Isolation and properties of cytochrome c oxidase from rat liver and quantification of immunological differences between isozymes from various rat tissues with subunit-specific antisera

Cytochrome c oxidase was isolated from rat liver either by affinity chromatography on cytochrome-c--Sepharose 4B or by chromatography on DEAE-Sepharose. Dodecyl sulfate gel electrophoresis of both preparations showed the same subunit pattern consisting of 13 different polypeptides. Kinetic analysis of the two preparations gave a higher Vmax for the enzyme isolated by chromatography on DEAE-Sephacel. Specific antisera were raised in rabbits against nine of the ten nuclear endoded subunits. A monospecific reaction of each antiserum with its corresponding subunit was obtained by Western blot analysis, thus excluding artificial bands in the gel electrophoretic pattern of the isolated enzyme due to proteolysis, aggregation or conformational modification of subunits. With an antiserum against rat liver holocytochrome c oxidase a different reactivity was found by Western blot analysis for subunits VIa and VIII between isolated cytochrome c oxidases from pig liver or kidney and heart or skeletal muscle. For a quantitative analysis of immunological differences a nitrocellulose enzyme-linked immunosorbent assay was developed. Monospecific antisera against 12 of the 13 subunits of rat liver cytochrome c oxidase were titrated with increasing amounts of total mitochondrial proteins from different rat tissues dissolved in dodecyl sulfate and dotted on nitrocellulose. The absorbance of a soluble dye developed by the second peroxidase-conjugated antibody was measured. From the data the following conclusions were obtained: (a) The mitochondrial encoded catalytic subunits I-III of cytochrome c oxidase are probably identical in all rat tissues. (b) All nine investigated nuclear encoded subunits of cytochrome c oxidase showed immunological differences between two or more tissues. Large immunological differences were found between liver, kidney or brain and heart or skeletal muscle. Minor but significant differences were observed for some subunits between heart and skeletal muscle and between liver, kidney and brain. (c) Between corresponding nuclear encoded subunits of cytochrome c oxidase from fetal and adult tissues of liver, heart and skeletal muscle apparent immunological differences were observed. The data could explain cases of fatal infantile myopathy due to cytochrome c oxidase deficiency.     

Analysis of yeast RNA polymerases with subunit-specific antibodies

Specific antibodies directed against each polypeptide component of yeast RNA polymerases A or B were prepared and their affinity spectrum determined by protein blot immunodetection. The majority of enzyme A or B subunits were specifically recognized by their respective antiserum. A direct correspondence was established between the polypeptides immunologically related in the three forms of RNA polymerases A, B, and C by reacting the different antibodies with enzymes subunits transferred to a nitrocellulose membrane. Subunit-specific antibodies and antibodies to native enzymes A and B were used to probe the activity of RNA polymerases A, B, and C. Based on DNA protection experiments, the largest subunit of enzymes A and B as well as the common subunit ABC23 appear to be involved in DNA binding.     

Beta 1 subunits of sodium channels. Studies with subunit-specific antibodies

The purified Na+ channel from rat brain consists of alpha (260 kDa), beta 1 (36 kDa), and beta 2 (33 kDa) subunits. Pure beta 1 subunits were prepared from purified rat brain Na+ channels which had been adsorbed to hydroxylapatite resin and used to prepare specific anti-beta 1 subunit antiserum. Antibodies purified from this antiserum by antigen affinity chromatography immunoprecipitate 125I-labeled, purified beta 1 subunits and purified Na+ channels (measured as high affinity [3H] saxitoxin binding sites) and recognize beta 1 subunits on immunoblots of solubilized rat brain membranes. The affinity-purified anti-beta 1 antibodies recognize beta 1 subunits in rat spinal cord, heart, skeletal muscle, and sciatic nerve, but do not detect immunoreactive beta 1 subunits in eel electroplax or eel brain. The developmental time course of expression of immunoreactive beta 1 subunits in rat forebrain was measured by immunoprecipitation followed by immunoblotting with affinity-purified anti-beta 1 antibodies. The amount of immunoreactive beta 1 subunits increased steadily to adult levels during the first 21 days of postnatal development.     

Rat liver alcohol dehydrogenase. Purification and properties

Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD(+) and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn(2+) at concentrations above 0.1mm.     

Purification and partial characterization of different forms of phosphofructokinase in man.

Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from human muscle, brain, heart and granulocytes has been purified using a two or three step purification procedure. The main step is Blue Dextran-Sepharose 4B chromatography with selective elution of phosphofructokinase by formation of the ternary complex ADP or ATP-fructose-6-P-enzyme. Muscle and heart contain only enzyme subunits with a molecular weight of 85,000. This type of subunit is predominnant in brain, where it co-exists with subunits of about 80,000 daltons. A single type of subunits is found in the granulocytes, with a molecular weight of 80,000. Anti-muscle phosphofructokinase antiserum reacts only with M-type enzyme. Anti-granulocyte enzyme antiserum, absorbed by pure brain phosphofructokinase, exhibits a narrow specificity against the so-called L-type enzyme. Anti-brain antiserum, absorbed by pure muscle phosphofructokinase and partly purified red cell enzyme, exhibits a narrow specificity against a phosphofructokinase form predominant in fibroblasts and present in brain (F-type).     

Glycoprotein nature of yeast alkaline phosphatase. Formation of active enzyme in the presence of tunicamycin.

The nonspecific alkaline phosphatase of yeast (Saccharomyces strain 1710) has been purified by ion exchange, hydrophobic, and affinity chromatography. This vacuolar enzyme has a molecular weight of 130,000 and is composed of subunits (probably of 66,000 molecular weight). It also has a small quantity of covalently associated carbohydrate; hydrolysis yielded mannose and glucosamine. The endo-beta-N-acetylglucosaminidase of Streptomyces plicatus released carbohydrate indicating that the latter was attached to protein through an N-acetylglucosaminylasparginyl bond. Synthesis of active alkaline phosphatase by yeast protoplasts is not depressed by tunicamycin, an inhibitor of dolichol-mediated protein glycosylation. Unlike the enzyme normally produced, the alkaline phosphatase which is formed in the presence of the antibiotic does not interact with concanavalin A and, therefore is deficient in or lacking carbohydrate. We infer that there is no regulatory link in yeast between the glycosylation of a protein and its synthesis. The fact that other Asn-GlcNAc-type glycoprotein enzymes of yeast such as acid phosphatase are not produced in their active forms by tunicamycin-treated protoplasts may mean that, as unglycosylated proteins, they cannot be correctly folded or processed. Protoplasts derepressed for phosphatase production contained substantial amounts of a second alkaline phosphatase which differed from the purified enzyme in substrate specificity, sensitivity to calcium, and reactivity with concanavalin A.     

Affinity chromatography isolation of human cytochrome oxidase and small-scale Western immunoblot probing of the enzyme complex in mitochondrial cytopathy patients.

Isolation of human cytochrome oxidase by a one-step affinity chromatography procedure on a Sepharose 4B-ferrocytochrome c matrix following solubilization with the nonionic detergent laurylmaltoside yields an enzyme isolate of adequate purity for producing polyclonal antisera. Such an antiserum produced a distinctive immunoreactive profile in Western immunoblot studies to that reported using the enzyme isolated with ionic detergents. A sensitive and highly reproducible Western immunoblotting method is described for probing mitochondrial fractions prepared from small frozen skeletal muscle biopsies with an antiserum against the human placenta cytochrome oxidase. Application of this method to mitochondrial cytopathy patients with partial cytochrome oxidase deficiency shows that the detected subunits are synthesized in these patients.     

Biochemical and immunological characterization of the STA2-encoded extracellular glucoamylase from saccharomyces diastaticus

In Saccharomyces diastaticus each one of three unlinked genes (STA1, STA2, STA3) encodes a glucoamylase (alpha-1,4 glucanglucohydrolase, EC 3.2.1.3) that allows yeast to grow on starch. The enzyme encoded by the STA2 gene (glucoamylase II) has been purified from culture medium to near homogeneity by ethanol precipitation, Trisacryl M DEAE chromatography, and HPLC gel filtration. Glucoamylase II consists of two identical subunits whose average size is 300 kDa. Under denaturing conditions, the native dimeric enzyme readily dissociates to a monomer. Enzymatic deglycosylation of denatured enzyme gives rise to intermediate, partially glycosylated forms and to a 56-kDa completely deglycosylated protein. Glucoamylase releases glucose units by cleaving alpha-1,4 bonds from the nonreducing end of different oligosaccharides, but has only a barely detectable alpha-1,6 hydrolyzing activity. The pH optimum for the purified enzyme was found to be 5.1. The enzyme has a greater affinity for maltohexaose (Km = 0.98 mM, V/Km = 2.39) than for maltotriose (Km = 2.38, V/Km = 0.68) or maltose (Km = 3.20, V/Km = 0.39). Both polyclonal and monoclonal antibodies have been raised against glucoamylase II. The polyclonal antibodies specifically inhibit yeast glucoamylase II activity in a dose-dependent manner, but are found to immunoblot other yeast glycoproteins as well. This oligosaccharide-specific reaction can be competed out by adding excess mannan without affecting glucoamylase reactivity. The cross-reactivity of the polyclonal antibodies with other amylolytic enzymes correlates well with evolutionary distance. Evidence is presented that monoclonal antibodies specific for either carbohydrate or protein epitopes have been obtained.     

Modified GMP-affinity chromatography for the purification of mutant hypoxanthine phosphoribosyltransferase

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) catalyzes the conversion of hypoxanthine and guanine to IMP and GMP, respectively, in the presence of 5-phosphoribosyl-1-pyrophosphate. Deficiencies of HPRT are associated with neurological abnormalities and gout. A human HPRT variant enzyme failed to bind to a GMP-affinity column under standard purification conditions. We developed a series of predictive tests for designing the affinity chromatography protocol which enabled purification of both normal and variant HPRT. The primary variable for the present variant was a difference in toleration of salt; other aspects recommended for evaluation are assessment of ligand-enzyme affinity, pH optimum, and tolerance of nonspecific ligands for washes. In addition, a method for determining the amount of GMP linked to the column material was developed and consisted of acid hydrolysis and HPLC quantitation of guanine.     

Subunit structure, expression, and function of NAD(H)-specific isocitrate dehydrogenase in Saccharomyces cerevisiae.

Mitochondrial NAD(H)-specific isocitrate dehydrogenase was purified from Saccharomyces cerevisiae for analyses of subunit structure and expression. Two subunits of the enzyme with different molecular weights (39,000 and 40,000) and slightly different isoelectric points were resolved by denaturing electrophoretic techniques. Sequence analysis of the purified subunits showed that the polypeptides have different amino termini. By using an antiserum to the native enzyme prepared in rabbits, subunit-specific immunoglobulin G fractions were obtained by affinity purification, indicating that the subunits are also immunochemically distinct. The levels of NAD(H)-specific isocitrate dehydrogenase activity and immunoreactivity were found to correlate closely with those of a second tricarboxylic acid cycle enzyme, malate dehydrogenase, in yeast cells grown under a variety of conditions. S. cerevisiae mutants with defects in NAD(H)-specific isocitrate dehydrogenase were identified by screening a collection of yeast mutants with acetate-negative growth phenotypes. Immunochemical assays were used to demonstrate that one mutant strain lacks the 40,000-molecular-weight subunit (IDH1) and that a second strain lacks the 39,000-molecular-weight subunit (IDH2). Mitochondria isolated from the IDH1 and IDH2 mutants exhibited a markedly reduced capacity for utilization of either isocitrate or citrate for respiratory O2 consumption. This confirms an essential role for NAD(H)-specific isocitrate dehydrogenase in oxidative functions in the tricarboxylic acid cycle.     

The rate of import and assembly of F1-ATPase in Saccharomyces cerevisiae

Subunit specific antiserum can be employed to study the course of ATPase assembly in mitochondria isolated from bakers' yeast. Comparing rates of subunit import with rates of enzyme assembly indicated that no substantial pool of unassembled subunits exists for the three largest ATPase peptides (alpha, beta, and gamma). Blocking import of specific ATPase subunits, however, did reveal a possible accumulation of unassembled alpha and gamma subunits in isolated mitochondria. The kinetic experiments also revealed a lag in the import of beta subunit relative to the uptake of alpha and gamma precursors. Experiments conducted in yeast cells confirmed that beta subunit is assembled soon after it is imported, but did not indicate a delay in import relative to the other subunits of F1.     

Purification and characterization of limonoate dehydrogenase from Rhodococcus fascians.

Limonoate dehydrogenase from Rhodococcus fascians has been purified to electrophoretic homogeneity by a procedure that consists of ion-exchange, hydrophobic, and affinity chromatography. The native enzyme has a molecular mass of around 128,000 Da and appears to be composed of four similar subunits (30,000 Da each). The isoelectric point is 4.9 as determined by isoelectric focusing. The homogeneous enzyme was used to determine the NH2-terminal amino acid sequence. The enzyme was purified from cells grown in either fructose or limonoate as a carbon source. Limonoate dehydrogenase activity was higher in limonoate-grown cultures. Additionally, the enzyme preparations differed in their affinity for limonoids but not for NAD+. In all cases limonoate dehydrogenase exhibited a higher catalytic rate and stronger affinity for limonoate A-ring lactone than for disodium limonoate, the limonoid traditionally used for in vitro activity assays. Our data confirm previous reports proposing that limonoate A-ring lactone is the physiological substrate for limonoate dehydrogenase. The increase in limonoate dehydrogenase activity observed in limonoate-grown cultures appears to be caused by a rise in protein levels, since chloramphenicol prevented such an effect.     

Purification of an insulin-sensitive cyclic AMP phosphodiesterase from rat liver

A low-Km cyclic nucleotide phosphodiesterase solubilised from rat liver membranes by mild proteolysis with chymotrypsin has been purified to apparent homogeneity. The purification included chromatography on cellulose phosphate, Ecteola-cellulose, hydroxyapatite, a theophylline affinity matrix and HPLC on a DEAE-substituted column. The purified enzyme has linear kinetic plots with a Km of 0.24 microM and a Vmax of 6.2 mumol mg-1 min-1 with cyclic AMP as a substrate. It also hydrolyses cyclic GMP with a Km of 0.17 microM and a Vmax which is about a third of that with cyclic AMP. Cyclic GMP is also a competitive inhibitor of cyclic AMP hydrolysis with a Ki of 0.18 microM. The proteolytically solubilised enzyme has a subunit molecular mass of 73 kDa by SDS gel electrophoresis and of 130 kDa by HPLC size-exclusion chromatography, suggesting that it exists as a dimer. A partially purified preparation of this enzyme was used to raise antiserum in a sheep. The antiserum immunoprecipitated activity from liver and adipose tissue of rat and mouse. It had little activity against phosphodiesterase from other rat tissues or other species. Insulin-activated phosphodiesterase from both adipocytes and hepatocytes was immunoprecipitated by the antiserum suggesting that the purified enzyme was an insulin-sensitive phosphodiesterase.     

Purifications of Rat Adrenal Dopamine-β-Hydroxylase: Immunological Analysis of Its Soluble and Membrane-Bound Forms with the Use of an Antibody Raised Against the Soluble Form

Soluble and membrane-bound dopamine-beta-hydroxylases (sDBH and mDBH, respectively) from rat adrenal glands have been purified through concanavalin A-Sepharose chromatography, gel filtration, and ion-exchange high-performance chromatographies. Both sDBH and mDBH were composed by four subunits of apparent molecular weight of 75,000 and showed a native molecular weight of 300,000. This procedure has not allowed us to obtain a sufficient amount of enzyme to immunize a rabbit. A second, more rapid procedure was designed to isolate sDBH, including concanavalin A-Sepharose chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A rabbit antiserum was raised against this purified protein. The specificity of the antiserum was demonstrated by neutralization of rat adrenal gland DBH activity, labeling of rat adrenal medulla on histological sections, and, after Western blot, labeling of the 75,000-molecular-weight band in the different fractions associated with DBH activity during purification. The antiserum had a higher affinity for the sDBH denatured by sodium dodecyl sulfate than for the native protein. It had a higher affinity for sDBH than for mDBH. These results strongly suggest the presence of specific hydrophilic epitopes on the sDBH, revealing structural differences between the two hydroxylase forms. Two protein bands were stained on Western blots of crude rat adrenal gland extract. One band had an apparent molecular weight of 75,000, and the other of 82,000. Our results showed that the two proteins contained similar epitopes, an observation suggesting a close structural relationship. The higher-molecular-weight protein could be the 75,000 protein before covalent modifications and cleavage.     

Molecular and immunological characterization of plastid and cytosolic pyruvate kinase isozymes from castor-oil-plant endosperm and leaf

1. Monospecific antiserum was raised in rabbits to homogeneous cytosolic pyruvate kinase isolated from 5-day-old germinating endosperm of the castor oil plant, Ricinus communis. An earlier study demonstrated that the purified enzyme is putatively heterotetrameric, composed of two subunits which migrate as 57-kDa and 56-kDa proteins upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis [Plaxton, W. C. (1988) Plant Physiol. (Bethesda) 86, 1065-1069]. Both proteins were detected on Western blots of extracts prepared under denaturing conditions from 4-8-day-old, but not 0-3-day-old, germinating-endosperm tissue. This suggests that both subunits exist in vivo, and that the large increase in pyruvate kinase activity which occurs around the fourth day of germination is due to an increase in pyruvate kinase concentration. 2. The cytosolic and plastidic pyruvate kinase isozymes (termed PKc and PKp, respectively) from castor-oil-plant developing endosperm and expanding leaf tissue were separated by anion-exchange chromatography on Q-Sepharose. The antigenic reaction of the partially purified enzyme preparations to rabbit polyclonal antibodies raised against homogeneous germinating-castor-bean PKc was tested by immunoprecipitation and Western blotting. Although developing-endosperm and leaf PKc appeared to be antigenically very similar to germinating-endosperm PKc, they differed from the heterotetrameric germinating-endosperm enzyme by being composed of a single type of subunit with a molecular mass of about 56 kDa. No cross-reactivity of the PKc antibodies was observed with either developing-endosperm or leaf PKp, nor with rabbit muscle or Bacillus stearothermophilus pyruvate kinase. Conversely, none of the castor-oil-plant pyruvate kinase preparations showed significant cross-reactivity with antibodies raised against purified yeast or rabbit muscle pyruvate kinases. 3. To investigate the structural relationship between the two germinating-endosperm-PKc subunits, each polypeptide was characterized by amino acid composition analysis and peptide mapping by CNBr fragmentation. The amino acid compositions and CNBr cleavage patterns of the two subunits were similar, but not identical, suggesting that these polypeptides are related, but distinct, proteins. Mild tryptic attack of native enzyme led to an approximate 6-kDa reduction in the apparent molecular mass of both subunits, further indicating sequence similarity between the two polypeptides. 4. Native molecular masses of the various castor-oil-plant pyruvate kinases were estimated by Superose-6 gel-filtration chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of acid trehalase from the yeast suc2 mutant

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.     

Studies on Metabolic Pathway of NAD in Yeast

The enzyme system of NAD degradation was extracted from autolysate of Saccharomyces oviformis.

The enzymes were partially separated by ammonium sulfate fractionation and DEAE-cellulose column chromatography, and then the metabolic pathway of NAD in yeast was presented, in which four enzymes were contained. It has been found that, among them, the 5?nucleotidase has more affinity for AMP and the nucleosidase has strict affinity for nicotinamide riboside.

In the degradation of NAD in the yeast, nucleotide pyrophosphatase was main enzyme, but NADase, nucleotide pyrophosphorylase and adenosine deaminase seemed not to play an important role.     

The yeast mitochondrial adenosine triphosphatase complex. Subunit stoichiometry and physical characterization

Immunoprecipitation of uniformly labeled yeast submitochondrial preparations using a subunit-specific or a holoenzyme antiserum has been employed to determine the subunit stoichiometry of the oligomycin-sensitive ATPase complex. The Triton-solubilized enzyme consists of 10 types of subunits. The number of copies of each subunit, in order of decreasing molecular weight, is 3:3:1:2:1:2:2:1:2:3. on the basis of the stoichiometry data, the ATPase complex has a molecular weight of 5.8 x 10(5) and contains a minimum of 20 polypeptide chains. Analysis of water-soluble ATPase (F1-ATPase) indicates that the stoichiometry of the three largest subunits of the enzyme is preserved in the absence of the other subunits. The molecular weights of both forms of the ATPase, derived from stoichiometry data, agree well with measurements obtained from gel filtration and sedimentation studies. The implications of these data for the structure, function, and assembly of the complex are discussed.     

The product of the his4 gene cluster in Saccharomyces cerevisiae. A trifunctional polypeptide.

The his4 region of yeast encodes the information for the third (phosphoribosyl-AMP cyclohydrolase), second (phosphoribosyl-ATP pyrophosphohydrolase), and tenth (histidinol dehydrogenase) steps in the histidine biosynthetic pathway. These three activities co-purify with a single protein which has a subunit molecular weight of 95,000 (95,000 protein), as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Extracts of yeast strains which carry nonsense or deletion mutations in various portions of the his4 region, purified in parallel by affinity chromatography on AMP-agarose columns, were examined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis slabs. All such mutant extracts examined were found to lack the 95,000 protein found in a strain carrying a wild type his4 allele. The presence of a protease inhibitor, phenylmethylsulfonyl fluoride, during the purification of the trifunctional enzyme prevented the degradation of the 95,000 protein to polypeptides of lower molecular weight. Monospecific antibody prepared against the 95,000 protein removed all three of the activities specified by his4 from solution; active 95,000 protein was recovered in the resuspended immunoprecipitates. All this evidence shows that the product of the his4 region is a trifunctional, 95,000-dalton protein. Preliminary evidence from two-dimensional gel electrophoresis, NH2-terminal analysis, and gel filtration column chromatography indicates that the native trifunctional enzyme is a dimer of identical 95,000-dalton subunits.     

Purification, subunit structure and immunological comparison of fructose-bisphosphate aldolases from spinach and corn leaves

The cytosol and chloroplast fructose-bisphosphate aldolases from spinach leaves were separated by ion-exchange chromatography on DEAE-cellulose, and were purified by subsequent affinity chromatography on phosphocellulose to apparent homogeneity as judged from polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two aldolases had specific activities of 7.2 and 7.8 units mg protein-1. Molecular weight determinations by electrophoresis in sodium dodecyl sulfate gels and by sedimentation velocity centrifugation in sucrose gradients showed that the aldolases contained four subunits of Mr 38 000 and 35 000, respectively. Antibodies against the cytosol and chloroplast aldolase from spinach leaves were raised in a guinea pig and in a rabbit, respectively. In the Ouchterlony double-diffusion test, the two aldolases did not cross-react. A small degree of cross-reaction was observed by a test in which immune complexes were adsorbed to a solid-phase support (Staphylococcus aureus Cowan I cells) and nonbound enzyme activity was determined after centrifugation. These results imply major structural differences between the two spinach leaf aldolases. Only one major aldolase could be resolved on DEAE-cellulose from corn leaves. The aldolase was purified and had a specific activity of 6.4 units X mg protein-1. The corn leaf aldolase cross-reacted with the antiserum raised against the chloroplast enzyme from spinach leaves, but not with the other antiserum. Thus, the corn leaf aldolase could be identified as a chloroplast enzyme. Since aldolase activity is mostly restricted to the bundle sheath cells of corn leaf, it was concluded that it is compartmentalized in the chloroplasts of these cells but not in chloroplasts of the mesophyll cells.