Biochemical and immunological studies of angiotensin converting enzymes from human, bovine, dog, hog, rabbit, rat and sheep kidneys

1. Molecular and kinetic properties of angiotensin converting enzymes from seven different species' kidneys were similar concerning apparent molecular weight, heat sensitivity, Km value, optimum pH, activation by chloride ion, and inhibition by specific converting enzyme inhibitors (captopril and SA 446). 2. Rabbit antibody against pure human kidney enzyme cross-reacted partially with each animal kidney enzyme except for the rabbit enzyme. Antigenic determinants of animal enzymes were variable from species to species and differed from those of the human enzyme in extent and specificity.     

Thiamine pyrophosphatase and nucleoside diphosphatase in rat brain

Two types of nucleoside diphosphatase were found in rat brain. One (Type L) had similar properties to those of the liver microsomal enzyme with respect to its isoelectric point, substrate specificity, Km values, optimum pH, activation by ATP and molecular weight. The other (Type B), which separated into multiple forms on isoelectric focusing, had lower Km values and a smaller molecular weight than the Type L enzyme, and was inhibited by ATP. The Type B enzyme catalyzed the hydrolysis of thiamine pyrophosphate as well as those of various nucleoside diphosphates at physiological pH, while Type L showed only nucleoside diphosphatase activity at neutral pH. These findings suggest that the two enzymes play different physiological roles in the brain.     

Comparison of dipeptidyl peptidase IV prepared from pig liver and kidney

Dipeptidyl peptidase IV (dipeptidylpeptide hydrolase, EC 3.4.14.-) has been purified from the microsomal fraction of pig liver, using an immunoaffinity chromatography, and its properties compared with those of the enzyme purified from pig kidney. The amino acid compositions of both enzymes were similar. The same kinds of carbohydrates were found in both enzymes, but there were differences in the molar concentrations of individual sugars. The liver enzyme had greater concentrations of mannose, fucose and sialic acid than the kidney enzyme, while the concentrations of galactose and glucosamine were greater in the kidney enzyme. The carbohydrates accounted for approx. 18.3 and 22.7% of the weight of the kidney and liver enzymes, respectively. The pH optima, molecular weights, substrate specificities and Km values of the two enzymes and the effects of diisopropylfluorophosphate on their activities were nearly identical. The liver enzyme was heat- and pH-sensitive, but not attacked by proteinases.     

Unique cathepsin D-type proteases in rat thoracic duct lymphocytes and in rat lymphoid tissues.

Two unique cathepsin D-type proteases apparently present only in rat thoracic duct lymphocytes and in rat lymphoid tissues are described. One, termed H enzyme, has an apparent molecular weight of similar to95,000; the other, termed L enzyme, has an apparent molecular weight of similar to45,000, in common with that of most cathepsins D from other tissues and species. Both enzymes differ from cathepsin D, however, by a considerably greater sensitivity to inhibition by pepstatin and by a smaller degree of inhibition by an antiserum which inhibits rat liver cathepsin D. H enzyme is converted to L enzyme by treatment with beta-mercaptoethanol; the relationship between the two enzymes remains unknown. H and L enzyme have been detected in rat lymphoid tissues and in mouse spleen, but they are not present in other rat tissues (liver, kidney, adrenals), rabbit tissues, calf thymus, bovine spleen, or human tonsils. As measured on acid-denatured bovine hemoglobin as substrate, both enzymes have pH activity curves identical with that of rat liver cathepsin D, with optimal activity at pH 3.6. Activity on human serum albumin is much less and also shows an optimum at pH 3.6; hence, neither enzyme has the properties of cathepsin E. Thiol-reactive inhibitiors have no effect on the activity of H and L enzyme; thus they do not belong to the B group of cathepsins. Additional information, discussed in this paper, leads us to conclude that partially purified H and L enzymes are cathepsin D-type proteases.     

Rat peptidylglycine alpha-amidating enzyme: the relation between activities at neutral and alkaline pH Values

A substantially high level of alpha-amidating activity at an alkaline pH (8-9.5), often seen as another pH optimum peak in addition to the neutral one, has been observed in various rat tissues. We have also found that crude enzymes from rat brain, pituitary, and small intestine showed a pH profile with two pH optima at neutral pH (6.5-7) and alkaline pH (8.5-9) when D-Tyr-Val-Gly was used as substrate. With a combination of ion-exchange and gel filtration chromatographies, we obtained two fractions, S-1 and S-2, from rat brain; S-1 contained an alpha-amidating enzyme of an apparent molecular weight of 36,000 (36K enzyme) exhibiting a single pH optimum at 8.5. On the other hand, S-2 apparently showed almost no or only marginal activity at either pH 7 or 8.5, but when S-2 was combined with S-1, a neutral pH optimum at 7 could be elicited. The factor in S-2 that was responsible for this combined action was a protein of an apparent molecular weight of 41,000 (41K protein). Both proteins were found to be colocalized in the same subcellular organelle, probably in the secretory granule. It seems likely, then, that the pH profiles characterized by two optimal peaks seen in crude rat enzymes can be attributed to the presence at an appropriate ratio of the 41K protein and 36K enzyme.     

Prostaglandin-E2-9-ketoreductase in rabbit kidney.

The 100,000 xg supernatant of rabbit kidney contains a prostaglandin-E2-9-ketoreductase which has an obligatory requirement for NADPH. This enzyme is localised in the renal cortex and is able to quantitatively convert PGE2 to PGF2alpha. A broad pH profile was evident with an optimum at pH 7 with 5. Kinetic studies indicated a Km of 3 with 2 x 10-4M PGE2. The isoelectric point was at pH 5 with 65 and the molecular weight, as estimated by gel filtration, was 21,800. These values differ from those obtained with enzyme from monkey brain tissue and suggest a tissue specificity of PGE2-9-ketoreductase. By combining isoelectric focussing techniques with sephadex filtration considerable purification of the renal enzyme was achieved.     

Organ distribution of rat kynureninase and changes of its activity during development

Kynureninase activity was measured in various organs of the rat by a sensitive assay method based on the use of an HPLC system. High activities were detected in liver, kidney and spleen, and much lower activities in adrenals, intestine, lung, heart, brain, skeletal muscle and pancreas. Kynureninase of liver, kidney and spleen showed the same molecular weight (110,000) and the same isoelectric point (pI 5.4). They also showed the same properties of heat stability and apparent optimum pH. The enzymes in kidney and spleen were localized in the cytosol. The developmental study showed increasing activities of the enzyme after birth and a maximum on the 60th day in both liver and spleen. The activity in kidney increased after birth and reached a plateau on the 30th day.     

Purification and characterization of kynurenine--2-oxoglutarate aminotransferase from the liver, brain and small intestine of rats.

1. Kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) was purified to homogeneity from the liver, brain and small intestine of rats by the same procedure. The three enzyme preparations had nearly identical pH optima, substrate specificities and molecular weights. Isoenzyme 1 was active with 2-oxoglutarate but not with pyruvate as amino acceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order to activity: L-aspartate greater than L-tyrosine greater than L-phenylalanine greater than L-tryptophan greater than 5-hydroxy-L-tryptophan greater than L-kynurenine. The molecular weight was approximately 88 000 as determined by sucrose-density-gradient centrifugation. The pH optimum was between 8.0 and 8.5. On the basis of substrate specificity, substrate inhibition, subcellular distribution and polyacrylamide-disc-gel electrophoresis, it is suggested that liver, brain and small intestinal kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) is identical with mitochondrial tyrosine-2-oxoglutarate aminotransferase and also with mitochondrial aspartate-2-oxoglutarate aminotransferase. 2. An additional kynurenine-2-oxoglutarate aminotransferase (isoenzyme 2) was purified from the liver. This enzyme was specific for 2-oxoglutarate and L-kynurenine. Sucrose-density-gradient centrifugation gave a molecular weight of approximately 100 000. The pH optimum was between 6.0 and 6.5. This enzyme was not detected in the brain or small intestine.     

Purification and properties of rat brain dipeptidyl aminopeptidase

Dipeptidyl aminopeptidase, which hydrolyzes the 7-(Gly-Pro)-4-methylcoumarinamide, has been purified from the brains of 3 week-old rats. It was purified about 2,600-fold by column chromatography on CM-cellulose, hydroxyapatite and Gly-Pro AH-Sepharose. This enzyme hydrolyzed Lys-Ala-beta-naphthylamide well with an optimum pH of 5.5. It was inhibited by diisopropyl fluorophosphate, phenyl-methanesulfonyl fluoride, some cations, and puromycin, but was not inhibited by p-chloromercuribenzoate, N-ethylmaleimide, dithiothreitol, EDTA, iodoacetic acid, and bacitracin, indicating that rat brain dipeptidyl aminopeptidase is a serine protease. This enzyme showed a molecular weight of 220,000 by gel filtration and of 51,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The properties of purified rat brain dipeptidyl aminopeptidase were similar to those of bovine pituitary dipeptidyl peptidase II, but the molecular weight and substrate specificity of these enzymes were different.     

Biochemical characterization, cloning, and sequencing of ADP-dependent (AMP-forming) glucokinase from two hyperthermophilic archaea, Pyrococcus furiosus and Thermococcus litoralis

The ADP-dependent (AMP-forming) glucokinases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus litoralis catalyze the phosphorylation of glucose using ADP as the essential phosphoryl group donor. Both enzymes were purified to homogeneity and characterized with regard to each other. The enzymes had similar enzymological properties as to substrate specificity, coenzyme specificity, optimum pH, and thermostability. However, a difference was observed in the subunit composition; while the T. litoralis enzyme is a monomer with a molecular mass of 52 kDa, the P. furiosus enzyme has a molecular mass of about 100 kDa and consists of two subunits with identical molecular masses of 47 kDa. The genes encoding these enzymes were cloned and sequenced. The gene for the P. furiosus enzyme contains an open reading frame for 455 amino acids with a molecular weight of 51,265, and that for the T. litoralis enzyme contains an open reading frame for 467 amino acids with a molecular weight of 53,621. About 59% similarity in amino acid sequence was observed between these two enzymes, whereas they did not show similarity with any ATP-dependent kinases that have been reported so far. In addition, two phosphate binding domains, and adenosine and glucose binding motifs commonly conserved in the eukaryotic hexokinase family were not observed.     

Organ secificity of rat sodium- and potassium-activated adenosine triphosphatase.

We compared several Na,K-ATPase preparations from various organs of the rat. The brain Na,K-ATPase differed from the enzymes of other organs in its pH dependence and responses to ouabain and N-ethylmaleimide in spite of similarities in the kinetic parameters of activation by Na+, K+, Mg2+, and ATP. The optimum pH of the brain MaI-enzyme was at 7.4 to 7.5 at 37 degrees D. The Lubrol extract of this brain enzyme preparation showed a lower optimum oH of 6.6. When the Lubrol extract of the brain was fractionated wtih (NH4)2SO4, the activity of the precipitate in the neutral pH region was restored. On the other hand, the optimum pH of the kidney NaI-enzyme was slightly affected by Lubrol and ammonium sulfate treatments (pH 7.5 leads to 7.3). The brain enzyme (K 1/2 = 0.9 microM) showed about 100-fold higher sensitivity to ouabain than the enzymes from other organs (I 1/2 = 100 microM) in the presence of 120 mM Na+ and 10 mM K+. In a Hill plot of the ouabain inhibition, the former failed to give a linear relationship, while the latter gave a straight line with a Hill coefficient of 1.0. The effect of K4 on the brain enzyme-ouabain interaction led us to consider that the brain enzyme might have two components as regards ouabain affinity, high and low affinity components. The time course of N-ethylmaleimide inhibition of the brain enzyme was rapid and biphasic, while the kidney enzyme showed only a slow phase following pseudo-first order kinetics. ATP protected the kidney enzyme activity completely agai,st N-ethylmaleimide inhibition, but the protection of the brain enzyme activity by ATP was only partial. We divided rat Na,K-ATPases into two groups, the brain type, which is restricted to the central nervous system, and the kidney type, which is found in most organs.     

Purification and properties of GM1 ganglioside beta-galactosidases from bovine brain

Two GM1-beta-galactosidases, beta-galactosidases I, and II, have been highly purified from bovine brain by procedures including acetone and butanol treatments, and chromatographies on Con A-Sepharose, PATG-Sepharose, and Sephadex G-200. beta-Galactosidase I was purified 30,000-fold and beta-galactosidase II 19,000-fold. Both enzymes appeared to be homogeneous, as judged from the results of polyacrylamide disc gel electrophoresis. Enzyme I had a molecular weight of 600,000-700,000 and enzyme II one of 68,000, as determined on gel filtration. On sodium dodecyl sulfate polyacrylamide slab gel electrophoresis under denaturing conditions, enzyme II gave a single band with a molecular weight of 62,000, while enzyme I gave two minor bands with molecular weights of 32,000 and 20,000 in addition to the major band at 62,000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide but not from galactosylceramide. Enzyme I showed a pH optimum of 4.0 and was heat stable, while enzyme II showed a pH optimum of 5.0 and lost 50% of its activity in 15 min at 45 degrees C. Enzyme I showed a pI of 4.2 and enzyme II one of 5.9.     

Purification of glutamate dehydrogenase from the rabbit liver and study of its major physico-chemical properties

Highly purified preparations of glutamate dehydrogenase were obtained from mitochondrial and cytoplasmic fractions of rabbit liver by affinity chromatography on CL-Sepharose 4B modified by adenosine diphosphate. Some physico-chemical properties of the purified enzymes (e. g., specific activity, molecular weight, quaternary structure, stability against denaturating effect of urea, pH optimum of catalyzed reactions, Km values for substrates and coenzymes) were found to be identical. The sole difference was detected in the ability of enzyme preparations to be activated by adenosine diphosphate. The activation of the cytoplasmic enzyme is 160%, that of mitochondrial glutamate dehydrogenase is 230-240% under the same conditions.     

Purification and characterization of gamma-glutamyl transpeptidase from human pancreas

Gamma-glutamyl transpeptidase was purified from human pancreas to an electrophoretically homogeneous state. The enzyme was separated into two active fractions on a DEAE-cellulose column. Both enzyme preparations had the same molecular weight (9 x 10(4)) and were composed of two nonidentical subunits (molecular weight, 61,000 and 27,000). While the optimum pH and pH- and thermal-stability range of both enzymes were identical, their isoelectric points were considerably different. Following incubation with neuraminidase, however, the isoelectric point of F-11 became similar to that of F-I, suggesting that this difference in electrophoretic mobility is due to a difference in the content of sialic acid moiety.     

The purification, properties and characterization of three forms of alpha-L-fucosidase from monkey brain.

alpha-L-Fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) has been purified to apparent homogeneity (about 22 000-fold over the crude homogenate) from monkey brain. Values of kinetic constants for the purified enzyme were as follows: pH optimum, 5.0; Km, 0.22 mM; V, 913 mumol/mg per h. alpha-L-Fucose was a competitive inhibitor (Ki, 0.275 mM) of the enzyme. Evidence for the involvement of sulphydryl group(s) and carboxyl group containing amino acid(s) in the catalytic process is presented. The purified enzyme was a tetramer of molecular weight of 285 000 of identical subunits of 73 500 held together by non-covalent forces. Gel filtration studies revealed the presence of three molecular forms of the activity in the purified preparation which appeared to be the tetramer, dimer and monomer. The existence of three types of activities was also aupported by a triphasic heat inactivation profile of the enzyme at 50 or 55 degrees C and the distinctly different pH activity profiles of the differentially heat-inactivated enzymes. Immunodiffusion studies using antibody developed against purified monkey brain alpha-L-fucosidase showed that the monkey brain enzyme had only partial immunological identity with the enzymes from the non-neural tissues of monkey as well as the human and rat liver and the rat brain. However, the monkey brain and liver enzymes appeared to be similar to the human brain and liver enzymes, respectively.     

Similarities in properties and a functional difference in purified leucyl-tRNA synthetase isolated from two developmental stages of Tenebrio molitor

In Tenebrio molitor, as well as in other biological systems, there are indications that differences in leucyl-tRNA synthetase activity may play a role in translational control. However, it has not been clear whether the difference in activity is due to the appearance of a multiplicity of enzymes during development or to the alteration of a single enzyme.The purification of leucyl-tRNA synthetase from day 1 and day 7 after the larval pupal molt of Tenebrio molitor is described. The enzyme from both developmental stages was purified over a 1000-fold. The two enzyme preparations are identical in molecular weight (99,000). They show the same characteristics after aging. The pH optimum, heat inactivation behavior, and dependency on divalent cations are the same for both enzymes. They also show identical kinetics with similar values of Km for leucine, ATP, Mg²⁺, and tRNA day 1. However, leucyl-tRNA synthetase purified from day 7 exhibits an additional function in recognizing a new species of isoaccepting tRNA in day 7 tRNA. We have tentatively concluded that the two enzymes are probably different forms of the same enzyme and the additional activity is due to alteration of the enzyme at the macromolecular level during development.     

Calmodulin-dependent protein kinases purified from rat brain and rabbit liver

A calmodulin-dependent protein kinase was purified from rat brain by the same protocol used previously for a rabbit liver calmodulin-dependent glycogen synthase kinase. The rat brain kinase readily phosphorylated rabbit skeletal muscle glycogen synthase at sites 1b and 2, the same sites phosphorylated by rabbit liver calmodulin-dependent kinase. The two kinases have other similarities: substrate specificity, potent inhibition by sodium fluoride, and nearly equal Ka's (10-20 nM) for calmodulin. Also, both enzymes have similar Stokes radii, 70 A (rabbit liver) and 75 A (rat brain), but quite different sedimentation coefficients, 10.6 S and 17.4 S, respectively. Consequently, the calculated molecular weights are also different: 560,000 for the brain enzyme and 300,000 for the liver enzyme. The major subunit of the rat brain kinase appears to be a single 51-kDa peptide, not a doublet pattern of 51- and 53-kDa subunits that is characteristic of the rabbit liver enzyme. Our findings are consistent with the hypothesis that the rat brain and rabbit liver enzymes belong to a class of closely related calmodulin-dependent protein kinases, possibly isozymes. This class of enzymes may be responsible for regulating several of the known calcium-dependent physiological functions.     

Reduced triphosphopyridine-liked aldehyde reductase. II. Species and tissue distribution

A 30,000 MW, barbiturate sensitive TPNH-linked aldehyde reductase which reduces aromatic aldehydes (3-pyridinecarboxaldehyde, 4-nitrobenzaldehyde, 4-cyanobenzaldehyde), d-glyceraldehyde, d-glucuronate and (+)-camphorquinone was found in liver, kidney, brain and heart tissue from a variety of animals. In livers, rabbit kidney, rabbit heart, and bovine brain a high molecular weight reductase was also found, which was less sensitive to barbiturate inhibition and had higher reactivity for cyclohexanone and d-ribose. The low molecular weight, TPNH-linked aldehyde reductases are probably homologous and should be classified under the systematic name alcohol:NADP oxidoreductase (EC 1.1.1.2). Since the substrate specificity of aldehyde reductase overlaps several previously described TPNH-linked reductases from the same tissues, reexamination of the properties of these enzymes for reclassification as EC 1.1.1.2 is necessary.     

Comparative studies of two types of acid proteases from rat gastric mucosa and spleen

Two types of acid proteases, cathepsin D and cathepsin E-like enzyme, from rat gastric mucosa and spleen were compared in their biochemical and immunochemical properties. The enzymes were partially purified by employing the same chromatographic procedures and they showed a single proteolytically active band in polyacrylamide gel electrophoresis. Two low molecular weight enzymes, cathepsins D, from both tissues showed the same molecular weight and the same sensitivities to various inhibitors, but slightly different electrophoretic mobilities. The rabbit antiserum raised against gastric mucosa cathepsin D precipitated both enzymes. On the other hand, high molecular weight enzymes, gastric mucosa cathepsin D-like acid proteinase and spleen cathepsin E-like acid proteinase, were similar to each other as judged by their chromatographic profiles, electrophoretic mobilities, and high stabilities in urea solution. Furthermore, the antiserum specific to gastric mucosa cathepsin D-like acid proteinase inhibited both enzyme activities in a similar manner. However, the antiserum specific to one type of enzyme did not react with the other type. These results indicate that: gastric mucosa cathepsin D is immunologically identical with spleen cathepsin D; gastric mucosa cathepsin D-like acid proteinase has biochemical and immunological properties quite similar to spleen cathepsin E-like enzyme; these two types of acid proteases are quite different proteins existing in the individual tissues.     

Prostaglandin-E2-9-ketoreductase in rabbit kidney

The 100,000 x g supernatant of rabbit kidney contains a prostaglandin-E₂-9-ketoreductase which has an obligatory requirement for NADPH. This enzyme is localised in the renal cortex and is able to quantitatively convert PGE₂ to PGF_2α. A broad pH profile was evident with an optimum at pH 7·5. Kinetic studies indicated a K_m of PGE₂. The isoelectric point was at pH 5·65 and the molecular weight, as estimated by gel filtration, was 21,800. These values differ from those obtained with enzyme from monkey brain tissue and suggest a tissue specificity of PGE₂-9-ketoreductase. By combining isoelectric focussing techniques with sephadex filtration considerable purification of the renal enzyme was achieved.