Modifications of aldolase during in vivo aging of rabbit red cells.

Crude hemolysates, partially purified aldolase and aldolase purified to homogeneity from reticulocytes and mature erythrocytes, were incubated with a specific antiserum raised against crystalline rabbit muscle aldolase. We show that the same aldolasic activity corresponds to a greater amount of antigen in older than in younger cells, in crude hemolysates as well as in the above mentioned preparations; that is to say, old-cell aldolase contains cross-reacting material (CRM). Properties of purified enzyme from reticulocytes and mature erythrocytes were compared to those of muscle crystalline aldolase: -- the molecular specific activity of purified aldolase from erythrocytes is lower than with crystalline muscle aldolase, i.e. CRM is maintained throughout the purification steps. -- the specific activity of red cell aldolase towards both substrates (FDP and F1P) is lower than that of crystalline muscle aldolase. However, the ratio of activity towards the two substrates FDP/F1P is decreased in erythrocytes and reticulocytes. -- no other difference was found: Michaelis constant towards FDP, thermodenaturation constant and C terminal extremities are identical as are the molecular weights.     

Site-directed mutagenesis of human aldolase isozymes: the role of Cys-72 and Cys-338 residues of aldolase A and of the carboxy-terminal Tyr residues of aldolases A and B

In order to elucidate the role of particular amino acid residues in the catalytic activity and conformational stability of human aldolases A and B [EC 4.1.2.13], the cDNAs encoding these isoenzyme were modified using oligonucleotide-directed, site-specific mutagenesis. The Cys-72 and/or Cys-338 of aldolase A were replaced by Ala and the COOH-terminal Tyr of aldolases A and B was replaced by Ser. The three mutant aldolases A thus prepared, A-C72A, A-C338A, and A-C72,338A, were indistinguishable from the wild-type enzyme with respect to general catalytic properties, while the replacement of Tyr-363 by Ser in aldolase A (A-Y363S) resulted in decreases of the Vmax of the fructose-1, 6-bisphosphate (FDP) cleavage reaction, activity ratio of FDP/fructose-1-phosphate (F1P), and the Km values for FDP and F1P. The wild-type and all the mutant aldolase A proteins exhibited similar thermal stabilities. In contrast, the mutant aldolase A proteins were more stable than the wild-type enzyme against tryptic and alpha-chymotryptic digestions. Based upon these results it is concluded that the strictly conserved Tyr-363 of human aldolase A is required for the catalytic function with FDP as the substrate, while neither Cys-72 nor Cys-338 directly takes part in the catalytic function although the two Cys residues may be involved in maintaining the correct spatial conformation of aldolase A. Replacement of Tyr-363 by Ser in human aldolase B lowered the Km value for FDP appreciably and also diminished the stability against elevated temperatures and tryptic digestion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of pig muscle aldolase. A comparative study

Aldolase with a specific activity of 10.8 units/mg protein was isolated from pig muscle. Its molecular weight was found to be 150,000. The optimum pH for the catalytic activity was 7.25 and the apparent temperature optimum was 313 K. The Km value was 2.9 X 10(-5) M with FDP as substrate, and 2.8 X 10(-3) M with F1P as substrate. The thermal stability of this pig muscle enzyme was higher than that of the rabbit muscle enzyme. The thermal inactivation of the pig aldolase did not show simple first-order kinetics. The higher conformational stability of the pig aldolase than that of the rabbit enzyme was demonstrated by its higher resistance to the denaturing effect of urea.     

Glyceraldehyde-3-phosphate dehydrogenase of horseshoe crab (Tachypleus tridentatus).

Glyceraldehyde-3-phosphate dehydrogenase [ED 1.2.1.12] was purified from the horseshoe crab, a living fossil, and its properties were examined. 1 The purified enzyme was homogeneous as judged by various tests. The enzyme, like enzymes from other sources, was a tetramer with a subunit molecular weight of 36,000. The kinetic parameters and pH optimum were also similar to those of other enzymes, though the enzyme was more stable against heat and pH denaturations. 2 Analysis of SH groups showed that there were 4 SH groups per subunit, one of which was essential for the enzyme activity and was highly reactive. 3. CD spectra of the enzyme suggested that the enzyme had a very high content of beta-structure (ca. 45 per cent). 4. The horseshoe crab enzyme could form a hybrid in vitro with the rabbit muscle enzymes in concentrated salt solution at acidic pH. 5. There results indicate that the enzyme has overall structural similarity to other enzymes and that the enzyme is highly conserved during a long period of evolution. Some discussions on the structure and activity of the horseshoe crab enzyme are made in comparison with the enzymes from other sources.     

Properties of fructose-1,6-diphosphate phosphatase and fructose-1,6-diphosphate aldolase fromPseudomonas putida

The fructose-1,6-P₂ (FDP) phosphatase, (FDPase) and FDP aldolase fromPseudomonas putida were partially purified by a combination of (NH₄)₂SO₄ fractionation and DEAE-Sephadex column chromatography. Michaelis-Menten kinetics were observed with, respect to FDP in both FDPase and FDP aldolase. TheK _m for FDP at pH 8.0 was 1.2×10⁻⁵M for FDPase and 3.0×10⁻⁵M for FDP aldolase. The specific activities of these two enzymes (assayed under optimal conditions in cell-free extracts ofP. putida grown ond-fructose), as well as their kinetic properties, are consistent with the suggestion that during growth ond-fructose most, of the FDP generated is converted to fructose-6-P (F-6-P), which is subsequently utilized via the Entner-Doudoroff pathway (EDP).     

Glucose and fructose uptake by <Emphasis Type="Italic">Limulus polyphemus</Emphasis> hepatopancreatic brush border and basolateral membrane vesicles: evidence for Na<Superscript>+</Superscript>-dependent sugar transport activity

[³H]-fructose and [³H]-glucose transport activities were determined in brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) from Limulus polyphemus (horseshoe crab) hepatopancreas. Glucose transport was equilibrative in the absence of sodium and sodium dependent in the presence of sodium in BBMV, suggesting GLUT-like and SGLT-like transport activity. Glucose transport by BLMV was equilibrative and sodium independent. Fructose uptake by BBMV and BLMV was equilibrative in the absence of sodium and sodium dependent in the presence of sodium. Western blot analysis using a rabbit anti-mouse SGLT-1 polyclonal antibody indicated the presence of a cross-reacting horseshoe crab BBMV protein of similar molecular weight to the mammalian SGLT1. Sequence alignment of the mouse SGLT-4 and SGLT1 with a translated, horseshoe crab-expressed sequence tag also indicated significant identity between species. Fructose and glucose uptake in the absence and presence of sodium by hepatopancreas BBMV and BLMV indicated the presence of sodium-dependent transport activity for each sugar that may result from the presence of transporters similar to those described for other species.     

Horseshoe crab (Limulus polyphemus) lectin but not garden snail (Helix pomatia) lectin elicits insulin-like activities in vitro

Lectins from the horseshoe crab (Limulus polyphemus) and the garden snail (Helix pomatia) were tested for insulinomimetic activities in isolated rat epididymal adipocytes. The sialic acid binding horseshoe crab lectin suppressed epinephrine-induced lipolysis and augmented lipogenesis from D-[3-3H]-glucose while the N-acetylgalactosamine binding snail lectin was inactive. The results suggest that the insulin receptor on rat adipocytes contains sialic acid in its carbohydrate moiety but does not possess non-reducing alpha-D-galactopyranosyl or 2-acetamido-2-deoxy-alpha-D-galactopyranosyl end groups.     

Comparative studies of fructose 1,6-diphosphate aldolase fromEscherichia coli 518 andLactobacillus casei var.rhamnosus ATCC 7469

A comparative study has been carried out with FDP aldolases fromEscherichia coli 518 andLactobacillus casei ATCC 7469, which had been purified 17.6- and 65-fold, respectively. The aldolase ofL.casei was stable only in the presence of mercaptoethanol, whereas that ofE.coli was strongly inhibited at low (1.0×10^–4 m) and activated at high concentrations (2.0×10^–1 m) of the same compound.p-Chloromercuric benzoic acid inhibited both aldolases, with 40% inhibition at 2×10^–5 m withE.coli aldolase against at 2×10^–4 m withL.casei aldolase. Significant differences were also observed in pH optima and Km values.E.coli aldolase exhibited a maximal activity at pH 9.0 and gave a Km value of 1.76×10^–3 m FDP with strong substrate inhibition above 7×10^–3 m, against pH 6.8–7.0 and a Km of 7.04×10^–3 m FDP forL.casei aldolase. Strong resistance ofL.casei aldolase against inhibition by EDTA, Ca²⁺ and Mn²⁺ was observed compared with complete inhibition at concentrations of 20mm, 40mm and 20mm, respectively, withE. coli aldolase. Polyacrylamide gel electrophoresis did not reveal any differences between the two enzyme preparations.The differences of the properties of FDP aldolases from different bacterial genera are discussed in relation to other Class II aldolases.     

Carbon starvation mediated changes in carbohydrate metabolism inNeurospora crassa

Carbon starvation conditions were found to increase the activities of gluconeogenic enzymes such as malic enzyme, cytosolic malate dehydrogenase and isocitrate lyase along with proteases and inhibition in glucose catabolic enzymes such as G6P dehydrogenase and FDP aldolase inNeurospora crassa     

Construction and expression of human aldolase A and B expression plasmids in Escherichia coli host

E. coli expression plasmids for human aldolases A and B (EC 4.1.2.13) have been constructed from the pIN-III expression vector and their cDNAs, and expressed in E. coli strain JM83. Enzymatically active forms of human aldolase have been generated in the cells when transfected with either pHAA47, a human aldolase A expression plasmid, or pHAB 141, a human aldolase B expression plasmid. These enzymes are indistinguishable from authentic enzymes with respect to molecular size, amino acid sequences at the NH2- and COOH-terminal regions, the Km for substrate, fructose 1,6-bisphosphate and the activity ratio of fructose 1,6-bisphosphate/fructose 1-phosphate (FDP/F1P), although net electric charge and the Km for FDP of synthetic aldolase B differed from those for a previously reported human liver aldolase B. In addition, both the expressed aldolases A and B complement the temperature-sensitive phenotype of the aldolase mutant of E. coli h8. These data argue that the expressed aldolases are structurally and functionally similar to the authentic human aldolases, and would provide a system for analysis of the structure-function relationship of human aldolases A and B.     

Purification and characterization of N-methylalanine dehydrogenase.

Cell free extracts of Pseudomonas MS previously have been shown to carry out the synthesis of a novel amino acid, N-methylalanine (Kung, H.F., and Wagner, C. (1970) Biochim. Biophys. Acta 201, 513-516). An enzyme has been isolated from this organism which is responsible for the synthesis of N-methylalanine. The stoichiometry of the reaction catalyzed by this enzyme leads to the following formulation: Methylamine + pyruvate + NADPH + H-+ yields N-methylalanine + NADP-+ + H2O. This enzyme has been physically separated from alanine dehydrogenase, which is also present in these extracts. This new enzyme has been named N-methylalanine dehydrogenase. It has been purified to near homogeneity as judged by disc gel electrophoresis. Gel filtration chromatography showed that N-methylalanine dehydrogenase has an apparent molecular weight of 77,000, while electrophoresis in sodium dodecyl sulfate gave rise to a single band with a molecular weight of approximately 36,500. The enzyme is optimally active in the pH range between 8.2 and 8.6. The apparent K-m values for pyruvate, NADPH, and methylamine, respectively, are 1-5 times 10 minus 2 M, 3-5 times 10 minus 5 M, and 7.5 times 10 minus 2 M.     

Correction

Phosphoribosylpyrophosphate synthetase (PRS; EC 2.7.6.1) from Hevea brasiliensis Mull. Arg. latex was located in the cytosol. After purification, its apparent molecular weight under nondenaturing conditions was estimated at 200,000 [plus or minus] 10,000; a single band at 57,000 [plus or minus] 3,000 was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme seemed to be a homotetramer. Its affinity constants were estimated at 200 [plus or minus] 30 [mu]M for adenosine triphosphate and 40 [plus or minus] 2 [mu]M for ribose-5-phosphate. The purified enzyme proved to be functional in a paraphysiological medium (cytosol deproteinized by ultrafiltration). Optimum pH was 7.5 in buffer and 6.5 in a paraphysiological medium. No PRS activity was detected in the absence of the Mg2+ ion. Of the numerous compounds tested, only Mn2+, phosphoribosylpyrophosphate and inorganic phosphate affected the enzymatic reaction. Mn2+ (inhibitor constant = 20 [mu]M) and phosphoribosylpyrophosphate (inhibitor constant = 30 [mu]M) were inhibitors. PRS responded allosterically (Hill's coefficient = 2.3) to ribose-5-phosphate in the presence of a physiological concentration of inorganic phosphate (10 mM). These results are set in the physiological context of laticifers.     

The non-catalytic nucleotide-binding site of mitochondrial ATPase is localised on the alpha-subunit(s) of factor F1

The incubation of isolated factor F1 with the di-aldehyde derivative of ADP (oxADP) which is formed as a result of ADP treatment by periodate, causes the covalent binding of 0.9--1 molecules of the oxADP with a molecule of the enzyme. This modification of factor F1 is not accompanied by any changes in the ATPase activity of the enzyme. The modification of factor F1 is preceded by the reversible binding of oxADP with the enzyme with a Kd of 80 micro M. ADP partly prevents factor F1 from modification by oxADP. The electrophoresis of modified factor F1 in polyacrylamide gel in the presence of sodium dodecyl sulphate showed that oxADP binds with the alpha-subunit(s) of factor F1. When submitochondrial particles are incubated with [3H]oxADP, the main part of the radioactive label may be discovered in the polypeptide with a molecular weight of some 30 000 which is probably the adenine nucleotides' translocase. The isolation of factor F1 from particles preincubated with [3H]oxADP showed that the membrane-bound factor F1 covalently binds 0.2--0.3 mol of oxADP per mol of enzyme. Here again, all the oxADP is bound with the alpha subunit(s) of factor F1. The modification of membrane-bound factor F1 by oxADP is accompanied by the partial inhibition of the particles' ATPase activity. The results obtained testify to the fact that the non-catalytic site of mitochondrial ATP ase located on the alpha-subunit(s) of factor F1 may participate in the mechanism of ATP hydrolysis by membrane-bound ATPase.     

Further studies on lipopolysaccharide-sensitive serine protease zymogen (factor C): its isolation from Limulus polyphemus hemocytes and identification as an intracellular zymogen activated by alpha-chymotrypsin, not by trypsin

An intracellular serine protease zymogen, factor C, is an initiator in the hemolymph coagulation system of horseshoe crab. We purified this zymogen from the hemocytes of the American horseshoe crab, Limulus (L.) polyphemus, the objective being to compare its properties with those of the Japanese horseshoe crab, Tachypleus (T.) tridentatus, factor C. The purified zymogen L.-factor C showed similar properties to those of T.-factor C, in terms of molecular mass (123,000), amino acid composition (1,011 residues), subunit structure (two chains), and antigenicity. Like the zymogen T.-factor C, this zymogen was also activated autocatalytically in the presence of bacterial lipopolysaccharide (LPS) and its synthetic lipid A analogue. A most interesting finding is that both protease zymogens are rapidly activated by alpha-chymotrypsin or rat mast cell chymase, but not by trypsin. The active enzyme factor C showed alpha-thrombin-like specificity toward synthetic tripeptide substrates. This factor C was also strongly inhibited by an alpha-thrombin inhibitor, D-Phe-Pro-Arg-chloromethyl ketone. Thus, the enzymatic properties of factor C are similar to those of mammalian alpha-thrombin. On the other hand, the coagulation cascade system present in the hemocyte lysate was activated when chymotrypsin, free from LPS, was added to the lysate used to detect the endotoxins. The implication of our findings is that the chymotrypsin-catalyzed initiation of the horseshoe crab coagulation system is unique, since all known mammalian coagulation, fibrinolysis and complement systems are initiated by trypsin-like enzymes.     

Fructose 1,6-bisphosphate aldolase activity ofRhizobium species

FDP aldolase was found to be present in the cell-free extracts of Rhizobium leguminosarum, Rhizobium phaseoli, Rhizobium trifolii, Rhizobium meliloti, Rhizobium lupini, Rhizobium japonicum and Rhizobium species from Arachis hypogaea and Sesbania cannabina. The enzyme in 3 representative species has optimal activity at pH 8.4 in 0.2M veronal buffer. The enzyme activity was completely lost by treatment at 60 degrees C for 15 min. The Km values were in the range from 2.38 to 4.55 X 10(-6)M FDP. Metal chelating agents inhibited enzyme activity, but monovalent or bivalent metal ions failed to stimulate the activity. Bivalent metal ions in general were rather inhibitory.     

Evidence for the spatial separation of the binding sites for substrate and for cytoskeletal proteins on the enzyme aldolase

The effect of the proteolysis of aldolase on both the substrate specificity of the enzyme and binding capacity for actin have been studied. Carboxypeptidase A, trypsin, chymotrypsin and pepsin, all acted to cleave peptides from the C-terminal portion of the enzyme, resulting initially in a marked loss of activity towards fructose-1:6-bisphosphate (FBP), without impairment of activity towards fructose-1-phosphate (F1P). In some cases, however, further proteolysis caused reductions in activity with F1P as well. By correlating the size of the peptide fragments released by these enzymes with the known sequence of aldolase, evidence has been provided that cleavage of His-359 and/or Tyr-361 lead to the loss of FBP activity, while further cleavage of up to six amino acids begin to affect activity against F1P, as well. In regard to the ability of the proteolysed aldolase to bind to F-actin, it was evident from these studies that binding ability was not impaired in the initial stages of proteolysis referred to above, but was retained until the enzyme was extensively degraded. This differential behaviour of the active and binding sites on aldolase clearly establish their separate topographical localization. These results have been discussed in relation to the positioning of these separate sites on the enzyme, the nature of the interaction between aldolase and actin and the phenomenon of enzyme ambiquity in cells and tissues.     

Plastid Class I and Cytosol Class II Aldolase of Euglena gracilis (Purification and Characterization)

The plastidic class I and cytosolic class II aldolases of Euglena gracilis have been purified to apparent homogeneity. In autotrophically grown cells, up to 81% of the total activity is due to class I activity, whereas in heterotrophically grown cells, it is only 7%. The class I aldolase has been purified to a specific activity of 20 units/mg protein by anion-exchange chromatography, affinity chromatography, and gel filtration. The native enzyme (molecular mass 160 kD) consisted of four identical subunits of 40 kD. The class II aldolase was purified to a specific activity of 21 units/mg by (NH4)2SO4 fractionation, anion-exchange chromatography, chromatography on hydroxylapatite, and gel filtration. The native enzyme (molecular mass 80 kD) consisted of two identical subunits of 38 kD. The Km (fructose-1,6-bisphosphate) values were 12 [mu]M for the class I enzyme and 175 [mu]M for the class II enzyme. The class II aldolase was inhibited by 1 mM ethylenediaminetetraacetate (EDTA), 0.8 mM cysteine, 0.5 mM Zn2+, or 0.5 mM Cu2+. Na+, K+, Rb+, and NH4+ (but not Li+ or Cs+) enhanced the activity up to 7-fold. After inactivation by EDTA, the activity could be partially restored by Mn2+, Cu2+, or Co2+. A subclassification of class II aldolases is proposed based on (a) activation/inhibition by Cys and (b) activation or not by divalent ions.     

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.     

Chemistry and biochemistry of 1-desoxysphinganine 1-phosphonate (dihydrosphingosine-1-phosphonate)

The chemical synthesis of labelled 1-desoxy-D,L-sphinganine 1-phosphonate has been elaborated. This compound is an analog of sphinganine 1-phosphate, a naturally occurring intermediate in the biological degradation of long chain bases.The phosphonate is highly toxic when administered intravenously due to its hemolytic effect. The microsomal sphingosine 1-phosphate lyase(aldolase) cleaves [3-³H] 1-desoxysphinganine 1-phosphonate to [1-³H] hexadecanal and aminoethyl phosphonate like sphinganine 1-phosphate however at a reduced rate. The phosphonate is a competitive inhibitor of the lyase (aldolase). K_i has been determined. The molecular dimensions of the phosphonate have been discussed with reference to the aldolase mechanism and known properties of the enzyme.     

Purification and Characterization of Phosphoribosylpyrophosphate Synthetase from Rubber Tree Latex

Phosphoribosylpyrophosphate synthetase (PRS; EC 2.7.6.1) from Hevea brasiliensis Mull. Arg. latex was located in the cytosol. After purification, its apparent molecular weight under nondenaturing conditions was estimated at 200,000 [plus or minus] 10,000; a single band at 57,000 [plus or minus] 3,000 was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme seemed to be a homotetramer. Its affinity constants were estimated at 200 [plus or minus] 30 [mu]M for adenosine triphosphate and 40 [plus or minus] 2 [mu]M for ribose-5-phosphate. The purified enzyme proved to be functional in a paraphysiological medium (cytosol deproteinized by ultrafiltration). Optimum pH was 7.5 in buffer and 6.5 in a paraphysiological medium. No PRS activity was detected in the absence of the Mg2+ ion. Of the numerous compounds tested, only Mn2+, phosphoribosylpyrophosphate, and inorganic phosphate affected the enzymatic reaction. Mn2+ (inhibitor constant = 20 [mu]M) and phosphoribosylpyrophosphate (inhibitor constant = 30 [mu]M) were inhibitors. PRS responded allosterically (Hill's coefficient = 2.3) to ribulose-5-phosphate in the presence of a physiological concentration of inorganic phosphate (10 mM). These results are set in the physiological context of laticifers.