Comparison of the Soluble and Membrane-Bound Forms of the Puromycin-Sensitive Enkephalin-Degrading Aminopeptidases from Rat

Enkephalin degradation in brain has been shown to be catalyzed, in part, by a membrane-bound puromycin-sensitive aminopeptidase. A cytosolic puromycin-sensitive aminopeptidase with similar properties also has been described. The relationship between the soluble and membrane forms of the rat brain enzyme is investigated here. Both of these aminopeptidase forms were purified from rat brain and an antiserum was generated to the soluble enzyme. Each of the aminopeptidases is composed of a single polypeptide of molecular mass 100 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography. The antisoluble aminopeptidase antiserum reacts with both enzyme forms on immunoblots and inhibits both with nearly identical inhibition curves. The isoelectric points (pI = 5.0) of both forms were shown to be identical. N-terminal sequencing yielded a common sequence (P-E-K-R-P-F-E-R-L-P-T-E-V-S-P-I-N-Y) for both enzyme forms, and peptide mapping yielded 26 peptides that also appeared identical between the two enzyme forms. Studies on the nature of the association of the membrane enzyme form with the cell membrane suggest that this enzyme form does not represent the soluble form trapped during the enzyme preparation. It is suggested that the membrane form of the puromycin-sensitive aminopeptidase is identical to the soluble enzyme and that it associates with the membrane by interactions with other integral membrane proteins.     

Some properties of partially purified preparations of cAMP phosphodiesterase from beef heart]

Beef heart cAMP phosphodiesterase (EC 3.1.4.17) was isolated and partially purified using fractionation by ammonium sulfate and gel filtration on the columns with Sephadex G-200 and Sepharose 6B. This method allowed to preserve the enzyme binding to the low-molecular weight thermostable protein regulator of the phosphodiesterase activity. The enzyme preparation was purified 130--180-fold as compared to the original homogenate. The pH-dependence of the enzyme activity in the imidazole and tris -- buffers for the fraction with maximal activity was carried out. The kinetic analysis of this fraction revealed an abnormal kinetic behaviour with two Km values. The enzyme is represented by four forms differing in their molecular weights and possessing different capacity for activation by Ca2+ and protein regulator. No activation was observed in the forms with higher molecular weights, whereas the activity of the forms with lower molecular weights depended on the presence of Ca2+ and protein regulator. It is assumed that some of the above-described forms are capable of interconversions.     

Multiple molecular forms of purified human erythrocyte acetylcholinesterase.

1. Human erythrocyte acetylcholinesterase was solubilized by Triton X-100 and purified by affinity chromatography to a specific activity of 3800 IU/mg of protein. The yield of the purified enzyme was 25--45%. 2. Gel filtration on Sepharose 4-B in the presence of Triton X-100 revealed one peak of enzyme activity with a Stokes' radius of 8.7 nm. Density gradient centrifugation in 0.1% Triton X-100 showed one peak of enzyme activity with an S4 value of 6.3S. 3. Isoelectric focusing in Triton X-100 resolved the enzyme into five molecular forms with isoelectric points of 4.55, 4.68, 4.81, 4.98 and 5.18. Upon incubation with neuraminidase the enzyme activity in the first four forms was decreased with a concommitant increase in activity in the form with the higher isoelectric point. 4. After removal of excess Triton X-100 on Bio-Gel HTP, polyacrylamide gel electrophoresis showed seven bands of protein and corresponding bands of enzyme activity. Density gradient centrifugation of the detergent-depleted enzyme at high ionic strength revealed five multiple molecular forms with S4 values of 6.3 S, 10.2 S, 12.2 S, 14.2 S and 16.3 S. At low ionic strength, higher aggregates were observed in addition to the other forms. Dodecylsulfate-polyacrylamide gel electrophoresis gave one subunit only with an apparent molecular weight of 80 000. 5. These results suggest that human erythrocyte acetylcholinesterase, solubilized by Triton X-100, exists in various forms differing in net charge but of apparently similar molecular dimensions. After removal of the detergent, forms with different molecular sizes are observed.     

Purification and characterization of homogeneous sunflower seed acid phosphatase

Acid phosphatase (EC 3.1.3.2) from sunflower seed was purified 1800-fold to homogeneity using both conventional and affinity chromatographic methods. The purified enzyme was a mixture of two enzyme forms distinguishable by polyacrylamide gel electrophoresis (PAGE). Gel exclusion chromatography, which did not distinguish between the two forms, gave an apparent M, of 103 000. Preparative PAGE permitted the separation of the two forms, and SDS-PAGE showed that they contained equivalent peptide subunits of apparent M, 56 000 and 52 000. Amino acid analysis indicated that both enzyme forms have similar amino acid compositions. Data on substrate specificity and pH dependence is presented. The kinetic constants for hydrolysis of p-nitrophenyl phosphate as catalysed by sunflower seed acid phosphatase were independent of pH in the range 3-5. The enzyme was competitively inhibited by inorganic phosphate and non-competitively inhibited by phosphomycin.     

Non-equivalency of active centers of alpha-ketoglutarate dehydrogenase detected by modification of histidine residues

Using selective chemical modification of histidine residues of the alpha-ketoglutarate dehydrogenase component within the alpha-ketoglutarate dehydrogenase complex, the existence of interconvertible forms of the enzyme was demonstrated. These forms are distinguished by kinetics of inactivation caused by diethylpyrocarbonate. The interconversion of the enzyme forms involves alpha-ketoglutarate. Studies on substrate effects on the inactivation kinetics of individual enzyme forms revealed the non-equivalency of the enzyme active centers within the dimeric molecule of the alpha-ketoglutarate dehydrogenase component. The accessibility of an essential histidine residue in the active center of a neighbouring substrate-free monomer to the modifier increases as a result of interaction of the enzyme active centers during alpha-ketoglutarate binding by one of the subunits. The non-equivalency of the active centers manifests itself in different rates of interaction and in the unequal stability of binding of alpha-ketoglutarate to the alternate sites of the dimer. It is assumed that the biphasic kinetics of inactivation of pigeon breast muscle alpha-ketoglutarate dehydrogenase is due to tight binding of alpha-ketoglutarate in one of active centers of the enzyme dimeric molecule.     

Comparative study on vertebrate liver AMP deaminases

Similar activity of AMP deaminase was found in rat, hen, turtle and flounder liver when estimated at high AMP concentration. The enzyme activity was of an order of magnitude higher in frog liver. Simple step by step phosphocellulose column chromatography revealed two forms of AMP deaminase in chicken and flounder liver and one form in the liver of rat and turtle. All enzymes (except for frog liver AMP deaminase) were activated by ATP. The enzymes from rat, frog and both forms from flounder were also activated by ADP. GTP exhibited a variety of effects. The enzyme from rat and turtle was inhibited, both forms from hen and flounder were activated and frog liver enzyme was not influenced.     

The pigeon heart 5''-nucleotidase responsible for ischaemia-induced adenosine formation.

The pH-induced reversible dissociation of pigeon liver malic enzyme (EC 1.1.1.40) was studied by combined use of chemical cross-linking and SDS/polyacrylamide-gel electrophoresis. The tetrameric enzyme showed a pH-dependent dissociation in an acidic environment. At pH values above 8.0 most molecules existed as tetramers. The enzyme was gradually dissociated at lower pH. When the pH was below 5.0 most of the enzyme was present as the monomeric forms. Reassociation of the subunits was accomplished by adjusting the pH to neutrality. The dissociation and reassociation were almost instantaneous. No trimer was detected. The pigeon liver malic enzyme was thus shown to have a double-dimer quaternary structure with D2 symmetry. In the presence of substrates, the monomer-dimer-tetramer equilibrium favours the direction of dissociation. Tartronate, an L-malate analogue, was found to be more effective than L-malate in this process. When the monomeric forms were immobilized, the enzyme subunits were found to be fully active in catalysis. A possible arrangement of the four identical subunits of the enzyme molecule is proposed to account for the results obtained in this investigation. The origin of the half-of-the-sites reactivity of pigeon liver malic enzyme is also discussed.     

Characterization of multiple forms of phosphoinositide-specific phospholipase C from bovine aorta.

Three forms (I, II and III) of phospholipase C were separated from the cytosol of bovine aorta by chromatography on Blue Sepharose. All three forms showed an increase of enzyme activity when free Ca2+ in the assay was raised between 40 microM and 9 mM. The pH optimum was in the range of 6.0 to 6.5 for each subtype. Marked differences in thermostability were found when the three enzyme forms were pre-incubated at 50 degrees C prior to the assay. All three forms were able to hydrolyse phosphatidylinositol as well as phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. In contrast, when phosphatidylcholine was used as substrate, no enzyme activity was observed. Spermine and spermidine, but not putrescine, were able to stimulate form I and III; neomycin sulphate inhibited all three subtypes.     

Partial purification and characterization of the interconvertible forms of trehalase from Saccharomyces cerevisiae

Cryptic trehalase from Saccharomyces cerevisiae was purified about 3000-fold. The recovery of 970% of the original "activity" indicated the removal of an inhibitor of the enzyme. Active trehalase, obtained through phosphorylation of cryptic trehalase by cAMP-dependent protein kinase, was isolated by chromatography on DEAE-cellulose. A major phosphorylated protein, with an apparent Mr of 86,000, was detected after SDS-polyacrylamide gel electrophoresis. This protein band correlated exactly with the elution profile of trehalase activity and 32Pi incorporation into the enzyme on DEAE-cellulose chromatography. Partially purified active trehalase showed absolute specificity towards trehalose with an apparent Km of 4.79 X 10(-3) M. Both forms of the enzyme showed an apparent molecular weight of 160,000, by gel filtration. Centrifugation on a glycerol density gradient indicated multiple forms of trehalase-c, with Mr of 320,000, 160,000, and 80,000. After activation of each of these forms by protein kinase, a single form of trehalase-a was observed, with a Mr of 160,000. Trehalase-c appears to be a totally inactive form of the enzyme. The only mechanism of activation seems to be phosphorylation by cAMP-dependent protein kinase. When the protein kinase concentration was varied, at a fixed trehalase-c concentration, a sigmoidal activation plot was obtained. This result suggests the occurrence of multiple forms of cryptic trehalase.     

Investigation of structure and rate of synthesis of ornithine decarboxylase protein in mouse kidney

An immunoblotting technique was used to study the forms of ornithine decarboxylase present in androgen-induced mouse kidney. Two forms were detected which differed slightly in isoelectric point but not in subunit molecular weight (approximately 55 000). Both forms were enzymatically active and could be labeled by reaction with radioactive alpha-(difluoromethyl)-ornithine, an enzyme-activated irreversible inhibitor. On storage of crude kidney homogenates or partially purified preparations of ornithine decarboxylase, the enzyme protein was degraded to a smaller size (Mr approximately 53 000) without substantial loss of enzyme activity. The synthesis and degradation of ornithine decarboxylase protein were studied by labeling the protein by intraperitoneal injection of [35S]methionine and immunoprecipitation using both monoclonal and polyclonal antibodies. The fraction of total protein synthesis represented by renal ornithine decarboxylase was increased at least 25-fold by testosterone treatment of female mice and was found to be about 1.1% in the fully induced androgen-treated female. Both forms of the enzyme were rapidly labeled in vivo, and the immunoprecipitable ornithine decarboxylase protein was almost completely lost after 4-h exposure to cycloheximide, confirming directly the very rapid turnover of this enzyme. Treatment with 1,3-diaminopropane which is known to cause a great reduction in ornithine decarboxylase activity did not greatly selectively inhibit the synthesis of the enzyme. However, 1,3-diaminopropane did produce an increase in the rate of degradation of ornithine decarboxylase and a general reduction in protein synthesis. These two factors, therefore, appear to be responsible for the loss of ornithine decarboxylase activity and protein in response to 1,3-diaminopropane.     

Purification and characterization of Aspergillus ficuum endoinulinase

Endoinulinase from Aspergillus ficuum, which catalyzes the hydrolysis of inulin via an endo-cleavage mode, was purified by chromatography from Novozym 230 as a starting commercial enzyme mixture on CM-Sephadex and DEAE-Sepharose, and by preparative electrophoresis under native conditions. The enzyme was estimated to be pure on the basis of its I/S ratio, whose value was infinite in our assay conditions. Two forms separated by using this method. SDS gel electrophoresis showed the two purified forms to respectively exhibit molecular weights of 64,000 +/- 500 and 66,000 +/- 1,000. The results of deglycosylation indicated that the two forms were originally the same protein but with different sugar contents. A molecular weight of 54,800 +/- 1,500 was found by gel filtration of the native enzyme, indicating the native functional protein to be a monomer. The enzyme showed nearly absolute substrate specificity towards inulin and inulooligosaccharides, and acted via an endo-attack to produce mainly inulotriose during the late stage of the reaction. The apparent Km and Vmax values for inulin hydrolysis were 8.1 +/- 1.0 mM and 773 +/- 60 U/mg, respectively. The internal peptides of the enzyme showed sequence homology to the endoinulinase of Penicillium purpurogenum.     

Interconversion of multiple forms of tyrosine aminotransferase in vitro and in vivo in cultured hepatoma cells.

Corticosteroid-induced tyrosine aminotransferase (EC 2.6.1.5) from cultured hepatoma cells was separated by carboxymethyl-Sephadex chromatography into three molecular forms resembling those described previously in the rat liver. Enzyme forms were isolated and used as purified substrates to examine their in vitro interconversion by various subcellular fractions. Isolated form III was converted to forms II and I, and isolated form II was converted to form I by the coarse particulate fraction sedimenting at 1000 X g. This activity was inhibited by the serine enzyme inhibitor phenylmethane sulfonyl fluoride or by raising the pH to 8.7. Conversion of enzyme forms in vitro in the opposite direction (I leads to II leads to III) could not be detected. The distribution of enzyme forms in vivo was examined by the use of experimental conditions that prevent their in vitro interconversion during cell extraction. Tyrosine aminotransferase extracted from cell subjected to various treatments that affect the rates of enzyme synthesis or degradation existed always predominantly as form III. It appears, therefore, that multiple forms of tyrosine aminotransferase are not related to the turnover of this enzyme in vivo.     

Endocytosis and degradation of native, cathepsin D-degraded, and glutathione-inactivated aldolase by perfused rat liver

The uptake and degradation of 125I-labeled (a) native aldolase, (b) cathepsin D-inactivated aldolase, and (c) aldolase inactivated by oxidized glutathione were studied in perfused rat liver. All three forms of aldolase were removed from the perfusion medium and degraded by the liver, but the uptake of the glutathione-inactivated enzyme (half-life in perfusate = 10 min) was much faster than that of the native enzyme (half-life = 30 min) or the cathepsin-inactivated enzyme (half-life = 42 min). The degradation of the enzyme was almost totally inhibited by leupeptin, indicating that thiol proteinases in lysosomes play an important role in the digestion process. Degradation of native and cathepsin D-inactivated aldolase appeared to be slower than that of the glutathione-inactivated enzyme but studies in which liver was preloaded with aldolase by perfusion at 19 degrees C and then warming to 37 degrees C indicated that the rate of degradation of all three forms was similar. It is concluded that the liver is capable of distinguishing between the glutathione-altered aldolase and native or partially degraded aldolase with regard to endocytosis, but that all three forms are degraded at similar rates once within lysosomes.     

Interconversion of multiple forms of tyrosine aminotransferase in vitro and in vivo in cultured hepatoma cells

Corticosteroi-induced tyrosine aminotransferase (EC 2.6.1.5) from cultured hepatoma cells was separated by carboxymethyl-Sephadex chromatography into three molecular forms resembling those described previously in the rat liver. Enzyme forms were isolated and used as purified substrates to examine their in vitro interconversion by various subcellular fractions. Isolated form III was converted to forms II and I, and isolated form II was converted to form I by the coarse particulate fraction sedimenting at 1000 × g. This activity was inhibited by the serine enzyme inhibitor phenylmethane sulfonyl fluoride or by raising the pH to 8.7. Conversion of enzyme forms in vitro in the opposite direction (I → II → III) could not be detected. The distribution of enzyme forms in vivo was examined by the use of experimental conditions that prevent their in vitro interconversion during cell extraction. Tyrosine aminotransferase extracted from cells subjected to various treatments that affect the rates of enzyme synthesis or degradation existed always predominantly as form III. It appears, therefore, that multiple forms of tyrosine aminotransferase are not related to the turnover of this enzyme in vivo.     

Intracellular proteolysis of pancreatic zymogens.

Activation of pancreatic digestive zymogens within the pancreatic acinar cell may be an early event in the development of pancreatitis. To detect such activation, an immunoblot assay has been developed that measures the relative amounts of inactive zymogens and their respective active enzyme forms. Using this assay, high doses of cholecystokinin or carbachol were found to stimulate the intracellular conversion of at least three zymogens (procarboxypeptidase A1, procarboxypeptidase B, and chymotrypsinogen 2) to their active forms. Thus, this conversion may be a generalized phenomenon of pancreatic zymogens. The conversion is detected within ten minutes of treatment and is not associated with changes in acinar cell morphology; it has been predicted that the lysosomal thiol protease, cathepsin B, may initiate this conversion. Small amounts of cathepsin B are found in the secretory pathway, and cathepsin B can activate trypsinogen in vitro; however, exposure of acini to a thiol protease inhibitor (E64) did not block this conversion. Conversion was inhibited by the serine protease inhibitor, benzamidine, and by raising the intracellular pH, using chloroquine or monensin. This limited proteolytic conversion appears to require a low pH compartment and a serine protease activity. After long periods of treatment (60 minutes), the amounts of the active enzyme forms began to decrease; this observation suggested that the active enzyme forms were being degraded. Treatment of acini with E64 reduced this late decrease in active enzyme forms, suggesting that thiol proteases, including lysosomal hydrolases, may be involved in the degradation of the active enzyme forms. These findings indicate that pathways for zymogen activation as well as degradation of active enzyme forms are present within the pancreatic acinar cell.     

Multiple forms of gamma-butyrobetaine hydroxylase (EC 1.14.11.1).

gamma-Butyrobetaine hydroxylase [4-trimethylaminobutyrate, 2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1] from human kidney was resolved into three forms by chromatofocusing. After further chromatography on an anion-exchanger, each form appeared as a single band on electrophoresis in polyacrylamide gel containing sodium dodecyl sulphate. The isoelectric points of isoenzymes 1, 2 and 3 were 5.6, 5.7 and 5.8 respectively, as estimated by isoelectric focusing. Their specific activities were 17-29 mu kat/g of protein. The concentrations of the three isoenzymes were about equal, possibly slightly lower for isoenzyme 1. The requirement for Fe2+ and the Km values for gamma-butyrobetaine and 2-oxoglutarate were about the same for the different enzyme forms. L- and D-Carnitine caused decarboxylation of 2-oxoglutarate to the same extent (8 and 29%) with the three forms. The enzyme forms had the same mass, 64 kDa, as determined by gel filtration in nondenaturing media. The same subunit mass, 42 kDa, was obtained for the multiple forms by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Isoenzyme 2 was resolved into two protein bands by isoelectric focusing in polyacrylamide gels containing urea. Isoenzyme 1 contained only one of these bands and isoenzyme 3 the other. The three enzyme forms of gamma-butyrobetaine hydroxylase thus appear to be dimeric combinations of two subunits differing in charge but not in size. gamma-Butyrobetaine hydroxylase from crude extracts of human, rat and calf liver was also separated into multiple forms by a chromatofocusing technique. The isoenzyme pattern was the same in human liver and kidney. The technique used to resolve the mammalian enzymes gave no evidence for the presence of multiple forms of the bacterial enzyme from Pseudomonas sp. AK 1.     

Pyroglutamyl-peptidase I: cloning,sequencing, and characterisation of the recombinant human enzyme

Pyroglutamyl-peptidase I (EC 3.4.19.3) is well known from bacteria and archaea, but has not previously been cloned or sequenced from any vertebrate. We describe the cloning and sequencing of the human (AJ278828) and mouse (AJ278829) forms of pyroglutamyl-peptidase I. The deduced amino acid sequences each consist of 209 residues and show approximately 30% identity with bacterial forms of the enzyme. They show clear homology to the enzyme from prokaryotes and place the mammalian forms of the enzyme in peptidase family C15 of the MEROPS database. The catalytic residues Glu81, Cys144, and His166 in the enzyme from Bacillus amyloliquefaciens are all conserved in the human sequence. A simple cartoon model of the human protein was constructed on the basis of the published crystal structures of pyroglutamyl-peptidase I forms from Thermococcus litoralis and B. amyloliquefaciens. The human enzyme was expressed by use of a baculovirus vector in Spodoptera frugiperda cells. The recombinant protein was enzymatically active and had properties similar to those described for the naturally occurring mammalian enzyme. Gel-filtration chromatography of the active enzyme gave a molecular mass of about 24kDa, showing that the enzyme is active as the monomer. This contrasted with indications that the prokaryotic enzymes may be tetrameric. Recombinant human pyroglutamyl-peptidase I was active on pGlu-aminomethylcoumarin in the range pH 6-9, with maximal activity being seen at pH 7.0-8.5; it showed an absolute requirement for a thiol-reducing agent. In crude preparations, the enzyme was completely stable for 90 min at 50 degrees C. The enzyme was inhibited by transition metal ions including Ni(2+), Zn(2+), and Cu(2+), and by sulfhydryl-blocking agents. Reversible inhibition was seen with 2-pyrrolidone (K(i)=50 microM), and surprisingly, with N-ethylmaleimide (K(i)=30 microM).     

The nature of the multiple forms of D-erythrodihydroneopterin triphosphate synthetase.

1. Three forms of the Lactobacillus plantarum enzyme D-erythro-dihydroneopterin triphosphate synthetase, the first enzyme in folate biosynthesis, have been demonstrated by polyacrylamide gel electrophoresis. The enzyme forms designated the alpha prime, alpha and beta forms have been shown to be conformers with molecular weights of approx. 200 000. Study of the subunit structure of the beta enzyme species by sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed a single protein with an estimated molecular weight of 20 000 which suggests that the enzyme molecule may be composed of ten polypeptide chains. 2. Of the three conformers only one form, the beta form, appears to be enzymatically active. The two other conformers must undergo conformational changes to the beta species before enzymatic activity can be demonstrated in reaction mixtures containing these enzyme forms. 3. The three enzyme species are interconvertible. The removal of phosphate ions from the enzymatically active beta form results in the formation of two inactive species which suggests that the conformation of the active enzyme is stabilized by non-covalently bound phosphate ions. Conversion of the inactive species to the beta enzyme form may be effected by the readdition of phosphate, substrate or certain nucleotides.     

beta-Galactosidase from Aspergillus niger. Separation and characterization of three multiple forms.

The enzyme beta-galactosidase (EC 3.2.1.23) from Aspergillus niger was purified and resolved into three multiple forms, using molecular sieving, ion-exchange, an hydrophobic chromatography. The isolated enzyme forms accounted for 83%, 8%, and 9% of the total beta-galactosidase activity, respectively. They were glycoproteins with estimated molecular weights of 124,000, 150,000 and 173,000, isoelectric points of about 4.6, and pH optima between 2.5 and 4.0. Amino acid and carbohydrate analyses showed that multiplicity was mainly due to dissimilar carbohydrate contents (about 12.5%, 20.5% and 29% neutral carbohydrates, respectively). The multiple form pattern might depend on the culture conditions. The beta-galactosidase forms were heat-stable up to about 60 degrees C. The Km values for lactose ranged from 85 mM to 125 mM, whereas those for the synthetic substrate o-nitrophenyl-beta-D-galactopyranoside were equal to about 2.4 mM. The V values obtained at 30 degrees C for lactose and o-nitrophenyl-beta-D-galactopyranoside were 104 units/mg enzyme protein and 121 units/mg enzyme protein, respectively (weighted averages for the three enzyme forms). The slight reactional dissimilarities between the three enzyme forms are unlikely to be physiologically relevant. The biological significance of A. niger beta-galactosidase multiplicity might be related to the observed differences in carbohydrate content, as suggested by recent reports on other microbial glycoprotein enzymes.