Independent temporal expression of two N-substituted aminoacyl-tRNA hydrolases during the development of Artemia salina.

Encysted embryos of the crustacean Artemia salina contain an enzymatic activity which hydrolyzes N-acetylphenylalanyl-tRNA to N-acetylphenylalanine and tRNA. The enzyme apparently does not hydrolyze other free or N-substituted aminoacyl-tRNAs. The levels of this enzyme do not significantly change during embryonic and early larval development. In contrast, an unspecific hydrolase active on several N-substituted aminoacyl-tRNAs is practically absent in the encysted embryos and during embryogenesis and appears abruptly during larval development. The independent temporal expression of these two hydrolases during Artemia salina differentiation makes this organism siuitable for the study of the physiological role of these enzymes.     

Sensitivity of ribosomal bound N-acetylphenylalanyl-tRNA to hydrolysis by a spectific hydrolase.

Binding of N-acetylphenylalanyl-tRNA, either enzymatically or non-enzymatically, to yeast 80-S ribosomes renders the substrate resistant to the hydrolytic action of a specific hydrolase present in yeast. In contrast, N-acetylphenylalanyl-tRNA bound to the 40-S ribosomal subunit is sensitive to enzymatic hydrolysis. The presence of the hydrolase in the aminoacyl-tRNA binding factor preparations greatly interferes with the formation of complexes between N-acetylphenylalanyl-tRNA and the small ribosomal subunit.     

Purification and initial characterization of peptidyl-tRNA hydrolase from rabbit reticulocytes.

We have identified an activity in rabbit reticulocyte lysate as peptidyl-tRNA hydrolase, based upon its ability to hydrolyze native reticulocyte peptidyl-tRNA, isolated from polyribosomes, and N-acylaminoacyl-tRNA, and its inability to hydrolyze aminoacyl-tRNA, precisely the same substrate specificity previously reported for peptidyl-tRNA hydrolase from bacteria or yeast. The physiological role of the reticulocyte enzyme may be to hydrolyze and recycle peptidyl-tRNA that has dissociated prematurely from elongating ribosomes, as suggested for the bacterial and yeast enzymes, since reticulocyte peptidyl-tRNA hydrolase is completely incapable of hydrolyzing peptidyl-tRNA that is still bound to polyribosomes. We have purified reticulocyte peptidyl-tRNA hydrolase over 5,000-fold from the postribosomal supernatant with a yield of 14%. The purified product shows a 72-kDa band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis that has co-purified with enzyme activity and comprises about 90% of the total stained protein, strongly suggesting that the 72-kDa protein is the enzyme. Sucrose density gradient analysis indicates an apparent molecular mass for the native enzyme of 65 kDa, implying that it is a single polypeptide chain. The enzyme is almost completely inactive in the absence of a divalent cation: Mg2+ (1-2 mM) promotes activity best, Mn2+ is partly effective, and Ca2+ and spermidine are ineffective. The hydrolase shows a Km of 0.60 microM and Vmax of 7.1 nmol/min/mg with reticulocyte peptidyl-tRNA, a Km of 60 nM and Vmax of 14 nmol/min/mg with Escherichia coli fMet-tRNA(fMet), and a Km of 100 nM and Vmax of 2.2 nmol/min/mg with yeast N-acetyl-Phe-tRNA(Phe). The enzyme has a pH optimum of 7.0-7.25, it is inactivated by heat (60 degrees C for 5 min), and its activity is almost completely inhibited by pretreatment with N-ethylmaleimide or incubation with 20 mM phosphate. The fact that the enzyme hydrolyzes E. coli but not yeast or reticulocyte fMet-tRNA(fMet) may be explained, at least in part, by structural similarities between prokaryotic tRNA(fMet) and eukaryotic elongator tRNA that are not shared by eukaryotic tRNA(fMet).     

Characterization of two carnosine-degrading enzymes from rat brain. Partial purification and characterization of a carnosinase and a beta-alanyl-arginine hydrolase

From rat brain extracts, two carnosine-degrading enzymes have been identified and partially purified by ion-exchange chromatography, hydrophobic interaction chromatography on phenyl-Sepharose CL-4B and gel filtration. These enzymes exhibit distinct differences in their chemical characteristics and substrate specificities. One enzyme, designated carnosinase, preferentially hydrolyzes carnosine and exhibits a low Km value (0.02 mM) towards this substrate. Carnosinase also degrades anserine but not homocarnosine or homoanserine. The other carnosine-degrading enzyme hydrolyzes beta Ala-Arg considerably faster than carnosine and, therefore, has been tentatively designated beta Ala-Arg hydrolase. This enzyme exhibits high Km values with carnosine (Km = 25 mM) and beta Ala-Arg (Km = 2 mM). Homocarnosine and gamma-aminobutyryl-arginine are not degraded by beta Ala-Arg hydrolase. Neither enzyme is inhibited by agents reactive on activated hydroxyl groups, such as diisopropyl fluorophosphate, and also not by a variety of peptidase inhibitors of microbial origin or from other sources. Carnosinase is also not inhibited by bestatin but beta Ala-Arg hydrolase, although not an aminopeptidase, is strongly inhibited by this aminopeptidase inhibitor (IC50 = 50 nM). While carnosinase is strongly inhibited by thiol-reducing agents such as dithioerythritol and 2-mercaptoethanol, beta Ala-Arg hydrolase is stabilized and activated by these substances. Both enzymes are strongly inhibited by metal-chelating agents. Carnosinase, however, is not dependent on exogeneously added metal ions and is strongly inhibited by Mn2+ as well as by heavy metal ions. In contrast, beta Ala-Arg hydrolase requires Mn2+ ions for full enzymatic activity. Based on these differences, selective incubation conditions could be evaluated in order to determine specifically both enzyme activities in crude tissue extracts. In rat, both enzymes are present in all tissues tested, except skeletal muscles, but considerable differences in their relative distribution among different tissues are also observed.     

Yeast aminopeptidase I. Chemical composition and catalytic properties.

An aminopeptidase (alpha-aminoacyl L-peptide hydrolase, EC 3.4.11.1) was purified to homogeneity from autolysates of brewer's yeast. The enzyme which is responsible for most of the yeast cell's aminopeptidase activity is a glycoprotein containing about 12% of conjugated carbohydrate and 0.02% Zn2+ and having a complex quaternary structure. The active species has a molecular weight of approx. 600000 and an isoelectric point of 4.7. The enzyme is remarkably stable, even in dilute solutions. All types of L-amino acid and peptide derivatives containing a free amino terminus are attacked, including amino acid amides and esters. As to its substrate specificity, the enzyme belongs to the so called leucine-aminopeptidases. It is strongly and specifically activated by Zn2+ and Cl- (or Br-) and inactivated by metal-chelating agents. The activation by Zn2+ seems to be mediated by a conformational transition which affects exclusively V and leads to a form of the enzyme which enhanced stability against heat. Halide anions, on the other hand, are acting as positive allosteric effectors, modulating both V and Km.     

Purification and properties of an inducible beta-galactosidase isolated from the yeast Kluyveromyces lactis.

beta-Galactosidase (EC 3.2.1.32) was purified 80-fold from the yeast Kluyveromyces lactis induced for this enzyme by growth on lactose. When the purified enzyme was subjected to electrophoresis on an acrylamide gel in the presence of sodium dodecyl sulfate, one protein with an apparent molecular weight of 135,000 was observed. The enzyme has a sedimentation coefficient of 9.6S. This beta-galactosidase and the one from Escherichia coli are not antigenically related. Maximal enzyme activity requires Na+ and Mn2+ and a reducing agent. beta-Galactosidase has Km values of 12 to 17 and 1.6 mM for lactose and o-nitrophenyl-beta-D-galactoside, respectively. The hydrolase and transgalactosylase activities of the enzyme are similar to those of E. coli beta-galactosidase.     

Metabolism of resorcinylic compounds by bacteria. Purification and properties of acetylpyruvate hydrolase from Pseudomonas putida 01.

Acetylpyruvate hydrolase, the terminal inducible enzyme of the pathway of orcinol catabolism in Pseudomonas putida, catalyzes the quantitative conversion of acetylpyruvate into acetate and pyruvate. The enzyme has been purified approximately 40-fold from extracts of Ps. putida grown on orcinol. Disc gel electrophoresis of the preparations show one major and one minor band of protein. The molecular weight of the enzyme is approximately 38,000 by sodium dodecyl sulfate electrophoresis. Acetylpyruvate is the only known substrate for the enzyme; maleylpyruvate, fumarylpyruvate, acetoacetate, oxalacetate, and acetylacetone are not hydrolyzed by acetylpyruvate hydrolase. Several divalent cations, includ-Mg2+, Mn2+, Co2+, Ca2+, and Zn2+, enhanced hydrolytic activity, but Cu2+ was inhibitory. The enzyme shows a sharp pH optimum at 7.4. Acetylpyruvate hydrolase has an apparent K-m of 0.1 mM for acetylpyruvate with a molecular activity of 36 min minus 1 at 25 degrees. Pyruvate, oxalacetate, and oxalate are competitive inhibitors of acetylpyruvate hydrolysis by the enzyme with K-i values of 6.0, 4.5, and 0.45 mM, respectively.     

Purification and properties of an inducible aminoacyl-tRNA hydrolase from Artemia larvae

This paper describes the purification and properties of an enzyme present in Artemia larvae which hydrolyzes aminoacyl-tRNA by splitting the ester bond between the amino acid and the tRNA chain. The hydrolase has a molecular weight of 55 000 as estimated by gel filtration in Sephadex G-150, is maximally active in the presence of a divalent cation (Mg²⁺, Mn²⁺) and has a pH maximum at around neutrality. The enzyme has a wide substrate specificity, hydrolyzing with practically the same efficiency aminoacyl-tRNAs with the amino group free or substituted. This property distinguishes this enzyme from the widely distributed peptidyl-tRNA hydrolase and other more specific aminoacyl-tRNA hydrolases. The expression of the hydrolase during Artemia larval development is blocked by inhibitors of protein synthesis.     

Differences between microsomal and mitochondrial-matrix palmitoyl-coenzyme A hydrolase, and palmitoyl-L-carnitine hydrolase from rat liver.

Palmitoyl-CoA hydrolase (EC 3.1.2.2) and palmitoyl-L-carnitine hydrolase (EC 3.1.1.28) activities from rat liver were investigated. 1. Microsomal and mitochondrial-matrix palmitoyl-CoA hydrolase activities had similar pH and temperature optima, although the activities showed different temperature stability. They were inhibited by Pb2+ and Zn2+. The palmitoyl-CoA hydrolase activities in microsomal fraction and mitochondrial matrix were differently affected by the addition of Mg2+, Ca2+, Co2+, K+ and Na+ to the reaction mixture. ATP, ADP and NAD+ stimulated the microsomal activity and inhibited the mitochondrial-matrix enzyme. The activity of both the microsomal and mitochondrial-matrix hydrolase enzymes was specific for long-chain fatty acyl-CoA esters (C12-C18), with the highest activity for palmitoyl-CoA. The apparent Km for palmitoyl-CoA was 47 microM for the microsomal enzyme and 17 microM for the mitochondrial-matrix enzyme. 2. The palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase activities of microsomal fraction had similar pH optima and were stimulated by dithiothreitol, but were affected differently by the addition of Pb2+, Mg2+, Ca2+, Mn2+ and cysteine. The two enzymes had different temperature-sensitivities. 3. The data strongly suggest that palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase are separate microsomal enzymes, and that the hydrolysis of palmitoyl-CoA in the microsomal fraction and mitochondria matrix was catalysed by two different enzymes.     

Purification and characterization of an aminoacyl-tRNA hydrolase from the filamentous fungus Fusarium culmorum

This paper describes the partial purification and characterization of an enzyme present in the fungus Fusarium culmorum which hydrolyzes aminoacyl-tRNA by splitting the ester linkage between the amino acid and the tRNA molecule. The enzyme has a molecular weight of 46 000 as estimated by gel filtration in Sephadex G-100, is maximally active in the presence of a divalent cation (Mg²⁺ or Mn²⁺) and has a pH maximum around neutrality. The enzyme is quite unspecific, hydrolyzing with practically the same efficiency aminoacyl-tRNAs with the amino group either free or substituted. The K_m of the enzyme for phenylalanyl-tRNA^Phe, and N-acetylphenylalanyl-tRNA is around 1 μM. Binding to the 80 S ribosomes but not to the 40 S ribosomal subunit renders the substrate resistant to the action of the hydrolase. The characteristics of this hydrolase are similar to those found for the aminoacyl-tRNA hydrolase of Artemia, and different from the more widely distributed peptidyl-tRNA hydrolases and other more specific aminoacyl-tRNA hydrolases found in different organisms.     

Purification of S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum: reversible inactivation by cAMP and 2'-deoxyadenosine

S-Adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum has been purified to homogeneity. It is composed of four subunits, each with a molecular mass of 47,000. In the hydrolysis direction, the enzyme has a pH optimum of 7.5, a Km for S-adenosyl-L-homocysteine (SAH) of 6 microM, and a Vmax of 0.22 mumol min-1 mg-1. In the synthesis direction, the pH optimum is 8.0, the Km for adenosine is 0.4 microM, and the Vmax is 0.30 mumol min-1 mg-1. Although the enzyme binds beta-nicotinamide adenine dinucleotide, as well as adenosine 3',5'-cyclic monophosphate and 2'-deoxyadenosine, these ligands have no effect on enzymatic activity when added to the assay mixture. However, preincubation of SAH hydrolase with NAD+ results in a 25% activation of the enzyme. In addition, this ligand has a striking effect on subunit-subunit interactions, as shown by stabilization of quaternary structure during polyacrylamide gel electrophoresis. Preincubation with cAMP or 2'-deoxyadenosine inactivates the enzyme. Although in both cases the activity is restored upon further incubation with NAD+, we show that inactivation by these two ligands proceeds by different mechanisms. NAD+-reversible inactivation by cAMP and 2'-deoxyadenosine was also observed with the SAH hydrolase from rabbit erythrocytes. Thus, these previously unreported properties of SAH hydrolase also occur with mammalian enzymes and are not restricted to D. discoideum.     

ADP-ribosylarginine hydrolases

ADP-ribosylation is a reversible post-translational modification of proteins involving the addition of the ADP-ribose moiety of NAD to an acceptor protein or amino acid. NAD: arginine ADP-ribosyltransferase, purified from numerous animal tissues, catalyzes the transfer of ADP-ribose to an arginine residue in proteins. The reverse reaction, catalyzed by ADP-ribosylarginine hydrolase, removes ADP-ribose, regenerating free arginine. An ADP-ribosylarginine hydrolase, purified extensively from turkey erythrocytes, was a 39-kDa monomeric protein under denaturing and non-denaturing conditions, and was activated by Mg²⁺ and dithiothreitol. The ADP-ribose moiety was critical for substrate recognition; the enzyme hydrolyzed ADP-ribosylarginine and (2-phospho-ADP-ribosyl)arginine but not phosphoribosylarginine or ribosylarginine. The hydrolase cDNA was cloned from rat and subsequently from mouse and human brain. The rat hydrolase gene contained a 1086-base pair open reading frame, with deduced amino acid sequences identical to those obtained by amino terminal sequencing of the protein or of HPLC-purified tryptic peptides. Deduced amino acid sequences from the mouse and human hydrolase cDNAs were 94% and 83% identical, respectively to the rat. Anti-rat brain hydrolase polyclonal antibodies reacted with turkey erythrocyte, mouse and bovine brain hydrolase. The rat hydrolase, expressed inE. coli, demonstrated enhanced activity in the presence of Mg²⁺ and thiol, whereas the recombinant human hydrolase was stimulated by Mg²⁺ but was thiol-independent. In the rat and mouse enzymes, there are five cysteines in identical positions; four of the cysteines are conserved in the human hydrolase. Replacement of cysteine 108 in the rat hydrolase (not present in the human enzyme) resulted in a thiol-independent hydrolase without altering specific activity. Rabbit anti-rat brain hydrolase antibodies reacted on immunoblot with the wild-type rat hydrolase and only weakly with the mutant hydrolase. There was no immunoreactivity with either the wild-type or mutant human enzyme. Cysteine 108 in the rat and mouse hydrolase may be responsible in part for thiol-dependence as wall as antibody recognition. Based on these studies, the mammalian and avian ADP-ribosylarginine hydrolases exhibit considerable conservation in structure and function.     

Purification and characterization of leukotriene A4 epoxide hydrolase from dog lung

Leukotriene A4 epoxide hydrolase from dog lung, a soluble enzyme catalyzing the hydrolysis of leukotriene A4 (LTA4) to leukotriene B4 (LTB4) was partially purified by anion exchange HPLC. The enzymatic reaction obeys Michaelis- Menten kinetics. The apparent Km ranged between 15 and 25 microM and the enzyme exhibited an optimum activity at pH 7.8. An improved assay for the epoxide hydrolase has been developed using bovine serum albumin and EDTA to increase the conversion of LTA4 to LTB4. This method was used to produce 700 mg of LTB4 from LTA4 methyl ester. The partial by purified enzyme was found to be uncompetitively inhibited by divalent cations. Ca+2, Mn+2, Fe+2, Zn+2 and Cu+2 were found to have inhibitor constants (Ki) of 89 mM, 3.4 mM, 1.1 mM, 0.57 mM, and 28 microM respectively Eicosapentaenoic acid was shown to be a competitive inhibitor of this enzyme with a Ki of 200 microM. From these inhibition studies, it can be theorized that the epoxide hydrolase has at least one hydrophobic and one hydrophilic binding site.     

Pteroylpolyglutamate hydrolase from human jejunal brush borders. Purification and characterization

Pteroylpolyglutamate hydrolase was solubilized with Triton X-100 from human jejunal mucosal brush borders and purified approximately 5,000-fold using organomercurial affinity chromatography, DEAE-cellulose chromatography, and gel filtration. The apparent molecular weight of the purified enzyme in the Triton micelle was estimated as 700,000 using Bio-Gel A-1.5m gel filtration. Sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis followed by Coomassie stain demonstrated two polypeptide bands at 145,000 and 115,000 daltons. The purified enzyme had an isoelectric point of 7.2, was maximally active at pH 5.5, and was stable above pH 6.5 and at temperatures up to 65 degrees C for at least 90 min. Human jejunal brush-border pteroylpolyglutamate hydrolase is an exopeptidase which liberated [14C]Glu as the sole labeled product of PteGlu2[14C]Glue (where PteGlun represents pteroylpolyglutamate), failed to liberate a radioactive product from PteGlu2[14C]GluLeu2, and released all possible labeled PteGlun products during incubation with Pte[14C]GluGlu6 with the accumulation of Pte[14C]Glu. PteGlu2, PteGlu3, and PteGlu7 were substrates, each with Km = 0.6 microM, whereas PteGlu was a weak inhibitor of the hydrolysis of PteGlu3 with Ki = 20 microM. Components of the pteroyl moiety, Glu, and short chain Glun in alpha or gamma linkages were not inhibitory. The enzyme was activated by Zn2+ or Co2+. The properties of brush-border pteroylpolyglutamate hydrolase are different from those described for the soluble intracellular pteroylpolyglutamate hydrolase in other species and in human mucosa, yet are consistent with previous data on the process of hydrolysis of PteGlun in the intact human intestine.     

Aldolase-like imine formation in the mechanism of action of phosphonoacetaldehyde hydrolase.

Phosphonoacetaldehyde hydrolase (EC 3.11.1.1), the bacterial enzyme that catalyses the reaction HCO-CH2-PO(OH)2+H2O leads to HCO-CH3+Pi, is inactivated by borohydride if either phosphonoacetaldehyde or acetaldehyde is present. This supports the suggestion that the substrate forms an imine with an amino group of the enzyme. Such imine formation would labilize the C-P bond in the same way that aldolase and related enzymes labilize C-C and C-H bonds (Scheme 1a).     

A novel type of short- and medium-chain acyl-CoA hydrolases in brown adipose tissue mitochondria

Acyl-CoA hydrolase activities were studied in brown adipose tissue from hamsters. A latent activity was observed in isolated mitochondria. Two peaks of activity were clearly visible in mitochondria, one with an optimum at propionyl-CoA ("short-chain hydrolase") and one with an optimum at nonanoyl-CoA ("medium-chain hydrolase"); there was only low activity toward palmitoyl-CoA and longer-chain acyl-CoAs. In subcellular fractionation experiments, the activity of the short-chain and the medium-chain hydrolase fully followed that of the mitochondrial matrix marker enzyme 2-oxoglutarate dehydrogenase. The specific activity of the hydrolases in the mitochondrial fraction was doubled after cold acclimation. beta-NADH inhibited the short- and medium-chain hydrolases; alpha-NADH, NADPH, and NAD+ were without effect. ADP stimulated the short- and medium-chain hydrolases; ATP and AMP were practically without effect. Evidence is presented to indicate that NADH and ADP interact on the enzyme at the same site and that ADP is essential for the maintenance of the short- and medium-chain enzyme activities. A positive effect of KCl was found on the short- and medium-chain hydrolase activities. Also, the divalent ions Ca2+ and Mg2+ were stimulatory, but only Ca2+ was able to overcome NADH inhibition, possibly due to interaction directly with NADH. It is concluded that brown adipose tissue mitochondria, besides a conventional type of acyl-CoA hydrolase, contain two species of a novel type of acyl-CoA hydrolases which are characterized by being regulated by ADP and NADH (interacting at a common site) and by having an obligatory requirement for ADP.     

A thiocyanate hydrolase of Thiobacillus thioparus. A novel enzyme catalyzing the formation of carbonyl sulfide from thiocyanate.

A thiocyanate hydrolase that catalyzes the first step in thiocyanate degradation was purified to homogeneity from Thiobacillus thioparus, an obligate chemolithotrophic eubacterium metabolizing thiocyanate to sulfate as an energy source. The thiocyanate hydrolase was purified 52-fold by steps involving ammonium sulfate precipitation, DEAE-Sephacel column chromatography, and hydroxylapatite column chromatography. The enzyme hydrolyzed 1 mol of thiocyanate to form 1 mol of carbonyl sulfide and 1 mol of ammonia as follows: SCN- + 2H2O----COS + NH3 + OH-. This is the first report describing the hydrolysis of thiocyanate to carbonyl sulfide by an enzyme. The enzyme had a molecular mass of 126 kDa and was composed of three different subunits: alpha (19 kDa), beta (23 kDa), and gamma (32 kDa). The enzyme exhibited optimal activities at pH 7.5-8.0 and at temperatures ranging from 30 to 40 degrees C. The Km value for thiocyanate was approximately 11 mM. Immunoblot analysis with polyclonal antibodies against the purified enzyme suggested that it was induced in T. thioparus cells when the cells were grown with thiocyanate.     

Overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Leishmania donovani

The gene encoding S-adenosylhomocysteine (AdoHcy) hydrolase in Leishmania donovani was subcloned into an expression vector (pPROK-1) and expressed in Escherichia coli. Recombinant L. donovani AdoHcy hydrolase was then purified from cell-free extracts of E. coli using three chromatographic steps (DEAE-cellulose chromatofocusing, Sephacryl S-300 gel filtration, and Q-Sepharose ion exchange). The purified recombinant L. donovani enzyme exists as a tetramer with a molecular weight of approximately 48 kDa for each subunit. Unlike recombinant human AdoHcy hydrolase, the catalytic activity of the recombinant L. donovani enzyme was shown to be dependent on the concentration of NAD+ in the incubation medium. The dissociation constant (Kd) for NAD+ with the L. donovani enzyme was estimated to be 2.1 +/- 0.2 microM. The Km values for the natural substrates of the enzyme, AdoHcy, Ado, and Hcy, were determined to be 21 +/- 3, 8 +/- 2, and 82 +/- 5 microM, respectively. Several nucleosides and carbocyclic nucleosides were tested for their inhibitory effects on this parasitic enzyme, and the results suggested that L. donovani AdoHcy hydrolase has structural requirements for binding inhibitors different than those of the human enzyme. Thus, it may be possible to eventually exploit these differences to design specific inhibitors of this parasitic enzyme as potential antiparasitic agents.     

Protein kinase-mediated phosphorylation of a purified sterol ester hydrolase from bovine adrenal cortex.

Sterol ester hydrolase (cholesterol esterase, E.C. 3.1.1.13) of bovine adrenal cortex has been extensively purified by ammonium sulfate fractionation, acid precipitation, hydroxylapatite chromatography, and Sephadex G-200 chromatography. During the purification sequence, the hydrolase activity was purified free of endogenous protein kinase. With this purified preparation, activation by cyclic AMP and ATP-Mg2+ did not occur unless exogenous protein kinase was included in the activating system. Using [gamma-32P]ATP, the transfer of the terminal phosphate to the enzyme protein was demonstrated by three separate experimental approaches. With pooled fractions from Sephadex G-200 chromatography, significant binding of 32P by the enzyme protein was observed only in the presence of exogenous protein kinase. Time course studies disclosed a close concurrence between the extent of activation of the purified enzyme by cyclic AMP-dependent protein kinase and the level of 32P transfer from [gamma-32P]ATP to the enzyme protein. Finally, assays carried out during Sephadex G-200 chromatography showed a correspondence in the peaks for activated sterol ester hydrolase and for 32P binding by protein. The data confirm that activation of adrenal sterol ester hydrolase by cyclic AMP and ATP-Mg2+ involves protein kinase-catalyzed phosphorylation of the enzyme protein.