The sulphatase of ox liver. XXV. Sulphatase A as a hysteretic enzyme

The kinetic behaviour of the system native--substrate-modified sulphatase A (arylsulphate sulphohydrolase, EC 3.1.6.1) has been investigated and it has been shown that the progress curve of the complete reaction, including both the inactivation and reactivation stages, can be treated as that of a simple hysteretic system in which the substrate-modified enzyme is activated by a product of the reaction. It has been concluded that the early suggestions that the modification of sulphatase A was accompanied by the exposure of a second ligand-binding site are incorrect. It has been shown that, in the absence of sulphate, the rate of reversion of substrate-modified to native sulphatase A is increased by 4-nitrocatechol but not by the same concentration of 2-nitrophenol. A detailed reaction sequence is proposed. This explains the kinetic behaviour of sulphatase A with nitrocatechol sulphate or 2-nitrophenyl sulphate as substrate and in the presence or absence of sulphate.     

The sulphatase of ox liver. XXIV. The glycosulphatase activity of sulphatase a

The rhodizonic acid method for the determination of SO2-4 has been used to investigate the glycosulphatase activity of the sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) of ox liver. Sulphatase A hydrolyses D-glucopyranose and D-galactopyranose 2-, 3-, 4- and 6-sulphates: glucose sulphates are hydrolysed more rapidly than galactose sulphates and the 3-sulphates more rapidly than the other isomers. 2-Acetamido-2-deoxyglucopyranose 6-sulphate is not hydrolysed, nor is 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranose 1-sulphate. Sulphate is a competitive inhibitor of the glycosulphatase activity. Hydrolysis proceeds through fission of the O-S bond. Evidence is given that the hydrolysis of glucose 3-sulphate is accompanied by the formation of substrate-modified sulphatase A, although this has not been isolated. Sulphatase A has no detectable alkylsulphatase activity.     

Purification of glutamate dehydrogenase from ox brain and liver. Evidence that commercially available preparations of the enzyme from ox liver have suffered proteolytic cleavage.

1. A rapid procedure, involving ion-exchange chromatography on DEAE-cellulose and affinity chromatography on GTP-Sepharose, was used to purify glutamate dehydrogenase from ox brain and liver. 2. Preparations purified in this way differed from those of the ox liver enzyme that were obtained from commercial suppliers in their mobilities on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. This difference appears to result from the occurrence of limited proteolysis during the preparation of the latter enzyme samples. 3. N-Terminal sequence analysis showed the presence of four amino acid residues in the enzyme prepared in this study that were not present in those obtained from the commercial sources and which have not been reported in previous studies on the sequence of the ox liver enzyme. 4. A preliminary examination of the enzyme prepared in this way indicated that the Michaelis constants for the substrates are similar to those obtained from the commercial preparation, but that the response to allosteric effectors was modified.     

Purification of the high-Km aldehyde reductase from rat brain and liver and from ox brain.

A procedure is described that yields an apparently homogeneous preparation of the high-Km aldehyde reductase from rat brain. This procedure is also applicable to the purification of this enzyme from rat liver and ox brain. In the latter case, however, the purified preparation could be resolved into two protein bands, both of which had enzyme activity, by polyacrylamide-gel electrophoresis. Since a sample of the ox brain enzyme from an earlier step in the purification procedure only showed the presence of a single band of activity after electrophoresis, this apparent multiplicity probably results from modification of the enzyme, possibly by oxidation, during the final step of the purification. A number of properties of the rat brain enzyme were determined and these were compared with those of the enzyme from rat liver. The two preparations were similar in their stabilities, behaviour during purification, kinetic properties, electrophoretic mobilities and amino acid compositions. Antibodies to the rat liver enzyme cross-reacted with that from brain and the inhibition of both these preparations by the antiserum was similar, further supporting the view that the enzymes from these two sources were closely similar if not identical.     

Reversible association of ox liver glutamate dehydrogenase with the inner mitochondrial membrane

A comparative study on the catalytic and allosteric properties of particulate and soluble forms of ox liver glutamate dehydrogenase has been carried out. The response of the bound enzyme to release by various effectors was investigated. The particulate enzyme was found to have catalytic activities similar to the free enzyme in contrast to its behaviour when bound to pure anionic phospholipids. Possible reasons for such outstanding differences are discussed.     

Characterization of vitamin K-dependent carboxylase from the livers from the adult ox and dicoumarol-treated calf.

The properties of the microsomal vitamin K-dependent carboxylase from the livers of the adult ox and dicoumarol-treated calf were investigated. The enzymes from both sources utilized glutamic residues of synthetic peptides as substrates and could be solubilized with Triton X-100 similarly to the enzyme from vitamin K-deficient rat liver. Under the optimal assay conditions, the microsomes from calf liver had peptide carboxylase activity comparable with that of the rat liver microsomes and 6.5-fold that of adult ox liver microsomes. The apparent Km for reduced vitamin K and the ionic strength optima of the calf and adult ox enzyme clearly differ from those of the rat enzyme. Pyridoxal phosphate activated the adult ox carboxylase only slightly, whereas the calf enzyme was activated by pyridoxal phosphate as effectively as was the enzyme from the vitamin K-deficient rat. Mn2+ activated the adult ox enzyme 9-fold and calf enzyme 22-fold under optimal conditions (no KCl). Three other divalent metal cations (Ca2+, Ba2+, and Mg2+) activated the adult ox and calf enzymes to about half the extent caused by Mn2+, KCl inhibited this activation. The vitamin K-dependent carboxylase from the dicoumarol-treated calf is apparently more tightly bound to the microsomal membrane than is the adult ox enzyme. In many other respects (pH optimum), temperature optimum, Km values for peptide substrate, substrate specificity, inhibitor effects), the properties of the adult ox and calf enzymes resemble closely those of the rat enzyme.     

Some properties of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) was purified from ox liver, and obtained essentially free from 3-oxoacyl-CoA thiolases. The kinetic behaviour, like that of the synthases from chicken liver and yeast, is compatible with a reaction pathway involving condensation of an acetyl-enzyme with acetoacetyl-CoA. The Km for acetoacetyl-CoA, less than 1 micronM at pH 7.8, may possibly be low enough to permit rapid ketogenesis under physiological conditions without the need for a binary complex between the synthase and oxoacyl-CoA thiolase.     

Purification and general properties of aspartate aminotransferase of ox heart

1. A five-step procedure for preparing highly purified aspartate aminotransferase from ox heart is described. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis in starch gel and in polyacrylamide gel. 3. The pure enzyme has an isoelectric point of about pH5, and E(1%) (1cm.) 14.40 at 278mmu. 4. The molecular weight of the pure enzyme was determined as 96000 by sedimentation equilibrium. 5. The pH optimum for the pure enzyme was about 8. It was determined by a new assay technique. 6. A difference in the electrophoretic migration rate between the enzyme from ox heart and brain and the enzyme from pig heart and brain suggests a species specificity rather than an organ specificity. 7. A new effect of deionization on the visible-absorption spectrum of the enzyme was observed.     

PURIFICATION AND CHARACTERIZATION OF OX BRAIN NAD+ -DEPENDENT ISOCITRATE DEHYDROGENASE

Abstract— The NAD⁺ -dependent isocitrate dehydrogenase from ox brain has been purified about 130-fold by a method involving affinity chromatography on an NAD⁺ -derivative of agarose. The enzyme preparation is not homogeneous but it is free from contaminating enzyme activities that could interfere with kinetic studies. The kinetic properties of the enzyme did not appear to have been altered by the purification procedure involved. The initial velocity of the reaction showed a sigmoid dependence on the concentration of isocitrate, and ADP behaved as an allosteric activator. The kinetics with NAD⁺ as the substrate were hyperbolic. The molecular weight of the purified enzyme was found to be 285,000 ± 25,000.     

The kangaroo cation-independent mannose 6-phosphate receptor binds insulin-like growth factor II with low affinity.

The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993) Endocrinology 133, 440-446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195-204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage.     

Folding domains and intramolecular ionic interactions of lysine residues in glyceraldehyde 3-phosphate dehydrogenase.

1. Treatment with methyl acetimidate was used to probe the topography of several tetrameric glyceraldehyde 3-phosphate dehydrogenases, in particular the holoenzymes from rabbit muscle and Bacillus stearothermophilus. During the course of the reaction with the rabbit muscle enzyme, the number of amino groups fell rapidly from the starting value of 27 per subunit to a value of approx. five per subunit. This number could be lowered further to values between one and two per subunit by a second treatment with methyl acetimidate. The enzyme remained tetrameric throughout and retained 50% of its initial catalytic activity at the end of the experiment. 2. Use of methyl [1-14C]acetimidate and small-scale methods of protein chemistry showed that only one amino group per subunit, that of lysine-306, was completely unavailable for reaction with imido ester in the native enzyme. This results is consistent with the structure of the highly homologous glyceraldehyde 3-phosphate dehydrogenase of lobster muscle deduced from X-ray-crystallographic analysis, since lysine-306 can be seen to form an intrachain ion-pair with aspartic acid-241 in the hydrophobic environment of a subunit-subunit interface. 3. Several other amino groups in the rabbit muscle enzyme that reacted only slowly with the reagent were also identified chemically. These were found to be located entirely in the C-terminal half of the polypeptides chain, which comprises a folding domain associated with catalytic activity and subunit contact in the three-dimensional structure. Slow reaction of these 'surface' amino groups with methyl acetimidate is attributed to intramolecular ionic interactions of the amino groups with neighbouring side-chain carboxyl groups, a conclusion that is compatible with the reported three-dimensional structure and with the dependence of the reaction of ionic stength. 4. Very similar results were obtained with the enzymes from B. stearothermophilus and from ox muscle and ox liver, supporting the view that the ion-pair involving lysine-306 and aspartic acid-241 will be a common structural feature in glyceraldehyde-3-phosphate dehydrogenases. The B. stearothermophilus enzyme was fully active after modification. 5. No differences could be detected between the enzymes from ox muscle and ox liver, in accord with other evidence that points to the identify of these enzymes. 6. The pattern of slowly reacting amino groups in the enzyme from B. stearothermophilus, although similar to that of the mammalian enzymes, indicated one or two additional intramolecular ionic interactions of lysine residues that might contribute to the thermal stability of this enzyme.     

The sulphydryl groups of ox brain and liver glutamate dehydrogenase preparations and the effects of oxidation on their inhibitor sensitivities

Glutamate dehydrogenase preparations from several sources have been shown to have suffered limited proteolysis during purification. This proteolysis has been previously shown to involve removal of the N-terminal tetrapeptide and to result in changes in the regulatory properties of the enzyme. In the present work the previously unidentified N-terminal residue of the unproteolysed enzyme from ox brain and liver is shown to be cysteine. The thiol group of this residue is masked in the native enzyme but it becomes accessible after reduction. Exposure of solutions of the unproteolysed enzyme to air oxidation causes large changes in its sensitivity to inhibition by the antipsychotic drug perphenazine, GTP and by high concentrations of NADH. No such changes occurred in the behaviour of preparations of the enzyme that had suffered proteolysis during purification under these conditions.Special issue dedicated to Dr. Santiago Grisolia.     

Kinetic studies on the major form of aldehyde reductase in ox kidney: a general kinetic mechanism to explain substrate-dependent mechanisms and the inhibition by anticonvulsants

The inhibition of the major form of ox kidney aldehyde reductase (AR 1) by sodium barbitone revealed linear mixed kinetics. This behaviour is distinct from the non-linear intercept effect we reported for valproate [Daly and Mantle (1982) Biochem. J. 205, 381]. 4-Carboxybenzaldehyde exhibits partial uncompetitive substrate inhibition. These results are discussed in terms of a model that involves nucleotide-induced isomerization and an additional flux (with some substrates and inhibitors) through an enzyme.nucleotide.substrate/inhibitor ternary complex.     

Function and transcript analysis of gibberellin-biosynthetic enzymes in wheat

The enzymes gibberellin (GA) 20-oxidase and 3-oxidase are major sites of regulation in GA biosynthesis. We have characterised one member of each of the gene families encoding these enzymes that are highly expressed in elongating stems and in developing and germinating grains of wheat and are therefore likely to have prominent developmental roles in these tissues. We mapped the three homoeologues of the GA 20-oxidase gene TaGA20ox1 to chromosomes 5BL, 5DL and 4AL. TaGA20ox1 is expressed mainly in the nodes and ears of the elongating stem, and also in developing and germinating embryos. Expression in the nodes, ears and germinating embryos is predominantly from the A and D genomes. Each homoeologous cDNA encodes a functional enzyme that catalyses the multi-step conversions of GA12-GA9, and GA53-GA20. Time course and enzyme kinetic studies indicate that the initial oxidation steps from GA12 and GA53 to the free alcohol forms of GA15 and GA44, respectively, occur rapidly but that subsequent steps occur more slowly. The intermediate GA19 has an especially low affinity for the enzyme, consistent with its accumulation in wheat tissues. The three homoeologous cDNAs for the 3-oxidase gene TaGA3ox2 encode functional enzymes, one of which was shown to possess low levels of 2beta-hydroxylase, 2,3-desaturase, 2,3-epoxidase and even 13-hydroxylase activities in addition to 3beta-hydroxylase activity. In contrast to TaGA20ox1, TaGA3ox2 is expressed in internodes, as well as nodes and the ear of the elongating stem. It is also highly expressed in developing and germinated embryos.     

Automated proton titrations of pancreatic phospholipase A2

A computer-controlled titration system has been developed, tested, and used for analysis of the proton-titration behavior of pancreatic phospholipase A₂ from ox, horse, and pig. The results revealed an error in the reported amino acid composition of the equine enzyme. A simple interface for the input of digitized data from a pH meter is described together with the software developed for controlling the titration experiments.     

Mapping of the active site of Escherichia coli methionyl-tRNA synthetase: identification of amino acid residues labeled by periodate-oxidized tRNA(fMet) molecules having modified lengths at the 3'-acceptor end.

Initiator tRNA molecules modified at the 3'-end and lacking either the A76 (tRNA-C75), the C75-A76 (tRNA-C74), the C74-C75-A76 (tRNA-A73), or the A73-C74-C75-A76 (tRNA-A72) nucleotides were prepared stepwise by repeated periodate, lysine, and alkaline phosphatase treatments. When incubated with trypsin-modified methionyl-tRNA synthetase (MTST), excess amounts of the dialdehyde derivative of each of these shortened tRNAs (tRNA-C75ox, tRNA-C74ox, tRNA-A73ox, and tRNA-A72ox) abolished both the isotopic [32P]PPi-ATP exchange and the tRNA aminoacylation activities of the enzyme. In the presence of limiting concentrations of the various tRNAox species, the relative extents of inactivation of the enzyme were consistent with the formation of 1:1 complexes of the reacting tRNAs with the monomeric modified synthetase. Specificity of the labeling was further established by demonstrating that tRNA-C75ox binds the enzyme with an equilibrium constant and stoichiometry values in good agreement with those for the binding of nonoxidized tRNA-C75. The peptides of MTST labeled with either tRNA-C75ox or tRNA-C74ox were identified. The chymotryptic digestion of the covalent MTST.[14C]tRNA-C75ox complex yielded four peptides (A-D). In the case of tRNA-C74ox, only two of the above peptides (C and D) were identified. Peptides A, B, C, and D corresponded to fragments Ser334-Phe340, Lys61-Leu65, Val141-Tyr165, and Glu433-Phe437, respectively, in the MTST primary structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heritability of dispersal in banner-tailed kangaroo rats, Dipodomys spectabilis

In a wild population of banner-tailed kangaroo rats, heritability of dispersal was estimated using two measures of dispersal tendency: distance moved from the natal site and likelihood of leaving the natal home range. Neither of the heritability measures was significantly different from 0. The results indicate that the main causes of variation in dispersal behaviour in this species are environmental, and suggest that there is no class of ‘innate’ dispersers. The possibility cannot be excluded that this population retains modest additive genetic variance for dispersal tendency. The survival consequences of dispersal in kangaroo rats are known to depend on population density and to change significantly between years, so that selection should maintain genetic variation in dispersal tendencies. Modest genetic variation for dispersal tendencies, especially if dispersal is a conditional trait, will be extremely difficult to detect in field studies.     

Parasite and predator risk assessment: nuanced use of olfactory cues

Foraging herbivores face twin threats of predation and parasite infection, but the risk of predation has received much more attention. We evaluated, experimentally, the role of olfactory cues in predator and parasite risk assessment on the foraging behaviour of a population of marked, free-ranging, red-necked wallabies (Macropus rufogriseus). The wallabies adjusted their behaviour according to these olfactory cues. They foraged less, were more vigilant and spent less time at feeders placed in the vicinity of faeces from dogs that had consumed wallaby or kangaroo meat compared with that of dogs feeding on sheep, rabbit or possum meat. Wallabies also showed a species-specific faecal aversion by consuming less food from feeders contaminated with wallaby faeces compared with sympatric kangaroo faeces, whose gastrointestinal parasite fauna differs from that of the wallabies. Combining both parasite and predation cues in a single field experiment revealed that these risks had an additive effect, rather than the wallabies compromising their response to one risk at the expense of the other.     

Some physical and immunological properties of ox kidney biliverdin reductase.

The liver, kidney and spleen of the mouse and rat and the kidney and spleen of the ox express a monomeric form of biliverdin reductase (Mr 34,000), which in the case of the ox kidney enzyme exists in two forms (pI 5.4 and 5.2) that are probably charge isomers. The livers of the mouse and rats express, in addition, a protein (Mr 46,000) that cross-reacts with antibodies raised against the ox kidney enzyme and may be related to form 2 described by Frydman, Tomaro, Awruch & Frydman [(1983) Biochim. Biophys. Acta 759, 257-263]. Higher-Mr forms appear to exist in the guinea pig and hamster. The ox kidney enzyme has three thiol groups, of which two are accessible to 5,5'-dithiobis-(2-nitrobenzoate) in the native enzyme. Immunocytochemical analysis reveals that biliverdin reductase is localized in proximal tubules of the inner cortex of the rat kidney. Biliverdin reductase antiserum also stains proximal tubules in human and ox kidney. The staining of podocytes in glomeruli of ox kidney with antiserum to aldose reductase is particularly prominent. The localization of biliverdin reductase in the inner cortical zone of rat kidney is similar to that described for glutathione S-transferase YfYf, and it is suggested that one function of this 'intracellular binding protein' may be to maintain a low free concentration of biliverdin to allow biliverdin reductase to operate efficiently.