Purification and partial characterization of arginine-specific ADP-ribosyltransferase from skeletal muscle microsomal membranes

Integral membrane-associated arginine-specific mono-ADP-ribosyltransferase was purified from rabbit skeletal muscle microsomes. The ADP-ribosyltransferase was solubilized from the 100,000 x g pellet with 0.3% sodium deoxycholate and purified to greater than or equal to 95% homogeneity by successive DE52, concanavalin A-agarose, 3-aminobenzamide-agarose, and size-exclusion high-performance liquid chromatography (HPLC) steps in the presence of detergents. Two molecular weight forms of the enzyme were isolated and partially characterized. The apparent Mr of the alpha-form of the enzyme purified to greater than or equal to 95% homogeneity was approximately 39,000 +/- 500 as estimated by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr of the beta-form purified to greater than or equal to 80% homogeneity was 38,500 +/- 500. The rapid procedure resulted in a 200-fold purification for the alpha-form and a 645-fold purification for the beta-form, relative to the microsomal fraction. Positive identification of the enzyme was confirmed by utilizing a zymographic in situ gel assay and by HPLC assay of polyacrylamide gel slice incubations with an NAD and guanylhydrazone substrate. The specificity of the mono-ADP-ribosyltransferase zymographic assay was characterized by time course incubations, hydroxylamine sensitivity, 3-aminobenzamide inhibition, and histone dependence. The ADP-ribosyltransferase is inactivated by reducing agents.     

Further characterization of collagen galactosyltransferase from chick embryos.

Optimum extraction of collagen galactosyltransferase activity from chick embryos required relatively high concentrations of detergent and salt. The activity was inhibited by concanavalin A, and the enzyme had a high affinity for columns of this lectin coupled to agarose; these results suggest the presence of carbohydrate units in the enzyme molecule. Collagen galactosyltransferase was highly labile, and only 1% of the originally bound enzyme activity could be eluted from the concanavalin A-agarose column with a buffer containing methyl glucoside and ethylene glycol. The purification of the activity over the original supernatant of chick embryo homogenate was 250-300-fold, with the optimum reaction conditions for the purified transferase differing somewhat from those for crude enzyme preparations. The reaction was inhibited by glucose-free basement-membrane collagen, UDP and galactosylhydroxylsine, and also by Co2+ and a number of compounds resembling UDP-galactose. Hydroxylysine was also a weak inhibitor. Immobilized hydroxylysine and UDP-glucuronic acid did not bind the collagen galactosyltransferase, but the enzyme was retarded in a column of UDP-galacturonic acid linked to agarose.     

Amino acid specific ADP-ribosylation: specific NAD: arginine mono-ADP-ribosyltransferases associated with turkey erythrocyte nuclei and plasma membranes

Turkey erythrocytes contain NAD:arginine mono-ADP-ribosyltransferases which, like cholera toxin and Escherichia coli heat-labile enterotoxin, catalyze the transfer of ADP-ribose from NAD to proteins, to arginine and other low molecular weight guanidino compounds, and to water. Two such ADP-ribosyltransferases, A and B, have been purified from turkey erythrocyte cytosol. To characterize further the class of NAD:arginine ADP-ribosyltransferases, the particulate fraction was examined; 40% of erythrocyte transferase activity was localized to the nucleus and cell membrane. Transferase activity in a salt extract of a thoroughly washed particulate preparation was purified 36,000-fold by sequential chromatography on phenyl-Sepharose, (carboxymethyl) cellulose, concanavalin A-Sepharose, and NAD-agarose. Subsequent DNA-agarose chromatography separated two activities, termed transferases C and A', which were localized to the membrane and nucleus, respectively. Transferase C, the membrane-associated enzyme, was distinguished from the cytosolic enzymes by a relative insensitivity to salt and histone; transferase C was stimulated 2-fold by 300 mM NaCl in contrast to a 20-fold stimulation of transferase A and a 50% inhibition of transferase B. Similarly, histones, which stimulate transferase A 20-fold, enhanced transferase C activity only 2-fold. Transferase A', the nuclear enzyme, was retained on DNA-agarose. It was similar to transferase A in salt and histone sensitivity. Gel permeation chromatography showed slight molecular mass differences among the group of enzymes: A, 24,300 daltons (Da); B, 32,700 Da; C, and A', 25,500 Da. The affinities of transferase C for NAD and agmatine were similar to those of the cytosolic transferases A and B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of ADP-ribosylarginine hydrolase from turkey erythrocytes

ADP-ribosylation of arginine appears to be a reversible modification of proteins with NAD: arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyzing the opposing arms of the ADP-ribosylation cycle. ADP-ribosylarginine hydrolases have been purified extensively (greater than 90%) (150,000-250,000-fold) from the soluble fraction of turkey erythrocytes by DE-52, phenyl-Sepharose, hydroxylapatite, Ultrogel AcA 54, and Mono Q chromatography. Mobilities of the hydrolase on gel permeation columns and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions are consistent with an active monomeric species of approximately 39 kDa. Insertion of an organomercurial agarose chromatographic step prior to Ultrogel AcA 54 resulted in the isolation of a hydrolase exhibiting approximately 35-fold greater sensitivity to dithiothreitol (Ka,sensitive = 41 +/- 16.7 microM, n = 4; Ka,resistant = 1.44 +/- 0.12 mM, n = 3). A similar dithiothreitol-sensitive hydrolase was generated by exposure of the purified resistant enzyme to HgCl2. At 30 degrees C, both thiol-sensitive (HS) and thiol-resistant (HR) hydrolases were relatively resistant to N-ethylmaleimide (NEM); incubation with dithiothreitol prior to NEM resulted in complete inactivation. Both HS and HR required Mg2+ and thiol for enzymatic activity. Mg2+ stabilized both HS and HR against thermal inactivation in the absence and presence of thiol. A purified NAD:arginine ADP-ribosyltransferase, in the presence of NAD, inactivated both HS and HR; Mg2+ and to a greater extent Mg2+ plus dithiothreitol protected both HS and HR from NAD- and transferase-dependent inactivation. Thus, activation of the hydrolase enhanced its resistance to inactivation by transferase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli by dye-ligand chromatography

Improved purification schemes are reported for the enzymes L-aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli. Dye-ligand chromatography on commercially available dye matrices are incorporated as key steps in these purifications. Red A-agarose has a high affinity for L-aspartase, which is then eluted as a homogeneous protein fraction with 1 mM L-aspartic acid. Green A-agarose shows a high binding affinity for the bifunctional enzyme aspartokinase-homoserine dehydrogenase I. Purification is accomplished by elution with NADP+, followed by formation of a ternary complex with NADP and cysteine, a good competitive inhibitor of the homoserine dehydrogenase activity, and rechromatography on Green A-agarose. The final specific activity of each purified enzyme equaled or exceeded previously reported values, the overall yield of enzymes obtained was significantly higher, and these improved purification schemes were found to be more amenable to being scaled up for the production of large quantities of purified enzyme.     

Mono-ADP-ribosylation of Gs by an eukaryotic arginine-specific ADP-ribosyltransferase stimulates the adenylate cyclase system

An arginine-specific ADP-ribosyltransferase, named ADP-ribosyltransferase A, was partially purified from human platelets using polyarginine as an ADP-ribose acceptor. When human platelet membranes were incubated with the transferase A in the presence of NAD+, Gs, a stimulatory guanine nucleotide-binding protein of the adenylate cyclase was specifically mono-ADP-ribosylated. ADP-ribose transfer to Gs by this enzyme was suppressed when membranes were pre-ADP-ribosylated by cholera toxin. Incubation of membranes with the transferase A resulted in activation of the adenylate cyclase system. This stimulatory effect of the transferase A on the adenylate cyclase system was inhibited by the presence of polyarginine. These results indicate a role of ADP-ribosyltransferase A in regulation of the adenylate cyclase system via endogenous mono-ADP-ribosylation of Gs.     

Inactivation of glutamine synthetases by an NAD:arginine ADP-ribosyltransferase

Glutamine synthetase from ovine brain has a critical arginine residue at the catalytic site (Powers, S. G., and Riordan, J.F. (1975) Proc. Natl. Acad. Sci. U.S. A. 72, 2616-2620). This enzyme is now shown to be a substrate for a purified NAD:arginine ADP-ribosyltransferase from turkey erythrocyte cytosol that catalyzes the transfer of ADP-ribose from NAD to arginine and purified proteins. The transferase catalyzed the inactivation of the synthetase in an NAD-dependent reaction; ADP-ribose and nicotinamide did not substitute for NAD. Agmatine, an alternate ADP-ribose acceptor in the transferase-catalyzed reaction, prevented inactivation of glutamine synthetase. MgATP, a substrate for the synthetase which was previously shown to protect that enzyme from chemical inactivation, also decreased the rate of inactivation in the presence of NAD and ADP-ribosyltransferase. Using [32P]NAD, it was observed that approximately 90% inactivation occurred following the transfer of 0.89 mol of [32P]ADP-ribose/mol of synthetase. The erythrocyte transferase also catalyzed the NAD-dependent inactivation of glutamine synthetase purified from chicken heart; 0.60 mol of ADP-ribose was transferred per mol of enzyme, resulting in a 95% inactivation. As noted with the ovine brain enzyme, agmatine and MgATP protected the chicken synthetase from inactivation and decreased the extent of [32P]ADP-ribosylation of the synthetase. These observations are consistent with the conclusion that the NAD:arginine ADP-ribosyltransferase modifies specifically an arginine residue involved in the catalytic site of glutamine synthetase. Although the transferase can use numerous proteins as ADP-ribose acceptors, some characteristics of this particular arginine, perhaps the same characteristics that are involved in its function in the catalytic site, make it a favored ADP-ribose acceptor site for the transferase.     

Isolation and properties of lung 15-hydroxyprostaglandin dehydrogenase from pregnant rabbits

A NAD-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was purified to a specific activity of over 25,000 nmol NADH formed/min/mg protein with 50 microM prostaglandin E1 as substrate from the lungs of 28-day-old pregnant rabbits. This represented a 2600-fold purification of the enzyme with a recovery of 6% of the starting enzyme activity. The lungs of pregnant rabbits were used because a 42- to 55-fold induction of the PGDH activity was observed after 20 days of gestation. The enzyme was purified by CM-cellulose, DEAE-cellulose, Sephadex G-75, octylamino-agarose, and hydroxylapatite chromatography. The enzyme could not be purified by affinity chromatography using NAD- or blue dextran-bound resins. The purified enzyme was specific for NAD and had a subunit molecular weight of 29,000. The optimal pH range for the oxidation of prostaglandin E1 was between 10.0 and 10.4 using 3-(cyclohexylamino)propanesulfonic acid as the buffer. The Km and Vmax values for prostaglandin E1 were 33 microM and 40,260 nmol/min/mg protein, respectively, while the Km and Vmax values for prostaglandin E2 were 59 microM and 43,319 nmol/min/mg protein, respectively. The Km for prostaglandin F2 alpha was four times the value for prostaglandin E1. The PGDH activity was inhibited by p-chloromercuriphenylsulfonic acid but the enzymatic activity was restored by the addition of dithiothreitol. n-Ethylmaleimide also produced a rapid decline in enzymatic activity but when NAD was included in the incubation system, no inhibition was observed.     

Nuclear envelope glycoprotein with poly(A) polymerase activity of rat liver: isolation, characterization, and immunohistochemical localization

A protein with poly(A) polymerase activity has been identified and isolated from hepatic nuclear envelopes of rats to near homogeneity. The ability of the enzyme to bind to concanavalin A-agarose and to be eluted from the column with methyl alpha-D-mannopyranoside (0.2 M) as well as the inhibitory effects of alpha-mannosidase suggested that it was a glycoprotein. Poly(A) polymerase has an absolute requirement for a divalent cation, ATP, and an oligonucleotide primer. The enzyme activity with Mn2+ was about 20-fold higher than that with Mg2+. Several known inhibitors adversely affected poly(A) polymerase activity. The enzyme has a molecular weight of 64,000 when analyzed by polyacrylamide gel electrophoresis under denaturing conditions and has a sedimentation coefficient of 4.5 S. Immunohistochemical studies using polyclonal antibodies raised against the purified enzyme revealed that the antigen was localized in the nuclear membranes.     

Binding of human fibroblast interferon to concanavalin A-agarose. Involvement of carbohydrate recognition and hydrophobic interaction.

Human fibroblast interferon binds to a concanavalin A-agarose (Con A-Sepharose) equilibrated with methyl alpha-D-mannopyranoside, or levan; in contrast, it is only partially retarded on a similar column equilibrated with ethylene glycol. Interferon does not bind, however, to a lectin column equilibrated with both methyl alpha-D-mannopyranoside and ethylene glycol. Thus, a hydrophobic interaction between fibroblast interferon and the immobilized lectin seems to account for a large portion of the binding forces involved. Other hydrophobic solutes, such as dioxane, 1, 2-propanediol, and tetraethylammonium chloride, were found equally or more efficient than ethylene glycol in displacing interferon from the lectin column. The elution pattern of interferon from a concanavalin A-agarose (Con A-Sepharose) column, at a constant ehtylene glycol concentration and with an increasing mannoside concentration, reveals the existence of four distinct interferon components. The selective adsorption to and elution from a concanavalin A-agarose (Con A-Sepharose) column resulted in about a 3000-fold purification of human fibroblast interferon and complete recovery of activity. The specific activity of the partially purified interferon preparation is about 5 X 10(7) units per mg of protein. The chromatographic behavior of human leukocyte interferon is remarkable in that it does not bind to concanavalin A-agarose at all indicating the absence of carbohydrate moieties recognizable by the lectin, or if present, their masked status. When concanavalin A was coupled to an agarose matrix (cyanogen bromide activated) at pH 8.0 and 6.0 human fibroblast interferon bound to both lectin-agarose adsorbents and could be recovered with methyl alpha-D-mannopyranoside. Concanavalin A, immobilized directly on agarose matrix at pH 8.0 and 6.0, thus displays only carbohydrate recognition toward interferon. By contrast, unless a hydrophobic solute was included in the solvent containing methyl mannoside, human fibroblast interferon could not be recovered from concanavalin A-agarose coupled at pH 9.0. When concanavalin A was immobilized via molecular arms, in tetrameric as well as dimeric forms, the binding of interferon again occurred exclusively through carbohydrate recognition. Thus, the hydrophobic interaction can be eliminated by appropriate immobilization of the lectin, and then adsorbed glycoproteins, as exemplified here by interferon, can be recovered readily with methyl mannoside alone.     

Eukaryotic mono(ADP-ribosyl)transferase that ADP-ribosylates GTP-binding regulatory Gi protein

An NAD:cysteine ADP-ribosyltransferase designated ADP-ribosyltransferase C was purified approximately 35,000-fold from human erythrocytes with an 11% yield. The purified ADP-ribosyltransferase C exhibited one predominant protein band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight (Mr) of 28,500. The Km values for NAD and cysteine methyl ester were determined to be 65 and 4,400 microM, respectively. By using human erythrocyte inside-out membrane vesicles, the transferase C was found to ADP-ribosylate the alpha subunit (Mr = 41,000) of Gi, which is a substrate for pertussis toxin. The ADP-ribosylation of Gi alpha catalyzed by ADP-ribosyltransferase C was inhibited by pre-ADP-ribosylation with pertussis toxin. The linkage of ADP-ribose-Gi alpha in the membranes formed by ADP-ribosyltransferase C was as stable to hydroxylamine as that formed by pertussis toxin. These data represent the first demonstration that eukaryotic cells contain an ADP-ribosyltransferase which can catalyze the ADP-ribosylation of a cysteine residue in Gi alpha.     

Modification of the ADP-ribosyltransferase and NAD glycohydrolase activities of a mammalian transferase (ADP-ribosyltransferase 5) by auto-ADP-ribosylation.

Mono-ADP-ribosylation, a post-translational modification in which the ADP-ribose moiety of NAD is transferred to an acceptor protein, is catalyzed by a family of amino acid-specific ADP-ribosyltransferases. ADP-ribosyltransferase 5 (ART5), a murine transferase originally isolated from Yac-1 lymphoma cells, differed in properties from previously identified eukaryotic transferases in that it exhibited significant NAD glycohydrolase (NADase) activity. To investigate the mechanism of regulation of transferase and NADase activities, ART5 was synthesized as a FLAG fusion protein in Escherichia coli. Agmatine was used as the ADP-ribose acceptor to quantify transferase activity. ART5 was found to be primarily an NADase at 10 microM NAD, whereas at higher NAD concentrations (1 mM), after some delay, transferase activity increased, whereas NADase activity fell. This change in catalytic activity was correlated with auto-ADP-ribosylation and occurred in a time- and NAD concentration-dependent manner. Based on the change in mobility of auto-ADP-ribosylated ART5 by SDS-polyacrylamide gel electrophoresis, the modification appeared to be stoichiometric and resulted in the addition of at least two ADP-ribose moieties. Auto-ADP-ribosylated ART5 isolated after incubation with NAD was primarily a transferase. These findings suggest that auto-ADP-ribosylation of ART5 was stoichiometric, resulted in at least two modifications and converted ART5 from an NADase to a transferase, and could be one mechanism for regulating enzyme activity.     

Affinity chromatography of lysyl hydroxylase on concanavalin A-agarose.

Lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) has a high affinity for columns of concanavalin A-agarose, which was markedly reduced in the presence of alpha-methyl-D-mannoside, suggesting that the enzyme is a glycoprotein. Once bound, the enzyme could not be eluted with the glycoside alone, whereas an effective elution was achieved by a combination of alpha-methyl-D-mannoside and ethylene glycol. The data thus suggest that hydrophobic interaction stabilized the complex of the enzyme with the column. This information was applied to obtain a lysyl hydroxylase purification of about 3000-fold with a recovery of more than 10% from extract of chick embryos by relatively simple steps.     

Polypeptide domains of ADP-ribosyltransferase obtained by digestion with plasmin

Proteolysis by plasmin inactivates bovine ADP-ribosyltransferase; therefore, enzymatic activity depends exclusively on the intact enzyme molecule. The transferase was hydrolyzed by plasmin to four major polypeptides, which were characterized by affinity chromatography and N-terminal sequencing. Based on the cDNA sequence for human ADP-ribosyltransferase enzyme [Uchida, K., Morita, T., Sato, T., Ogura, T., Yamashita, R., Noguchi, S., Suzuki, H., Nyunoya, H., Miwa, M., & Sugimura, T. (1987) Biochem. Biophys. Res. Commun. 148, 617-622], a polypeptide map of the bovine enzyme was constructed by superposing the experimentally determined N-terminal sequences of the isolated polypeptides on the human sequence deduced from its cDNA. Two polypeptides, the N-terminal peptide (Mr 29,000) and the polypeptide adjacent to it (Mr 36,000), exhibited binding affinities toward DNA, whereas the C-terminal peptide (Mr 56,000), which accounts for the rest of the transferase protein, bound to the benzamide-Sepharose affinity matrix, indicating that it contains the NAD+-binding site. The fourth polypeptide (Mr 42,000) represents the C-terminal end of the larger C-terminal fragment (Mr 56,000) and was formed by a single enzymatic cut by plasmin of the polypeptide of Mr 56,000. The polypeptide of Mr 42,000 still retained the NAD+-binding site. The plasmin-catalyzed cleavage of the polypeptide of Mr 56,000-42,000 was greatly accelerated by the specific ligand NAD+. Out of a total of 96 amino acid residues sequenced here, there were only 6 conservative replacements between human and bovine ADP-ribosyltransferase.     

Isolation to homogeneity and partial characterization of a histo-blood group A defined Fuc alpha 1----2Gal alpha 1----3-N-acetylgalactosaminyltransferase from human lung tissue

The soluble histo-blood group A glycosyltransferase (Fuc alpha 1----Gal alpha 1----3-N-acetylgalactosaminyltransferase) was purified approximately 600,000-fold to homogeneity from human lung tissue. The enzyme was solubilized in 1% Triton X-100, partially purified by affinity chromatography on Sepharose 4B, and eluted with UDP. Final purification was obtained by twice repeated fast protein liquid chromatography ion exchange (Mono STM) with NaCl gradient elution and reverse-phase chromatography (proRPC) with acetonitrile gradient elution. Identity of the purified protein was established by (i) demonstration of the putative A transferase protein only in affinity-purified extracts of A but not O individuals, and (ii) specific immunoprecipitation of enzyme activity and putative protein with monoclonal antibodies. Sodium dodecyl sulfate electrophoresis revealed a single protein band with apparent Mr of approximately 40,000 under both reducing and nonreducing conditions. Digestion with N-glycanase yielded a reduction in Mr of approximately 6,000 (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), suggesting that the A transferase is a glycoprotein with N-linked carbohydrate chains. Amino acid composition and N-terminal amino acid sequence of the intact transferase, as well as of peptides released by endolysyl peptidase digest or cyanogen bromide cleavage, are presented.     

Interaction of thyroid peroxidase with concanavalin A covalently coupled to agarose.

We have investigated the interaction between concanavalin A-agarose (Con A-agarose) and thyroid peroxidase, an integral membrane protein found in the 105,000 X g, 1-h particulate fraction of thyroid tissue. An intact form of porcine thyroid peroxidase was obtained by solubilization with the nonionic detergent Triton X-100 and two fragmented, hydrophilic forms of the enzyme were prepared by trypsin treatment of the membrane. The three types of thyroid peroxidase bind to Con A-agarose and can be eluted with alpha-methyl-D-mannoside. The alpha-methyl-D-mannoside eluate of the most purified thyroid peroxidase preparation has been analyzed by polyacrylamide gel electrophoresis. Peroxidase activity corresponds with a glycoprotein band. The binding of thyroid peroxidase to Con A-agarose can be inhibited by sugars in the following order: alpha-methyl-D-mannoside greater than D-mannose greater than alpha-methyl-D-glucoside greater than D-glucose greater than D-galactose. This order of specificity is typical of Con A-sugar interactions. Furthermore, inactivation of the carbohydrate binding site of Con A by demetallization greatly reduces the extent of thyroid peroxidase binding. Reactivation of the carbohydrate binding site by the addition of Ca2+ and Mn2+ to demetallized Con A-agarose restores thyroid peroxidase binding. These and other experiments suggest that htyroid peroxidase is, like several other peroxidases, a glycoprotein. In addition, the interaction between thyroid peroxidase and Con A-agarose may provide a new purification tool for thyroid peroxidase.     

Purification and properties of a high affinity guanosine 3′: 5′-cyclic monophosphate-binding phosphatase from silver beet leaves

A cyclic nucleotide-binding phosphatase was purified from silver beet leaves by a procedure involving chromatography on CM-Sepharose CL-6B, DEAE-Sephacel, casein-Sepharose 4B, concanavalin A-agarose and Ultrogel AcA44. The enzyme is eluted from concanavalin A-agarose by 0.5 M α-methylglucoside at high ionic strength. The enzyme is monomeric, having a subunit molecular weight (M_r) of 28 000; the native M_r is 31 000 as determined from gel filtration. The enzyme catalyzes the hydrolysis of a range of phosphomonoesters including various nucleotides and O-phosphotyrosine but not O-phosphoserine or O-phosphothreonine. The leaf phosphatase is competitively inhited by guanosine 3′ : 5′-cyclic monphosphate (cGMP) and adenosine 3′ : 5′-cyclic monophosphate (cAMP) (K_i-values: 0.4 μM and 3.3 μM, respectively). The leaf phosphotase has the highest affinity for cGMP yet reported for a plant protein.     

Partial purification and characterization of chick-embryo prolyl 3-hydroxylase.

Prolyl 3-hydroxylase was purified up to about 5000-fold from an (NH4)2SO4 fraction of chick-embryo extract by a procedure consisting of affinity chromatography on denatured collagen linked to agarose, elution with ethylene glycol and gel filtration. The molecular weight of the purified enzyme is about 160000 by gel filtration The enzyme is probably a glycoprotein, since (a) its activity is inhibited by concanavalin A, and (b) the enzyme is bound to columns of this lectin coupled to agarose and can be eluted with a buffer containing methyl alpha-D-mannoside. The Km values for Fe2+, 2-oxoglutarate, O2 and ascorbate in the prolyl 3-hydroxylase reaction were found to be very similar to those previously reported for these co-substrates in the prolyl 4-hydroxylase and lysyl hydroxylase reactions.     

Purification of Meerrettich-peroxidase by means of affinity chromatography on concanavalin A-agarose]

Crude preparations of horse-radish peroxidase were purified by means of affinity chromatography on Concanavalin A-agarose. The peroxydase was bound to Concanavalin A, whereas the majority of other proteins of the preparation pass through the column. Subsequently the peroxidase was eluted by means of 1 M sucrose with high purity. The purified enzyme is convenient for the immunoenzyme technique.     

Purification and characterization of dipeptidyl peptidase I from human spleen.

The lysosomal hydrolase, dipeptidyl peptidase I (DPPI), was purified from human spleen and its enzymatic activity characterized. The enzyme was purified to apparent homogeneity by a combination of differential pH solubility, heat-treatment, affinity chromatography on concanavalin A-agarose and p-hydroxymercuribenzoate-agarose, and gel filtration chromatography on Sephacryl S-300. This procedure resulted in a 1100-fold purification of DPPI protein with a yield of approximately 2% of the total DPPI activity. The enzyme was characterized as a glycoprotein with a pI of 5.4, a molecular mass of 200,000 Da as determined by gel filtration under nondenaturing conditions, and a subunit size of 24,000 Da. Amino acid sequence analysis of peptides isolated from cyanogen bromide and trypsin digests of the 24,000-Da subunit revealed extensive sequence similarity between human and rat DPPI. Purified DPPI exhibited both hydrolytic and transpeptidase (polymerase) activity. DPPI exhibited activity against a variety of dipeptide substrates including peptides with either non-polar or polar residues in the P1 position. In contrast to the reported substrate specificity of bovine and murine DPPI, the human enzyme exhibited a modest preference for peptides with nonpolar residues in the P1 position. DPPI content was found to be highest among cytotoxic lymphocytes and myeloid cells. The high level of DPPI expression in these cell populations correlates with their sensitivity to the toxic effects of leucyl-leucine methyl ester, a substrate for DPPI.