PURIFICATION OF L-GLUTAMIC ACID DECARBOXYLASE FROM CATFISH BRAIN

—L-Glutamic acid decarboxylase (GAD) from brain of the channel catfish (Ictalurus punctatus) has been purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, calcium phosphate gel and preparative polyacrylamide gel electrophoresis. The purity of the enzyme preparation was established by showing that on both 7.5% regular and 3.7–15% gradient polyacrylamide gel electrophoresis the enzyme migrated as a single protein band which contained all the enzyme activity. The molecular weight of the purified GAD was estimated by gel filtration and gradient polyacrylamide gel to be 84,000 ± 2000 and 90,000 ± 4000, respectively. SDS-polyacrylamide gel electrophoresis revealed three major proteins with molecular weights of 22,000 ± 2000, 40,000 ± 5000 and 90, 000 ± 6000 which may represent a monomer, dimer, and tetramer. Antibodies against the purified enzyme were obtained from rabbit after four biweekly injections with a total of 80 μg of the enzyme. A double immunodiffusion test using these antibodies and a crude extract from catfish brains showed only a single, sharp precipitin band which still retained the enzyme activity, suggesting that the precipitin band was indeed a GAD-anti-GAD complex. In an enzyme inhibition study, a maximum inhibition of 60–70% was obtained at a ratio of GAD protein/anti-GAD serum of about 1:1.6. Furthermore, the precipitate from the GAD-anti-GAD incubation mixture also contained the enzyme activity, suggesting that the antibody was specific to GAD and that the antigen used was homogeneous. Advantages and drawbacks of the purification procedures described here and those used for mouse brain preparations are also discussed.     

A thermostable protein inhibitor of protein kinase from the swine brain. Isolation of the inhibitor and interaction with the enzyme

A heat-stable protein inhibitor of cAMP-dependent protein kinase has been isolated from pig brain tissue. During gel filtration the protein is eluted in three peaks corresponding to the tetramer, dimer and monomer. The monomer fraction was purified 609-fold. The molecular mass of the monomeric form as determined by gel filtration and electrophoresis is equal to 11,000 Da and 8000 Da, respectively. The inhibition of the phosphotransferase reaction with respect to ATP occurs via a non-competitive mechanism, while that for histone--via a competitive mechanism. A formal kinetic analysis of various modes of the inhibitor binding to different protein kinase forms, e. g., the cAMP-dependent protein kinase catalytic subunit, protein kinase holoenzyme in the presence and absence of cAMP as well as of holoenzyme preparations modified by dimethylsuberimidate and cupric 1,10-phenanthroline, has been carried out. It was demonstrated that 3-4 inhibitor molecules are involved in the interaction with protein kinase.     

Saturating Irradiance-Induced Photoinhibition without Monomerisation of Photosystem 2 Dimer in Soybean Leaves

The oligomeric state of photosystem 2 (PS2) complex in soybean leaves treated with saturating irradiance was studied by non-denaturing polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography. PS2 dimers resolved by non-denaturing PAGE accounted for about 75 % of total PS2 complex and there was no significant difference in the ratio of PS2 dimer to monomer between samples from saturating irradiance-treated and fully dark-adapted leaves. Furthermore, BBY particles were resolved into four chlorophyll-enriched fractions by gel filtration chromatography. From their molecular masses and protein components, these fractions were deduced to be PS2 dimer, PS2 monomer, oligomeric light-harvesting complex 2 (LHC2), and monomeric LHC2. Also, no change in the proportion of PS2 dimer in total PS2 was observed in the granal region of thylakoid membranes from soybean leaves after saturating irradiation. Hence the dimer is the predominant natural form of PS2 in vivo and no monomerisation of PS2 dimer occurs during saturating irradiance-induced photoinhibition in soybean leaves.     

Some molecular properties of plant starch phosphorylases and the levels of activity of the multiple forms

A number of plant tissues have two phosphorylase fractions (I and II) that can be separated by DEAE-cellulose chromatography. When only one is detectable, it corresponds to enzyme II. Peas differed from other legumes in showing an increase in enzyme I during seed germination. Examination of the I type enzymes, by sodium dodecyl sulphate polyacrylamide gel electrophoresis, sector cell ultracentrifugation and gel filtration, indicated that these were dimers composed of similar sub-units of MW near 90 000. When phosphorylase II enzymes were examined, the sub-unit MW was found to be higher, near 110 000 and, whereas ultracentrifugation techniques indicated a dimer of similar sub-units for the native enzyme, gel filtration gave higher MW values. Phosphorylase II from Victory Freezer peas differed from the other samples, in being able to form mixtures of dimer and tetramer.     

Properties of ascorbate oxidase isozymes

The ascorbate oxidase of two squash cultivars was resolved into five molecular forms by gel electrophoresis; that of cucumber was resolved into three forms. Molecular weight estimates by Sephadex gel filtration and interconversions of these forms strongly suggest the presence of a monomeric form of MW 30 000 for the cucumber enzyme and 35 000 for that of the squashes. The other two forms in the cucumber appear to be a dimer and a tetramer, whilst a tetramer, an octamer, a dodecamer, and a polymer of MW between 670 000 and 2 000 000 are likely to be the other four forms present in the squashes. The monomer was the most abundant form in the cucumber and the tetramer in the two squashes. The peel of these fruits was higher in activity than the flesh, but the isozyme pattern was the same in peel and flesh. The tetramer of the squashes and the dimer of cucumbers were the most resistant forms to heat inactivation. The enzyme is soluble and not associated with subcellular particles.     

Biochemical and mass spectrometric evidence for quaternary structure modifications of plant threonine deaminase induced by isoleucine

Arabidopsis thaliana threonine deaminase (TD) is a tetramer composed of identical approximately 59600 Da subunits. TD activity has been shown to be inhibited by isoleucine. This effect is reversed by a large excess of valine. Nondenaturant gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry experiments demonstrated that binding of isoleucine on TD induces dimerization of the enzyme, whereas tetramerization is restored by addition of a high valine concentration. Nondenaturant gel filtration and electrospray ionization mass spectrometry of the enzyme in the presence of increasing amounts of isoleucine suggest a fast equilibrium between the tetramer and the dimer. Finally, study of TD mutants allowed us to focus on the specific role of each isoleucine-binding site.     

Electrophoretically monodisperse cytochrome c oxidases

A discontinuous gradient polyacrylamide gel electrophoresis under nondenaturing conditions has been used to demonstrate monodispersity of procaryotic and eucaryotic cytochrome c oxidase preparations. Alkaline treated bovine enzyme which contains nine subunits as analysed by subsequent discontinuous SDS-polyacrylamide gel electrophoresis is a monodisperse dimer in 0.1% Triton X-100 and a monomer in 0.1% dodecyl maltoside. The Mr-values corrected for bound detergent are 286,000 in Triton X-100 and 152,000 in dodecyl maltoside respectively. The two-subunit bacterial cytochrome c oxidase of Paracoccus denitrificans is proved to be a monomer with a corrected Mr of 76,000 in both nonionic detergents Triton X-100 and dodecyl maltoside.     

Comparison of Methods for Separating Proteins Using Bisalbuminemia as a Model

Sera from bisalbuminemic chicken-turkey hybrids contain two albumins in equal amounts. These are observed as inherited electrophoretic variants and originate from the respective chicken and turkey parents. Sera from the hybrid birds served as a model system by which fractionating and identification procedures for evaluating serum albumin variants were compared.

The two albumins in the hybrid were isolated with preparative polyacrylamide gel electrophoresis (PAGE) and starch block preparative electrophoresis. Isoelectric focusing of the hybrid albumins resulted in the isolation of the turkey albumin. Interference of ampholinea prevented the complete isolation of the chicken albumin.

The two albumins in the hybrid have identical molecular weights and cannot be identified by sedimentation coefficient, gel filtration behavior, or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Because of the close relatedness the chicken and turkey albumins in the hybrid cross reacted with rabbit anti-hybrid serum as well as with rabbit anti-chicken and anti-turkey sera.     

Covalent coupling of bilirubin to albumin.

A method for covalent coupling of bilirubin to albumin is described. Human serum albumin-bilirubin (1:1 complex) has been treated with water soluble carbodiimide in order to obtain covalent coupling of bilirubin to albumin. The reaction conditions have been varied with respect to pH, reaction time and concentration of reagent to obtain the optimal coupling. The prepared albumin-bilirubin compounds were investigated by spectrophotometry, gel filtration and gel electrophoresis to ascertain the covalent nature of the bond and to characterize the products further. Gel electrophoresis and gel filtration showed that a monomer fraction could be prepared, and this fraction was a suitable material for further studies.     

Gramicidin S-synthetase. Electrophoretic characterization of the multienzyme.

1. A method characterizing the fully active gramicidin S-synthetase (EC. 6.3.2.-) multienzyme in protein mixtures by a combination of sedimentation and polyacrylamide gel electrophoretic mobility data has been described. 2. The molecular weight of 280000 has been reevaluated by gradient centrifugation, gel filtration, and polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate. The size of the multienzyme is not changed by sodium dodecyl sulfate treatment. 3. In polyacrylamide gel electrophoresis dimerisation occurs in Tris, while two bands, which may represent monomer and dimer, are observed in phosphate. 4. Reliability of molecular weight determinations of sodium dodecyl sulfate-protein complexes of sizes up to 300000 daltons has been determined, correlating either mobilities or retardation coefficients.     

Structure and function of amylases. II. Multiple forms of bacillus subtilis -amylase

The subunit structure of Bacillus subtilis α-amylase has been studied by gel filtration and by SDS-gel electrophoresis. The crystalline enzyme was found to be a 96,000 dalton zinc tetramer. Incubation of the 96,000 species at pH 5.5 or with EDTA produced a 48,000 zinc-free dimer; incubation with 100 mm sodium chloride produced a 72,000 zinc trimer; incubation at pH 8.5 produced a 48,000 zinc dimer and a 24,000 zinc-free monomer. Incubation of the 48,000 zinc dimer with EDTA produced a 24,000 monomer. After standing, the 48,000 zinc dimer formed insoluble aggregates that could be dissolved by treatment with EDTA. The aggregates had molecular weights between 125,000 and 400,000. The 72,000 zinc trimer also aggregated to form a single 144,000 species. All of the forms were enzymatically active, although with widely differing specific activities. Schematic diagrams for the structures of the multiple forms and their interconversions are presented.     

Development of sheep embryos in vitro in a medium supplemented with different batches of serum albumin

Variability in different lots of commercial serum albumin affects mammalian embryo development in culture. The composition of commercial preparations of ovine, bovine and defatted bovine serum albumin and a fraction of ovine serum containing proteins with a mean molecular weight of 65 kDa (fraction 3) was examined by polyacrylamide gel electrophoresis. All preparations were heavily contaminated with serum proteins other than albumin. Day-6 sheep morulae were cultured for 48 h in a basal bicarbonate-buffered salt solution supplemented with the commercial preparations of ovine, bovine or defatted bovine serum albumin. These three albumin preparations differed in their abilities to support the development of morulae into expanded blastocysts, but these differences disappeared when the basal medium was also supplemented with a component of ovine serum containing substances with molecular weights of less than 10 kDa. In the latter case, the three commercial albumin preparations and fraction 3 of ovine serum all supported full development in about 40-60% of morulae.     

Purification and properties of a high specific activity protein kinase from wheat germ

A protein kinase was extensively purified to near-homogeneity from wheat germ by a procedure involving affinity chromatography on casein-Sepharose 4B, gel filtration, and repeated chromatography on carboxymethyl-Sepharose CL-6B. The protein kinase preparations have the highest specific activities (up to 656 nanomoles phosphate incorporated per minute per milligram of protein) yet reported for plant protein kinases. The major polypeptides in purified preparations were revealed as two barely-resolved bands (molecular weight 31,000) on polyacrylamide gel electrophoresis in subunit-dissociating conditions. The molecular size of the protein kinase as determined from gel filtration is 30,000. The protein kinase catalyzes the phosphorylation of casein, phosvitin, and the wheat germ cyclic AMP-binding protein cABPII but not of bovine serum albumin and histones nor of the wheat germ cytokinin-binding protein CBP. The protein kinase has a pH optimum of 7.9 and a K_m value for ATP of 10 micromolar. The protein kinase differs from wheat germ CBP kinase in molecular weight, differential sensitivity to inhibitors, and in substrate specificity.     

Separation of biomacromolecules by electrofiltration through gel layers

A straightforward method for concomitant separation and isolation of biomacromolecules from a mixture in solution was developed. Three gel layers that comprise a middle separation layer of 10% polyacrylamide gel were constructed. This gel system was formed in an electroconcentration apparatus above a collection chamber surrounded at the bottom by a dialysis membrane. The mixture is applied over the gel layers where biomacromolecules are caused to migrate by electrophoresis through the gel system, where they are separated into discrete bands and electroeluted into the collection chamber without dismantling the apparatus. The isolated biomacromolecules are removed from the chamber in a highly pure and concentrated form ready for further investigations. Cooling can be applied throughout the whole process, and the setup and conditions of run can be modified according to the characteristics of the biomacromolecules to be purified. The components of a mixture containing the glycoprotein ovalbumin and bovine serum albumin monomer, dimer, and tetramer were successfully isolated as concentrated and highly pure fractions with good recoveries ranging from 70 to 89%. Other proteins were successfully isolated under denaturing conditions in the presence of sodium dodecyl sulfate (SDS) or 6 M urea.     

Spinach Thylakoid Polyphenol Oxidase : ISOLATION, ACTIVATION, AND PROPERTIES OF THE NATIVE CHLOROPLAST ENZYME

Polyphenol oxidase activity (E.C. 1.14.18.1) has been found in two enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified >900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. The higher molecular weight enzyme is the predominant form in freshly isolated preparations but on aging or further purification, the amount of lower molecular weight enzyme increases at the expense of the higher.     

Hidden self-association of proteins

Sedimentation equilibrium measurements were carried out on solutions of bovine serum albumin, aldolase, and ovalbumin in phosphate-buffered saline, pH 7.2, at 10 degrees C. The data obtained for each protein were analyzed to yield the dependence of apparent weight-average molecular weight upon protein concentration, over a concentration range of ca 1-200 g/L. Using the approximate theory of Chatelier and Minton [1987) Biopolymers 26, 507-524), models are formulated for the dependence of apparent weight-average molecular weight upon concentration in non-ideal solutions containing proteins which may self-associate according to a monomer/n-mer or a monomer/dimer/tetramer scheme. The concentration dependence data for serum albumin may be accounted for, assuming either no self-association or weak monomer/dimer association. The data for aldolase may be accounted for assuming either weak monomer/dimer or weak monomer/trimer association. The data for ovalbumin may be accounted for assuming either weak monomer/trimer or weak monomer/dimer/tetramer association. The associations do not approach saturation at the highest concentrations studied, and the standard-state free energy changes accompanying self-association amount to less than 4 kcal/mol of intermolecular contacts, suggesting that non-specific clustering of protein molecules at high concentration rather than the formation of specific complexes is being observed.     

Use of electrophoresis and gel filtration to characterize human pituitary somatotropin preparations after storage.

Fresh and stored preparations of human pituitary somatotropin examined by different polyacrylamide gel electrophoresis techniques and by gel exclusion chromatography (Ultrogel) clearly demonstrated that the hormone undergoes a structural alteration during storage for 3 years. The formed aggregates were readily quantitated from the gel filtration profile and from polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE). According to gel filtration the amount of aggregates in fresh and stored hormone was determined to be 2 and 10%, respectively, while SDS-PAGE indicated 6 and 14%.     

Cross-linking of hemoglobin, haptoglobin, and hemoglobin-haptoglobin complex with bifunctional imidoesters.

Dimethyl adipimidate was used to cross-link the polypeptides within hemoglobin, haptoglobin, and hemoglobin-haptoglobin complex. Cross-linked hemoglobin retained considerable ability to bind haptoglobin, although the amounts bound were reduced and the haptoglobin reaction could be used to fractionate the modified hemoglobin. With cross-links limited to intramolecular sites, hemoglobin showed four bands on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, identified, with reference to the subunit polypeptides, as monomer, dimer, trimer, and tetramer. The dimer region consisted of at least two separable species. When hemoglobin-haptoglobin complex was cross-linked, a band of hemoglobin dimer was present, which demonstrates that at least two hemoglobin subunits have a close spatial relation when bound to haptoglobin. Some comparisons with adipimidate-reacted hemoglobin were made using malonimidate and suberimidate and some marked differences were noted.     

Degradation of human normal immunoglobulin preparations and the level of measles virus antibodies

Seventy-four batches of normal human immunoglobulin of placental origin (NHIp) and 25 batches of the preparation derived from the serum (NHIs) were tested. The batches were divided into groups (A-E) according to production date and presence of sediment. The degree of degradation of NHI preparations was determined measuring the components soluble in trichloroacetic acid (TCASC), performing molecular filtration or carrying out electrophoresis in polyacrylamide gel. The level of antibodies to measles virus was determined with the enzymatic immunoadsorption test (ELISA) and haemagglutination inhibition test (HIT). It was found that NHI preparations were undergoing significant degradation before their expiration date. The content of TCASC ranged from 1.83% for NHIs preparations recently produced (group E) to 10.57-10.63% respectively in NHIp preparations with sediment (groups B and A). Molecular filtration made possible isolation of a polymer, a monomer, and degradation products. The level of antibodies against measles virus was the lower (7,400) the greater was the degree of degradation of NHIp preparations in the relation of NHI preparations recently produced (21,500).     

Isolation of lysyl hydroxylase, an enzyme of collagen synthesis, from chick embryos as a homogeneous protein.

Two procedures are reported for the purification of lysyl hydroxylase, both procedures involving (NH4)2SO4 fractionation, affinity chromatography on concanavalin A-agarose and elution of the column with ethylene glycol. The additional steps in procedure A consist of gel filtration and chromatography on a hydroxyapatite column, and in procedure B of affinity chromatography on collagen linked to agarose and gel filtration. The best preparations obtained with either of the two procedures were pure when examined by sodium dodecyl sulphate-polyacrylamide-disc-gel or slab-gel electrophoresis, but about half of the preparations obtained by procedure A had minor contaminants. The specific activity of a typical preparation purified by procedure B was 13 4000 times that of the 15 000 g supernatant of the chick-embryo homogenate, with a recovery of about 4%. The molecular weight of the pure enzyme was bout 200 000 by gel filtration, and that of the enzyme subunit about 85 000 by sodium dodecyl sulphate/polyacrylamide-disc-gel or slab-gel electrophoresis. It is suggested that the active enzyme is a dimer consisting of only one type of monomer, and that a previously described enzyme form with an apparent molecular weight of about 550 000 is a polymeric form of this dimer. The catalytic-centre activity of the pure enzyme, as determined with a saturating concentration of a synthetic peptide substrate and under conditions specified, was about 3-4 mol/s per mol.