous unfolding of the α-helices.     

Affinity chromatography of thyroxine-binding proteins from human serum

The affinity matrix prepared by the attachment of L-thyroxine (T4) to epichlorohydrine-activated Sepharose 4B biospecifically absorbs the T4-binding globulin (TBG), 25K and 80/27K proteins, immunoglobulin G (IgG) and albumin (HSA) from human normal and retroplacental sera. The absorbed protein patterns were shown to depend on the immobilized T4 concentration, pH, temperature and incubation time. The potent eluents desorbing 85-100% of the protein are 1 mM NaOH, 3 M NH4SCN, 10(-5) M T4 or 3 mM 8-anilinonaphthalene-1-sulfonic acid (ANS) for TBG; NaOH, NH4SCN, 3 mM MgCl2 or 12mM sodium cholate for 25K protein and HSA; NaOH, NH4SCN or MgCl2 for the 80/27K and 25K proteins and IgG. Moreover, T4 desorbs small amounts (6-8%) of the 80/27K and 25K proteins, while sodium cholate elutes about 6% of TBG. The eluted from T4-Sepharose 4B and further purified TBG, 25K and 80/27K proteins display different [125I]T4-binding activities within the pH range from 2 to 9 and differ by their resistance to thermal inactivation at 50-80 degrees C. Double radial immunodiffusion analysis with the use of antisera to TBG, 25K, 80/27K, HSA and IgG demonstrated that the proteins share no common antigenic determinants. It was concluded that the novel 25K and 80/27K proteins represent endogenous components of the human blood thyroid hormone-binding protein system rather than fragments or aggregates of the known T4-binding proteins.     

Affinity chromatography of trypsin and related enzymes. V. Basic studies of quantitative affinity chromatography.

A detailed study of the quantitative affinity chromatography of trypsin [EC 3.4.21.4] is reported here. Frontal chromatography using an enzyme solution of very low concentration on an affinity adsorbent gave the dissociation constant of the enzyme-immobilized ligand complex (Kd). Kd values determined under various conditions enabled us to discuss in detail the interaction of trypsin and affinity adsorbents (mainly Gly-Gly-Arg Sepharose). The pH dependence of Kd was consistent with that of the interaction of trypsin and product-type compounds. The effects of changes in temperature, ionic strength, dielectric constant, etc., were also studied. The Ki values of soluble competitive inhibitors can be determined by analysis of their effects on the elution volume of the enzyme. The values obtained were in good agreement with those obtained by kinetic analysis. The present method proved to be useful as a general procedure to investigate the interaction of a protein and a specific ligand.     

Affinity chromatography of lipoxygenases.

A number of aminohexyl agarose derivatives of unsaturated fatty acids have been prepared and evaluated as materials for the affinity chromatography of soybean and pea lipoxygenases. A practical method for a one-stage purification of soybean lipoxygenase-1, with a purification factor of 16, is described, using either linolenate or docosa-4,7,10,13,16,19-hexaenoate as ligands. Results show that alleged competitive inhibitors do not cause sharp elution from the affinity column, and that there is an increasing specificity of binding and sharpness of elution as the proportion of unsaturation in the ligand is increased. These results are discussed in terms of the relative importance of the types of bonding involved in enzyme-substrate binding.     

Affinity chromatography of the phosphatidylcholine exchange protein from bovine liver

Affinity chromatography has been used to purify the phosphatidylcholine exchange protein from bovine liver. The affinity resin consisted of 1-acyl-2-(9-carboxy)nonyl-glycero-3-phosphocholine linked to AH-Sepharose 4 B via the carboxyl group. Application of a crude exchange protein fraction to the affinity column resulted in a complete adsorption of the phosphatidylcholine exchange protein. The exchange protein eluted with a buffer containing 0.15% sodium deoxycholate. The most active fraction was 130-fold purified and accounted for 62% of the activity.     

Affinity chromatography of phosphofructokinase.

The behavior of mammalian phosphofructokinase on immobilized adenine nucleotides was investigated. Three different insolubilized ligands were compared using a pure rabbit muscle phosphofructokinase. N⁶-[(6-aminohexyl)-carbamoyl-methyl]-ATP-Sepharose bound at least 90 times more enzyme than either N⁶-(6-aminohexyl)-AMP-agarose or ATP-adipic acid hydrazide-Sepharose. The elution of phosphofructokinase from the ATP-Sepharose with various metabolites and combinations of metabolites was investigated. The enzyme is eluted specifically from N⁶-[(6-aminohexyl)-carbamoyl]-ATP-Sepharose with a mixture of 25 μm each of fructose 6-phosphate and ADP (±Mg²⁺). The enzyme is not eluted either with ATP (25 μm), fructose 1,6-diphosphate (1 mm), ADP (25 μm), fructose 6-phosphate (1 mm) alone, or with a mixture of fructose 1,6-diphosphate (25 μm) and ATP (25 μm). The recovery of bound enzyme was usually greater than 90%. A mixture of glucose 6-phosphate and ADP or a mixture of IDP and fructose 6-phosphate also elutes the enzyme, but the recovery with these eluants was only about 40%. It was concluded that the “dead-end” complex is the most effective in the elution. Using this method, phosphofructokinase has been prepared in an essentially homogeneous form from muscle and brain of rabbit and rat. The overall isolation procedure involves a high speed centrifugation of crude extracts which sediments phosphofructokinase as a pellet, followed with adsorption on N⁶-[(6-aminohexyl)-carbamoyl-methyl]-ATP-Sepharose and specific elution with the mixture of fructose 6-phosphate and ADP.