Isolation and characterization of the 165 000 dalton protein component of the M-line of rabbit skeletal muscle and its interaction with creatine kinase

The M-line protein component of molecular weight 165 000 was isolated and purified from rabbit skeletal muscle using ion exchange chromatography. Gel electrophoresis, in the presence and absence of sodium dodecyl sulfate, revealed the protein to be homogeneous. Sodium dodecyl sulfate gel electrophoresis and low speed sedimentation equilibrium studies in 0.5 M KCl, 50 mM potassium phosphate gave a molecular weight of 165 000 suggesting the protein to be made up of a single polypeptide chain. Circular dichroism spectra revealed the presence of two negative dichroic bands located at 216 and 208 nm, indicative of the presence of some beta-structure. Ellipticity values at these two wavelengths were --6500 +/- 400 and --7500 +/- 400 deg . cm2 . dmol-1, respectively. Addition of 165 000 component lowered the enzymatic activity of creatine kinase M-line protein and the nature of the inhibition was found to be a competitive one. When the protein was mixed with creatine kinase in a 1 : 1 mole ratio in a medium consisting of 0.2 M KCl, 25 mM Tris, 1 mM dithiothreitol (pH 8.0), low speed sedimentation equilibrium studies gave a molecular weight of 260 000 +/- 10 000 for the complex, indicative of an interaction of the two components of the M-line.     

Purification and some properties of cytoplasmic aspartate aminotransferase from sheep liver

1. A procedure for the purification of the cytoplasmic isoenzyme of aspartate aminotransferase from sheep liver is described. 2. The purified isoenzyme shows a single component in the ultracentrifuge at pH7.6 and forms a single protein band on agar-gel electrophoresis at pH6.3 or 8.6, as well as when stained for protein or activity after polyacrylamide-gel or cellulose acetate electrophoresis at pH8.8. 3. Immunoelectrophoresis on agar gel yields only one precipitin arc associated with the protein band, with rabbit antiserum to the purified isoenzyme. By immunodiffusion, cross-reaction was detected between the cytoplasmic isoenzymes from sheep liver and pig heart, but not between the cytoplasmic and mitochondrial sheep liver isoenzymes. 4. The s(20,w) of the enzyme is 5.69S and the molecular weight determined by sedimentation equilibrium is 88900; 19313 molecules of oxaloacetate were formed/min per molecule of enzyme at pH7.4 and 25 degrees C. 5. The amino acid composition of the isoenzyme is presented. It has about 790 residues per molecule. 6. The holoenzyme has a maximum of absorption at 362nm at pH7.6 and 25 degrees C. 7. A value of 2.1 was found for the coenzyme/enzyme molar ratio. 8. The purified enzyme revealed two bands of activity on polyacrylamide-gel electrophoresis at pH7.4 and an extra, faster, band in some circumstances. These bands occurred even when dithiothreitol was present throughout the isolation procedure. 9. Three main bands were obtained by electrofocusing on polyacrylamide plates with pI values 5.75, 5.56 and 5.35. 10. Structural similarities with cytoplasmic isoenzymes from other organs are discussed.     

IUB commission of editors of biochemical journals the citation of bibliographic references in biochemical journals recommendations (1971)

A pentose-rich acidic glycoprotein was isolated from protease digested bovine vitreous humor by fractionation on an AG1-X2 column using NaCl solution gradient.The material eluted at 0.35 M NaCl (glycoprotein) was electrophoretically heterogeneous at pH 8.6 after partial purification on Sephadex G-25. Gel filtration on G-100 resolved the glycoprotein into two fractions. These fractions differ in molecular weight; mol. wt approx. 95 000 material consisted of two components on electrophoresis and mol. wt approx. 28 000 material showed only a single component on electrophoresis. The lower molecular weight component was re-chromatographed on Sephadex G-100 yielding a single orcinol positive component which gave a homogeneous band on gel electrophoresis.Quantitative analysis of this material gave 30% protein, 7.0% pentose, 18.7% glucosamine, 9.2% galactosamine, 10.9% hexuronic acid and 16.1% hexose.Treatment with 0.5 M NaOH at 20°C for 24 h resulted in a 50% decrease in the threonine content suggesting the possible involvement of this amino acid in the protein-carbohydrate linkage group.Paper chromatography of the fraction hydrolysate demonstrated the presence of glucurone, xylose, arabinose, glucose and galactose.     

Purification and properties of NADP-dependent isocitrate dehydrogenase from the bovine adrenal cortex

NADP-dependent isocitrate dehydrogenase (EC 1.1.42) was isolated and 430 times purified from the hyaloplasm fraction of bull adrenal cortex using fractionation by ammonium sulphate and acetone, heat treatment, chromatography on DEAE-Sephadex A-50, gel-filtration on Sephadex G-200 and affinity chromatography on 2',5'-ADP-sepharose 4B. The specific activity of homogeneous enzyme is 60 units per 1 mg of protein at 30 degrees C, yield--34%, pH optimum--8.0, molecular weight, determined by gel filtration on Sephadex G-200, is 96 kDa. The preparation electrophoresis in PAAG in the presence of DS-Na reveals one protein fraction with the mobility corresponding to that of protein having molecular weight of 46 kDa. The data obtained evidence for a dimer structure of the isocitrate dehydrogenase molecule from bull adrenals.     

Purification and characterization of human factor II

Human plasma Factor II has been purified approximately 800-fold by a combination of barium citrate adsorption, ion-exchange chromatography and preparative polyacrylamide gel electrophoresis. The procedure is relatively simple and results in excellent yields of purified Factor II essentially free of Factor X activity. The purified factor behaved as a single component by analytical polyacrylamide gel disc electrophoresis at pH 8.9. No Factor V, VII or IX activity was detected in the purified Factor II. Its molecular weight was 7200±3000 as determined by analytical ultracentrifugation, electrophoresis in the presence of sodium dodecyl sulfate and gel filtration on Bio-Gel P-200. An apparent molecular weight of 90 000–100 000 was observed on calibrated columns of Sephadex G-100, G-150, and G-200. The specific activity of human factor II was approximately 1300 N.I.H. units/mg as determined by the two-stage assay and 7 Ortho units/mg by the one stage assay. The purified protein contained by weight 2.8% neutral hexose, 2.3% sialic acids and 3.1% hexosamines.     

Production of specific antisera and monoclonal antibodies to choline acetyltransferase: characterization and use for identification of cholinergic neurons.

Choline acetyltransferase (ChAT) has been purified from pig brain to greater than 95% homogeneity (purification factor: 646 000, specific activity of the purified enzyme: 128 mumol acetylcholine formed/min/mg). Gel electrophoresis of the purified enzyme in the presence of sodium dodecylsulphate and beta-mercaptoethanol revealed a single protein band at 68 000 daltons. Immunoprecipitation and double immunodiffusion tests showed that antisera raised against this protein specifically recognize ChAT. A monoclonal antibody prepared against the enzyme specifically binds a protein from crude pig brain supernatants which has a mol. wt. of 68 000 and a specific activity of 153 mumol/min/mg. This antibody shows no species cross-reactivity. The specificity of the immunohistochemical localization of ChAT has been established by comparing the labeling of pig retina using the antiserum with that obtained using the monoclonal antibody. Both probes specifically identify the same retinal structures: labeled cell bodies are found in the inner nuclear layer and the ganglion cell layer, while a double band is stained in the inner plexiform layer. In rat spinal cord, the antiserum labels the motoneurons and the preganglionic sympathetic neurons, located in the intermedio-lateral nucleus, the intercalated region, and the central autonomic area.     

Heterogeneity of streptococcus group A cell wall protein components

Cell wall surface proteins of group A streptococcus (M 29) were isolated by mild chemical extraction with 1 M hydroxylamine pH 6.0 (37 degrees C). The proteins were purified by ammonium sulfate fractionation, gel filtration on Sephadex G-150 and ion-exchange chromatography on DEAE-Trisacryl M. Using two independent methods (disc electrophoresis in 7.5% PAAG pH 8.9 and high pressure gel filtration), it was shown that after chromatography on Sephadex G-150 the original protein fraction contains up to 8 protein components, while SDS-PAAG electrophoresis performed according to Laemmli revealed up to 25 protein components in the same fraction. During SDS-PAAG electrophoresis six protein fractions performed after ion-exchange chromatography were resolved into 40 protein components whose molecular masses vary from 13 to 80 kDa. Possible reasons for the heterogeneity of surface proteins of group A streptococcus cell wall are discussed.     

Guanidoacetate methyltransferase. Purification and molecular properties.

Guanidoacetate methyltransferase has been purified about 140-fold from pig liver. Polyacrylamide gel electrophoresis of the purified enzyme showed four protein bands, each of which is associated with guanidoacetate methyltransferase activity. During gel electrophoresis at pH 3 in 8 M urea, guanidoacetate methyltransferase migrated as a single component. The molecular weight of the purified guanidoacetate methyltransferase was estimated to be 31,000 by sodium dodecyl sulfate-gel electrophoresis, which also showed only one protein component with guanidoacetate methyltransferase activity. This molecular weight is in agreement with that estimated by Sephadex G-75 chromatography. Guanidoacetate methyltransferase is inhibited by adenosylhomocysteine, 3-deazaadenosylhomocysteine, and sinefungin with Ki values of 16 microM, 39 microM, and 18 microM, respectively.     

Water-soluble acetylcholine receptor from Torpedo californica. Solubilization, purification and characterization

The nicotinic acetylcholine receptor from electrogenic tissue of Torpedo californica was solubilized by tryptic digestion of membrane fragments obtained from autolysed tissue, without use of detergent. The water-soluble acetylcholine receptor was purified by affinity chromatography on a cobra-toxin-Sepharose resin. The purified receptor bound 4000-6000 pmol per mg protein of alpha-[125I]bungarotoxin, and toxin-binding was specifically inhibited by cholinergic ligands. Gel filtration revealed a single molecular species of Stokes radius 125 +/- 10 A and on sucrose gradient centrifugation one major peak was observed of 20-22 S. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and beta-mercaptoethanol revealed two major polypeptides of mol. wt. 30 000 and 48 000.     

Experimental allergic aspermatogenic orchitis. II. Some chemical properties of the AP1 protein of the sperm acrosome.

The AP1 protein, a unique aspermatogenic protein localized in the sperm acrosome, exists as a single polypeptide chain of 136 amino acids, as shown by a single band on gel electrophoresis in sodium dodecyl sulfate and the recovery of the expected 21 to 22 tryptic peptides on peptide mapping. The AP1 protein appears to exist in a compact, highly stable conformation, as shown by its resistance to trypsin hydrolysis. Its aspermatogenic acitivity is not affected by trypsin treatment, by heating at 99 degrees C for 1 h, by 8 M urea, or by acid conditions. After reduction and alkylation, however, the molecule appears to open up, since it becomes hydrolyzable by trypsin and migrates more slowly on gel electrophoresis at pH 2.7 and 8.6. After alkylation, the AP1 protein still migrates as a single band at pH 2.7. The AP1 protein shows microheterogeneity near its isolectric point at pH 8.6; each of five bands shows the same amino acid analysis. Aggregation was not observed following treatment with dimethylsuberimidate. The molecular weight of 15 000, obtained from gel electrophoresis consists of 136 amino acids with a relatively high content of proline, half cystine, glycine, histidine and tryptophan. No galactose, mannose, fucose, glucose, or hexosamines were found; the AP1 protein is thus not a glycoprotein.     

Immunological evidence that the nonhormone binding component of avian steroid receptors exists in a wide range of tissues and species

A monoclonal antibody to a fungal protein has been used to demonstrate the presence of the nonhormone binding component of molybdate-stabilized steroid receptors in a variety of vertebrate tissues. We recently identified a steroid receptor in the aquatic fungus Achlya ambisexualis where sexual morphogenesis of the male is directed by the steroid antheridiol. This receptor resembles receptors of higher organisms in exhibiting an 8S, molybdate-stabilized form. In the chick oviduct, a 90 000 molecular weight protein has previously been shown to be associated with the molybdate-stabilized complex of the progesterone receptor. We have isolated a similar protein of molecular weight about 88 000 from A. ambisexualis and have obtained a hybridomal-derived monoclonal antibody directed against it. This mouse anti-Achlya immunoglobulin G1 (IgG1) cross-reacts with the 90 000 molecular weight protein in chick oviduct cytosol and was used to detect analogous 90 000 molecular weight proteins in mammalian tissues. Tissue cytosols were incubated with antibody, and the complexes were isolated onto protein A-Sepharose. The resin-bound proteins were then analyzed by gel electrophoresis. This procedure revealed the presence of 90 000 molecular weight proteins in several mammalian tissues including rat liver, mouse liver and uterus, pig ovarian granulosa cells, human endometrium, and HeLa cells. These results demonstrate that the 90 000 molecular weight protein is not peculiar to the chick oviduct but is present in several different tissues from a variety of animals. This antibody should be a useful probe for further studies on the biological role of these proteins.     

Characteristics of subunit composition of H+-ATPase from the anaerobic bacterium Lactobacillus casei

In the presence of Mg2+ or Ca2+ the membranes of the anaerobic glycolytic bacterium Lactobacillus casei hydrolyze 0.1-0.2 mumole ATP/min/mg of protein with a pH optimum 6.4. This activity is inhibited by N,N'-dicyclohexylcarbodiimide and is insensitive to oligomycin, ouabain, vanadate and hydroxylamine. A soluble ATPase was isolated and purified from L. casei membranes. The specific activity of this ATPase is 3.0-4.0 mumole ATP/min/mg of protein. The enzyme homogeneity was established by analytical polyacrylamide gel disc electrophoresis and by analytical centrifugation (S20, omega = 12 +/- 0,5). The molecular weight of the enzyme is 270 000. Polyacrylamide gel electrophoresis of ATPase denaturated by 1% SDS and 8 M urea in the presence of SDS revealed one type of subunits with Mr = 43 000. These subunits could not be separated by isoelectrofocusing in polyacrylamide gel in the presence of 8 M urea and migrated as a single peptide with pI at 4.2. The experimental results suggest that the soluble ATPase from L. casei consists of six identical subunits with Mr of 43 000.     

The stimulus-secretion coupling of glucose-induced insulin release. Environmental influences on L-glutamine oxidation in pancreatic islets.

Rat ovarian luteinizing hormone/human choriogonadotropin binding sites were labelled with 125I-choriogonadotropin in vivo, and the resulting 125I-choriogonadotropin-receptor complexes were solubilized by Triton X-100 and purified by use of antibodies to choriogonadotropin immobilized to agarose. The purified 125I-choriogonadotropin-receptor complex was treated with glutaraldehyde to crosslink radiolabelled hormone to the receptor. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the crosslinked product revealed a labelled Mr 130 000 major band in addition to the hormone and its alpha-subunit, indicating that a single receptor component was linked to the hormone. Unoccupied binding sites for luteinizing hormone were also solubilized by Triton X-100 from pseudopregnant rat ovaries, and attached to choriogonadotropin-agarose. The agarose gel was washed, and eluted with 0.1 M-sodium acetate, pH 4. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the pH 4 eluate revealed an Mr 90 000 major band which was abolished when ovaries presaturated with choriogonadotropin were used as starting material. These observations suggest that the hormone-binding component of the luteinizing hormone receptor is a polypeptide of Mr 90 000. This polypeptide was isolated and labelled with Na 125I. The labelled polypeptide showed a single band on sucrose density gradient centrifugation and on gel filtration on agarose.     

A cyclic adenosine 3',5'-monophosphate-dependent histone kinase from pig brain. Purification and some properties of the enzyme.

A cyclic adenosine 3',5'-monophosphate-dependent histone kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was isolated from pig brain. The enzyme has been purified 1140-fold; it is homogeneous on polyacrylamide gel electrophoresis and gel filtration. The estimated molecular weight of the enzyme is 120 000. Histone kinase dissociates into a catalytic subunit and a regulatory one (molecular weights 40 000 and 90 000, respectively). The catalytic subunit has been obtained in homogeneous state as evidenced by sodium dodecylsulphate-polyacrylamide gel electrophoresis. At all purification steps, enzymatic activity is stimulated 5-fold by cyclic AMP. An apparent Km value for cyclic AMP is about 3.3 - 10- minus 7 M. In the presence of cyclic AMP(5 - 10- minus 6 M), the Km value for ATP and F1 histone were 1.2 - 10- minus five and 3 - 10- minus 5 M, respectively. Optimum pH value for histone kinase is 6.5, its isoelectric point is situated at pH 4.6. The purified enzyme displays high specificity for the lysine-rich and moderately lysine-rich histones F1, F2a2 and F2b. Arginine-rich histones and other known protein substrates for cyclic AMP-dependent protein kinases (casein, Escherichia coli RNA polymerase, etc.) are extremely poor substrates for this enzyme.     

Reconstitution into liposomes of membrane proteins involved in ribosome binding on rough endoplasmic reticulum. Ribosome-binding capacity.

Platelet-derived growth factor was purified from fresh platelets by a large-scale procedure not involving the use of SDS (sodium dodecyl sulphate). The product, 0.5 mg of platelet-derived growth factor, obtained from about 3 x 10(13) platelets migrated as a single component in analytical gel electrophoresis in the presence of SDS and showed no inhomogeneity on sedimentation-equilibrium analysis in the ultracentrifuge. It had a high specific activity, 2 ng of platelet-derived growth factor/ml (70pM) being equivalent to 1% (v/v) human serum in an assay for multiplication-stimulating activity. Amino acid analysis revealed that platelet-derived growth factor contains all the common amino acids, except tryptophan, but no hexosamine. The molecular weight of platelet-derived growth factor, as determined by sedimentation-equilibrium analysis, was about 33 000. A similar value was obtained by gel electrophoresis in SDS under non-reducing conditions. In the presence of reducing agents the factor molecule was converted into two distinct components of lower molecular weight (17 000 and 14 000 respectively), as demonstrated by protein staining. The molecular model implicated by these findings is that platelet-derived growth factor consists of two different polypeptides chains, linked by disulphide bridges.     

Experimental allergic aspermatogenic orchitis. 1. Isolation of a spermatozoal protein (AP1) which induces allergic aspermatogenic orchitis.

A unique highly soluble aspermatogenic protein (AP1) was isolated from guinea pig testes and was shown by immunofluorescence to occupy the outer surface of the sperm acrosome. This protein is a potent inducer of allergic orchitis and aspermatogenesis; as little as 0.2 mug induced orchitis in 60 percent of guinea pig tested. The AP1 protein, relatively small and neutral, is stable under acid conditions, but at pH 8.6 shows a variety of forms due either to aggregation or polymorphism. The purified AP1 protein appeared homogeneous by polyacrylamide gel electrophoresis at pH 2.7 and in sodium dodecyl sulfate and by immunoelectrophoresis using rabbit antisera to either the purified protein or the testes extract. It also showed a single band on immunodiffusion over a wide concentration range. The purification procedure consisted of delipidation with chloroform/methanol (2/1); acid extraction at pH 3.0; precipitation with 85 percent saturated ammonium sulfate; trichloroacetic acid extraction and gel filtration on Bio-Gel A-1.5; gel filtration on Bio-Gel P-10; chromatography on CM52 cellulose; and preparative gel electrophoresis at pH 2.7. Approximately 20 mg of purified AP1 protein were obtained from 5000 g of wet guinea pig testes. The AP1 protein induced an autoimmune disease characterized by infiltration of mononuclear cells around and within the seminiferous tubules (orchitis), followed by extensive damage and destruction of the germinal cells (aspermatogenesis). The course of the disease induced by this protein (0.5 to 1 mug) was essentially identical with that seen with whole testicular tissue or other purified fractions.     

Covalent labelling of the lutropin binding site. Evidence for a single Mr 90000 sialoglycopolypeptide.

Membrane-associated sialoglycopolypeptides of rat ovaries were oxidized with NaIO4, reduced with NaB3H4 and solubilized with Triton X-100. The solubilized proteins carrying the 3H label were subjected to affinity chromatography on human choriogonadotropin coupled to agarose. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate followed by fluorography revealed a single component of apparent Mr 90000. This component was abolished when ovaries saturated with choriogonadotropin were used as starting material. The above result is identical to that obtained previously by conventional detection methods [ Metsikk ö & Rajaniemi (1982) Biochem. J. 208, 309-316] and indicates that the 3H-labelled lutropin/choriogonadotropin sialoglycopolypeptide was observed. The affinity-purified 3H-labelled protein co-eluted with the choriogonadotropin-binding activity solubilized with Triton X-100 from rat ovarian particles, showed a Stokes' radius of 6.2 nm and sedimented as a single band with a sedimentation coefficient of 5.1 S. The sedimentation coefficient of this 3H-labelled protein was not significantly altered when boiled in 1% sodium dodecyl sulphate, indicating that non-covalently associated subunits were not present. The 3H-labelled protein cosedimented with the choriogonadotropin-binding activity solubilized with Triton X-100 from rat ovary. When 125I-choriogonadotropin-receptor complex was covalently crosslinked with glutaraldehyde, an Mr 130000 component was produced as detected by sodium dodecyl sulphate gel electrophoresis. This component was extracted from the polyacrylamide gel and subjected to sucrose-density-gradient centrifugation in 0.1% Triton X-100. A single band sedimenting at the position of the 125I-choriogonadotropin-receptor complex solubilized from a prelabelled ovary was observed, exhibiting a sedimentation coefficient of 6.5S. These data suggest that the lutropin-binding site is a single sialoglycopolypeptide of Mr 90000, which binds one molecule of hormone resulting in an apparent Mr 130000 complex. The large Stokes' radius (6.2 nm) of the binding site is accounted for by bound detergent.     

Endogenous proteolytic activity and constituent polypeptide chains of sheep and pig 19 S thyroglobulin.

Porcine and ovine 19-S thyroglobulins prepared from frozen glands in several buffers using slice extraction or homogenization, ammonium sulfate precipitation and DEAE-cellulose chromatography or Sepharose 6B gel filtration were contaminated with protease activity of pH optima 4.5 and 8.6, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimum temperatures of autodigestion were 37 degrees C at pH 4.5 and 25 degrees C at pH 8.6. Thyroglobulins prepared from unfrozen glands pH 7.2 in 0.1 M sodium phosphate using slice extraction, ammonium sulfate precipitation and Sepharose 6B gel filtration were devoid of acid proteolytic activity but still underwent autodigestion at pH 8.6. Diisopropylfluorophosphate was a potent inhibitor of the alkaline protease activity of ovine thyroglobulin preparations. In contrast to thyroglobulin obtained from frozen glands the proteins purified from fresh unfrozen glands at pH 7.2 only showed the 19-S and the 12-S species by electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Very few bands migrating faster than 12-S were visible. After full reduction and S-alkylation of porcine and ovine thyroglobulins, no qualitative changes were observed in the gel electrophoresis pattern as compared to the unmodified proteins. Species of apparent mol. wt. corresponding to the native 12 S were the major component, strongly suggesting a mol. wt. of about 330 000 for the elementary peptide chains of pig and sheep thyroglobulins.     

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. Purification and properties of the high-activity form of the enzyme.

1. The high-activity form of aminolaevulinate synthetase has been prepared from extracts of semi-anaerobically grown cells of Rhodopseudomonas spheroides, which were allowed to become activated in air. Specific activity was 130 000--170 000 nmol of aminolaevulinate/h per mg of protein at 37 degree C. 2. Enzyme fraction Ia prepared on DEAE-Sephadex was a mixture of four active enzymes, pI5.55, 5.45, 5.35 and 5.2, when prepared in either Tris or phosphate buffers and when extracts were activated by air or by cystine trisulphide. 3. The enzyme was further purified by preparative polyacrylamide-gel electrophoresis in imidazole/veronal buffer, pH 7.6, followed by gel filtration on Sephadex G-100 and concentration with DEAE-Sephadex. 4. The most active enzyme, pI 5.55, ran as a single protein band, mol.wt. 49 000, in sodium dodecyl sulphate and 2-mercaptoethanol. The apparent molecular weight under non-denaturing conditions was 62 000--68 000 on Sephadex G-100 or G-200, pH 7.5, and on polyacrylamide-gel electrophoresis, pH 8.5, at enzyme concentrations below 10 000 units/ml, i.e. less than 60 microgram of protein/ml, and the enzyme was mainly monomeric. 5. The enzyme was homogeneous by gel disc electrophoresis at pH 8.9 and 7.6, but a slightly more diffuse band of protein was obtained during electrophoresis in glycine buffer, pH 7.4. 6. Enzyme samples possessed an intrinsic yellow fluorescence when viewed under u.v. light and this fluorescence coincided exactly with enzymic activity on gel electrophoresis. Fluorescence maxima were 420 nm (excitation) and 495 nm (emission). 7. Radioactive 35S-labelled enzyme had 14 atoms of sulphur/mol of protein (or/40 leucine residues) of which 5--6 residues were cyst(e)ine and 8--9 residues were methionine. 8. Mo carbohydrate was detected apart from glucose, which prevented accurate determination of tryptophan with methanesulphonic acid and tryptamine.     

Pig kidney phosphofructokinase. Purification to homogeneity and qualitative basic characterization.

Phosphofructokinase has been purified from pig kidney by extraction with phosphate buffer at pH 8, followed by alcohol treatment, affinity chromatography on matrix-bound Cibacron blue F3G-A, and gel chromatography on Sepharose 6B. Using sodium dodecyl sulphate electrophoresis the enzyme was found to be homogeneous and to have a specific activity of about 80 units/mg protein. Like other phosphofructokinases, at pH 7.0 the enzyme exhibits a sigmoidal dependence in its activity on the fructose 6-phosphate concentration and is strongly inhibited by ATP. The degree of citrate inhibition is influenced by the concentration of the two substrates. ATP strengthens and fructose 6-phosphate relieves the inhibition by citrate. AMP and cAMP are able to overcome the ATP inhibition. The ADP activation curve is biphasic. The molecular weight of the subunit of pig kidney phosphofructokinase was determined to be 88 000 by means of sodium dodecyl sulphate electrophoresis.