Correlation of an immobilized digestive enzyme assay with poultry true amino acid digestibility for soybean meal.

The immobilized digestive enzyme assay (IDEA) was run on 6 soybean meal (SBM) samples and compared with true amino acid digestibility (TAAD) content, as determined using cecectomized roosters. The IDEA values were excellent TAAD predictors as evidenced by R(2) values of 0.90 and 0.88 for lysine and cystine, respectively. The original IDEA took 2.5 d to run, and therefore we modified the protocol to reduce the time to 18 h. This modified IDEA procedure was run on 17 SBM samples, and IDEA values were shown to be excellent predictors of TAAD content. This IDEA SBM kit was validated by predicting the TAAD of 5 SBM not included in the 17-sample set above and the comparison of the predicted vs. determined TAAD. Finally, the IDEA SBM kit was used to compare the predicted TAAD of 338 SBM samples from around the world. The predicted lysine digestibility on the world survey samples ranged from 70.6 to 95.5% with an average of approximately 89%, and the ranges and means of the other amino acid digestibilities were also calculated.

Fish meals, fish components, and fish protein hydrolysates as potential ingredients in pet foods.

An experiment to determine the chemical composition and protein quality of 13 fish substrates (pollock by-products, n = 5; fish protein hydrolysates, n = 5; and fish meals, n = 3) was conducted. Two of these substrates, salmon protein hydrolysate (SPH) and salmon meal with crushed bones (SMB), were used to determine their palatability as components of dog diets. Pollock by-products differed in concentrations of CP, crude fat, and total AA by 71, 79, and 71%, respectively, and GE by 4.1 kcal/g. Fish protein hydrolysates and fish meals were less variable (approximately 18, 14, and 17%, and 1.4 kcal/g, respectively). Biogenic amine concentrations were much higher in fish protein hydrolysates as compared with pollock by-products and fish meals. Pollock liver and viscera had the highest total fatty acid concentrations; however, red salmon hydrolysate and SMB had the highest total PUFA concentrations (49.63 and 48.60 mg/g, respectively). Salmon protein hydrolysate had the highest protein solubility in 0.2% KOH. Based on calculations using immobilized digestive enzyme assay values, lysine digestibility of fish meal substrates was comparable to in vivo cecectomized rooster assay values and averaged approximately 90.3%. Also, pollock milt, pollock viscera, red salmon hydrolysate, and sole hydrolysate had comparable values as assessed by immobilized digestive enzyme assay and rooster assays. A chick protein efficiency ratio (PER) assay compared SMB and SPH to a whole egg meal control and showed that SMB had high protein quality (PER = 3.5), whereas SPH had poor protein quality (PER value less than 1.5). However, using whole egg meal as the reference protein, both fish substrates were found to be good protein sources with an essential AA index of 1.0 and 0.9 for SMB and SPH, respectively. In the dog palatability experiments, a chicken-based control diet and 2 diets containing 10% of either SPH or SMB were tested. Dogs consumed more of the SPH diet compared with the control, and similar amounts of the SMB and control diets. The intake ratios for each were 0.73 and 0.52, respectively. Salmon protein hydrolysate was especially palatable to dogs. These data suggest that chemical composition and nutritional quality of fish substrates differ greatly and are affected by the specific part of the fish used to prepare fish meals and fish protein hydrolysates.

Chemical composition, protein quality, palatability, and digestibility of alternative protein sources for dogs.

The chemical composition and protein quality of 11 alternative protein sources (chicken products, blood products, enzyme-hydrolyzed fish protein concentrate, soybean meal, and spray-dried pork liver) were determined, and an experiment was conducted to determine palatability and digestibility of processed red blood cell-containing diets. Chicken protein sources differed in concentrations of CP, acid-hydrolyzed fat, and total AA (TAA) by 20, 31, and 24%, respectively, and GE by 1.7 kcal/g. Blood protein sources varied little in acid-hydrolyzed fat and GE concentrations, but concentrations of CP and TAA differed by 11 and 8%, respectively. Protein solubility of chicken and blood protein source categories averaged 57 and 69%, respectively. Protein solubility of enzyme-hydrolyzed fish protein concentrate, soybean meal, and spray-dried pork liver was 53, 67, and 26%, respectively. Based on calculations from immobilized digestive enzyme assay values, lysine digestibility averaged approximately 80.4 and 81.7% for blood and chicken protein sources, respectively. Lysine digestibility values for soybean meal and spray-dried pork liver were 89 and 77%, respectively. A chick protein efficiency ratio (PER) assay showed that chicken protein sources had high protein quality values, as the PER ranged from 2.7 to 5.3, whereas blood protein sources had poor protein quality (PER values less than 1.5). Enzyme-hydrolyzed fish protein concentrate, spray-dried pork liver, and soybean meal had high protein quality (PER values greater than 2.8). In the dog palatability and digestibility experiments, a corn and chicken-based diet supplemented with either 0 or 3% processed red blood cells was tested. The palatability test showed that dogs consumed more of the diet that contained 0% vs. 3% processed red blood cells. The intake ratio for the 3% processed red blood cells diet was 0.34. Nutrient digestibilities did not differ, except for CP, where the digestibility was greater (P = 0.01) for dogs consuming the 0% processed red blood cells diet. These data suggest that chemical composition and quality of alternative protein sources differ greatly among ingredients within the same category. Palatability data suggest that a processed red blood cells-containing diet is not highly palatable but, when this diet was offered as only one food, dogs demonstrated no aversion response but some decrease in protein digestion.

Inhibition of in vivo myocardial ischemic and reperfusion injury by a synthetic manganese-based superoxide dismutase mimetic.

We determined whether an organic superoxide dismutase mimetic could reduce myocardial injury resulting from a 90-min occlusion of the left circumflex coronary artery, followed by 18 hr of reperfusion in an anesthetized canine. The superoxide dismutase-mimetic studied (SC-52608) was a synthetic Mn-based macrocyclic compound. SC-52608 or the inactive analog SC-54385 was administered as four doses of 4 mg/kg i.v. Drug, inactive analog or vehicle was administered 30 and 15 min before ischemia and 15 min and immediately before reperfusion. To ensure parity of left circumflex coronary artery occlusion-induced ischemia, only animals with ischemic zone blood flow of less than 0.15 ml/min/g were included in the final analysis. Ischemic zone blood flow was 0.069 +/- 0.016 ml/min/g in control animals (n = 10), 0.072 +/- 0.010 ml/min/g in SC-52608-treated animals (n = 11) and 0.053 +/- 0.011 ml/min/g in SC-54385-treated (n = 9) animals. A transient hypotensive effect was observed upon SC-52608 administration. Hemodynamic parameters were otherwise unaffected by SC-52608 or SC-54385. The areas at risk of infarct were 39.6 +/- 1.9%, 38.7 +/- 1.1% and 39.4 +/- 1.1% in control, SC-52608-treated and SC-54385-treated animals, respectively. Myocardial infarct sizes (% of area at risk of infarct) were 44.2 +/- 5.6%, 25.7 +/- 4.3% and 35.1 +/- 4.9% in control, SC-52608-treated and SC-54385-treated animals, respectively (P < .05 control vs. SC-52608-treated). Therefore, the synthetic superoxide dismutase mimetic protected the regionally ischemic and reperfused myocardium from injury, implicating oxygen-derived radicals in the tissue-injury process.

Protective effects of the SOD-mimetic SC-52608 against ischemia/reperfusion damage in the rabbit isolated heart.

An experimental model of myocardial ischemia/reperfusion injury was used to assess the cardioprotective effects of SC-52608, a low molecular weight superoxide dismutase mimetic. Langendorff perfused rabbit isolated hearts were subjected to 30 min of global ischemia followed by 45 min of reperfusion. Hearts perfused in the presence of 20 microM SC-52608 exhibited a decrease in the release of creatine kinase and intracellular potassium compared to hearts receiving vehicle (control). A progressive increase in left ventricular end-diastolic pressure developed upon reperfusion in all hearts, but was significantly greater in control hearts when compared to hearts treated with SC-52608 (P < 0.05). In addition, results obtained with a radiolabeled monoclonal antibody to the intracellular protein myosin, indicate an increased degree of irreversible damage in vehicle-treated hearts. Myocardial protection was not significant in an additional group of hearts treated with 10 microM SC-52608. The hemodynamic, biochemical, morphological, as well as the antimyosin binding data, demonstrate that pretreatment with SC-52608 protects the myocardium from damage associated with global ischemia and reperfusion. The mechanism by which SC-52608 mediates the observed protective effect is most likely related to its ability to scavenge superoxide.

A model of human small intestinal absorptive cells. 1. Transport barrier.

The Caco-2 cell culture model of human small intestinal absorptive cells was used to investigate transepithelial transport. Transport of permeability markers such as mannitol demonstrated that Caco-2 monolayers became less permeable with increasing age in culture. Cells were routinely used for transport studies between day 18 and day 32. A transport index was determined for each compound by calculating the ratio of transport of the molecules under investigation to transport of an internal standard such as the permeability marker mannitol. Comparison of transport rates at 4 and 37 degrees C was a simple approach for differentiating primary transport mechanisms (passive paracellular, passive transcellular, or transporter-mediated) but must be coupled with additional experimental manipulations for definitive determination of transport pathways. Compounds predicted to undergo predominantly paracellular transport (mannitol, FITC, PEG-900, and PEG-4000), transporter-mediated transcellular transport (glucose, biotin, spermidine, or alanine), or lipophilic transcellular transport (alprenolol, propranolol, clonidine, or diazepam) showed differential effects of temperature on rates of transport as well as the transport index.

Physiological and cytotoxic effects of Ca(2+) ionophores on Caco-2 paracellular permeability: relationship of 45Ca(2+) efflux to 51 Cr release.

The human intestinal cell line, Caco-2, and the Ca2+ ionophores, A23187 and ionomycin, were used to determine the interrelationships of 45Ca(2+) efflux, transepithelial electrical resistance (Rt), and [3H]-mannitol flux to changes in 51Cr release and lactate dehydrogenase (LDH) activity. Treatment of Caco-2 monolayers with ionomycin at concentrations of between 0.25 and 2.50 mumol/l showed similar 45Ca(2+) efflux rate constants and coefficients. Analysis of the control and ionomycin-induced 45Ca(2+) efflux values showed the data to best fit a three Ca(2+) compartmental model. All changes in Caco-2 Rt and [3H]-mannitol flux were reversible with no significant increases in 51Cr release with ionomycin concentrations of less than or equal to 2.5 mumols/l. Caco-2 monolayers treated with ionomycin at concentrations of between 5.0 and 50.0 mumols/l showed rapid non-exponential 45Ca(2+) effluxes with irreversible changes in Rt, [3H]-mannitol flux, and significant increases in 51Cr release. There was no changes in media LDH activity using either ionomycin or A23187 at concentrations of up to 50 mumols/l for 60 min. The results of our study show that: (1) disruption of Ca(2+) homeostasis in Caco-2 cells will occur with the addition of Ca(2+) ionophores at concentrations of greater than 2.50 mumols/l; (2) high concentrations (greater than 2.5 mumols/l) of ionomycin will cause non-exponential 45Ca(2+) efflux rates with irreversible changes in intracellular Rt and 14C-mannitol flux, and (3) early signs of Ca(2+) ionophore-induced damage can be detected by 51Cr release from Caco-2 cells into the media and not by changes in LDH media activity.

Synthetic peptide inhibitors of complement serine proteases--III. Significant increase in inhibitor potency provides further support for the functional equivalence hypothesis.

Synthetic peptides based on functionally equivalent (as defined by similar patterns of chemically equivalent amino acids) serine protease inhibitor (serpin) C-terminal sequences inhibit both classical and alternative pathways of complement activation. Inhibition was also found with hybrid peptides consisting of the cleavage site of one serpin (antithrombin III, alpha-1-antitrypsin, or antichymotrypsin) attached to the short and long functionally equivalent protease binding cores of the other two serpins. A hybrid peptide composed of the sequence at the site of cleavage of C4 by C1s attached to the long binding core of antithrombin III was selective in inhibiting the classical pathway with no effect on the alternative pathway at a concn of 10 microM. Extension of the functional equivalence hypothesis has produced inhibitors of complement activation named generic and generic +, whose sequences differ by 77% or 87%, respectively, from those of all three serpin sequences. A hybrid peptide composed of the antithrombin III cleavage site attached to the generic peptide is an inhibitor of complement activation at 500 nM, the most potent inhibitor found in this study.

Identification of a serpin-enzyme complex receptor on human hepatoma cells and human monocytes.

Formation of the covalently stabilized complex of alpha 1-antitrypsin (alpha 1-AT) with neutrophil elastase, the archetype of serine proteinase inhibitor serpin-enzyme complexes, is associated with structural rearrangement of the alpha 1-AT molecule and hydrolysis of a reactive-site peptide bond. An approximately 4-kDa carboxyl-terminal cleavage fragment is generated. alpha 1-AT-elastase complexes are biologically active, possessing chemotactic activity and mediating increases in expression of the alpha 1-AT gene in human monocytes and macrophages. This suggested that structural rearrangement of the alpha 1-AT molecule, during formation of a complex with elastase, exposes a domain that is recognized by a specific cell surface receptor or receptors. To test this hypothesis, the known three-dimensional structure of alpha 1-AT and comparisons of the primary structures of the serpins were used to select a potentially exteriorly exposed and highly conserved region in the complexed form of alpha 1-AT as a candidate ligand (carboxyl-terminal fragment, amino acids 359-374). We show here that synthetic peptides based on the sequence of this region bind specifically and saturably to human hepatoma cells and human monocytes (Kd = 4.0 X 10(-8) M, 4.5 X 10(5) plasma membrane receptors per cell) and mediate increases in synthesis of alpha 1-AT. Binding of peptide 105Y (Ser-Ile-Pro-Pro-Glu-Val-Lys-Phe-Asn-Lys-Pro-Phe-Val-Tyr-Leu-Ile) is blocked by alpha 1-AT-elastase complexes, antithrombin III (AT III)-thrombin complexes, alpha 1-antichymotrypsin (alpha 1-ACT)-cathepsin G complexes, and, to a lesser extent, complement component C1 inhibitor-C1s complexes, but not by the corresponding native proteins. Binding of peptide 105Y is also blocked by peptides with sequence corresponding to carboxy-terminal fragments of the serpins AT III and alpha 1-ACT, but not by peptides having the sequence of the extreme amino terminus of alpha 1-AT. The results also show that peptide 105Y inhibits binding of 125I-labeled alpha 1-AT-elastase complexes. Thus, these studies demonstrate an abundant, relatively high-affinity cell surface receptor which recognizes serpin-enzyme complexes (SEC receptor). This receptor is capable of modulating the production of at least one of the serpins, alpha 1-AT. Since the ligand specificity is similar to that previously described for in vivo clearance of serpin-enzyme complexes, the SEC receptor may also be involved in the clearance of certain serpin-enzyme complexes.

Identification of a second active site in laminin for promotion of cell adhesion and migration and inhibition of in vivo melanoma lung colonization.

Previously we reported that a pentapeptide (Tyr-Ile-Gly-Ser-Arg or YIGSR) from domain III of the B1 chain of laminin is a cell attachment site with the ability to stimulate cell adhesion and migration and to block experimental metastases. Here we report studies on the activities of synthetic peptides that cover domain III and report a second biologically active peptide PDSGR from this domain with activities similar to YIGSR. We also show that cyclic YIGSR is more potent in these assays than the linear peptide as expected since this sequence on laminin is bracketed by cysteines. Due to their proximity and similar spectrum of activities, it is possible that these sequences act in concert in the native laminin molecule.

Synthetic peptide inhibitors of complement serine proteases--I. Identification of functionally equivalent protease inhibitor sequences in serpins and inhibition of C1s and D.

Sequence homology comparisons between serum serine protease inhibitors led to the prediction that the C-terminal sequences are functionally equivalent and represent an essential protease binding domain. Inhibition of complement serine protease D cleavage of factor B and of C1s cleavage of C4 by synthetic peptides containing sequences from the C-termini of three serum serine protease inhibitors supports this prediction. These functionally equivalent peptides represent a new class of inhibitors of D and C1s as well as other serum serine proteases.

Synthetic peptide inhibitors of complement serine proteases--II. Effects on hemolytic activity and production of C3a and C4a.

Synthetic peptides based on the amino acid sequence at the site of cleavage of C3 by classical and alternative pathway convertases were found to be poor inhibitors of hemolysis except at concns of 1 mM and higher. Synthetic peptides of a second type, based on the C-terminal sequence of antithrombin III, were more effective; the best among them caused significant inhibition of hemolysis at a concn of 5 microM. A hybrid peptide composed of the sequence at the site of cleavage of C4 by C1s attached to an antithrombin III sequence was selective, inhibiting the classical pathway with no effect on the alternative pathway at a concentration of 25 microM. Several of the antithrombin III peptides that inhibited hemolysis did not inhibit C4 activation by the classical pathway or activation of C3 by the classical and alternative pathways suggesting that these peptides affect hemolysis by inhibiting enzymes other than C1s and C4b2a of the classical pathway and C3bBb of the alternative pathway.

Interactions of S-(2-haloethyl)-mercapturic acid analogs with plasmid DNA.

A series of related S-(2-haloethyl)-L-cysteine analogs were synthesized and their interaction with DNA was studied with plasmid pBR322. Both S-(2-chloroethyl)-L-cysteine (CEC) and S-(2-bromoethyl)-L-cysteine (BrEC) rapidly induced relaxation of the supercoiled plasmid as determined by agarose gel electrophoresis and electron microscopy, whereas S-(2-fluoroethyl)-L-cysteine did not interact with DNA. The relaxation was most probably due to strand scission at alkylated labile sites in the DNA. When 35S-labeled CEC or BrEC was used as the substrate, covalent binding of 35S to DNA was obtained; CEF displayed a somewhat higher binding than BrEC. No binding of 35S was obtained with (2-hydroxyethyl)-L-[35S]cysteine, [35S]cysteine, or [35S]cystine, substrates which did not induce relaxation of the DNA. Esterification of the carboxyl group resulted in a somewhat lower rate of DNA strand scission, whereas N-acetylation prevented the cysteine analogs from inducing DNA strand breaks. S-(2-Chloroethyl)-glutathione (GSH) did not interact with DNA as determined by lack of effect on the superhelicity of DNA, a finding which is in agreement with the hypothesis that the primary amine groups of CEC or BrEC may participate in the formation of reactive intermediates which can interact with DNA. S-(2-Hydroxyethyl)-GSH and S-(2-hydroxyethyl)-L-cysteine were unable to induce DNA strand breaks. Neutral denaturation of supercoiled pBR322 treated with the analogs revealed that compounds which were able to induce DNA strand breaks also interfered with denaturation of double-stranded circular DNA. No such interference was observed when double-stranded linear DNA (obtained by BamH1 restriction digestion) was treated with the analogs prior to denaturation. These data indicate that a marked difference exists between S-(2-chloroethyl)-L-cysteine and S-(2-chloroethyl)-glutathione in their reaction with supercoiled plasmid DNA. Either a major difference exists in the reactivity of the corresponding episulfonium ions of these conjugates or a separate mechanism of alkylation based on a free alpha-amino of the cysteine conjugate is participating in DNA strand breakage and possible crosslinking. In vivo toxic effects of these S-(2-chloroethyl) conjugates are predicted to be distinctly different.

The hydrolysis and alkylation activities of S-(2-haloethyl)-L-cysteine analogs--evidence for extended half-life.

A series of S-(2-haloethyl)-L-cysteine derivatives, which are analogs of the proposed glutathione half-mustard metabolites of dihaloethanes, were synthesized and studied with respect to their hydrolysis and alkylation rates in aqueous solution. The trend of relative hydrolysis rates, Br greater than Cl much greater than F, paralleled their respective leaving group abilities; however, a dramatic rate increase was seen at pH 8 versus pH's 6 or 4. Hydrolysis of S-(2-chloroethyl)-L-cysteine analogs, where the ionizable groups were blocked (carboxyl esterified and/or N-acetylated), revealed that the amine moiety was responsible for the increased hydrolysis of mustard gas (beta, beta'-dichlorodiethyl sulfide) gave similar results with S-(2-chloroethyl)-L-cysteine, a finding which is consistent with the reaction intermediate being a highly charged species. The alkylation rates with 4-(p-nitrobenzyl)-pyridine were not affected by blocking the ionizable groups. A mechanism of internal cyclization is proposed to explain the accelerated alkaline hydrolysis rates noted with S-(2-haloethyl)-L-cysteines but not with the N-acetylated analogs (mercapturic acids). This scheme proposes the formation of 3-(thiomorpholine)-carboxylic acid as an alternative pathway to the generally accepted hydrolysis reaction. This compound and not S-(2-hydroxyethyl)-L-cysteine was the identified product following pH 10 hydrolysis. Increased hydrolysis half-time of amine-blocked cysteine analogs versus parent cysteine analogs may exist with S-(2-haloethyl)-glutathione derivatives which may explain the substantial nucleic acid alkylation seen with S-(2-haloethyl) derivatives of glutathione.

The binding mechanism of glutathione and the anti-tumor drug L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125;NSC-163501) to gamma-glutamyltransferase.

The glutathione-protein binding interactions of rat renal gamma-glutamyltransferase (gamma GT) were studied by examining the effect of phenylglyoxal (PGO), a chemical modifying agent for arginyl residues. PGO inactivation of gamma GT followed pseudo-first order kinetics and the rate was dependent upon the concentration of PGO. Glutathione (GSH) protected the enzyme from inactivation by PGO. The anti-tumor drug L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) inactivated purified gamma GT. The inactivation capability of AT-125 was abolished by esterification of the carboxyl moiety and was regained upon incubation of AT-125 methyl ester with a carboxyl esterase. AT-125 and glutathione may bind to gamma GT via the electrostatic interaction of their respective carboxyl group(s) and an arginyl residue at the active site.

Involvement of arginine residues in glutathione binding to yeast glyoxalase I.

Yeast glyoxalase I was inactivated by arginine-specific reagents. Inactivation by 2,3-butanedione, phenylglyoxal and camphorquinone 10-sulfonic acid followed pseudo first-order kinetics with the rate dependent upon modifier concentration. Extrapolation to complete inactivation showed modification of approx. two of the ten total arginyl residues in the native enzyme, with approx. one residue protected by glutathione (GSH) as determined by [ring-14C]phenylglyoxal incorporation. GSH protected the enzyme from inactivation, whereas methylglyoxal, glutathione disulfide (GSSG) and dithiothreitol afforded partial protection. The hemimercaptal of methylglyoxal and GSH and the catalytic product, S-lactoylglutathione provided substantial protection from inactivation. A methyl ester placed on the glycyl carboxyl moiety of GSH abolished all protective capability which suggests that this functionality is responsible for binding to the enzyme. These results provide the first evidence concerning the molecular binding mode of GSH to an enzyme. Arginyl residues are proposed as anionic recognition sites for glutathione on other GSH-utilizing enzymes.

Phenol sulfotransferase. I. Purification of a rat liver enzyme by affinity chromatography.

A phenol sulfotransferase (3-phosphoadenylylsulfate:phenol sulfotransferase, EC 2.8.2.1) has been purified from rat liver using an affinity chromatography system consisting of a p-hydroxyphenylacetic acid-agarose conjugate. The ligand was separated from the insoluble matrix using a spacer of approx. 25 A degrees. When this affinity system was used in conjunction with other chromatography procedures, e.g., DEAE-cellulose and Sephacryl S-200, a 630-fold purification of the enzyme was achieved. The enzyme had a molecular weight of 69 000 as determined by gel filtration and 70 000 as determined by SDS-polyacrylamide gel electrophoresis. The enzyme readily sulfates p-nitrophenol, 2-naphthol, 1-naphthol, and salicylamide, as well as naturally occurring catecholamines. Using p-nitrophenol as the substrate, the pH optimum was determined to be in the range of 5.5 to 6.4. The Km value determined for p-nitrophenol was 3.6 microM, and that for 3'-phosphoadenosine 5'-phosphosulfate was 2.5 microM. Adenosine 3',5'-bisphosphate was found to be a strong product inhibitor with Ki = 0.4 microM.

Phenol sulfotransferase. II. Inactivation by phenylglyoxal, N-ethylmaleimide and ribonucleotide 2',3'-dialdehydes.

Phenylglyoxal, a chemical modifying agent for arginine residues, produced rapid inactivation of a rat liver phenol sulfotransferase (3-phosphoadenylylsulfate:phenol sulfotransferase, EC 2.8.2.1). Enzyme inactivation was accompanied by incorporation of 1.5 mol [7-14C]phenylglyoxal per mol enzyme. 3'-Phosphoadenosine 5'-phosphosulfate (PAPS), the sulfate donor, prevented inactivation and decreased [7-14C]phenylglyoxal incorporation to 0.78 mol/mol enzyme. The sulfhydryl-modifying agent, N-ethylmaleimide, also caused rapid inactivation of phenol sulfotransferase with concomitant incorporation of 2.35 mol N-[3H]ethylmaleimide per mol enzyme. These results suggest a possible role for arginine residues as anionic recognition sites for the sulfate donor PAPS, and indicate the presence of essential sulfhydryl residues on phenol sulfotransferase. Ribonucleotide dialdehydes (ATPDA, ADPDA, AMPDA, APSDA), but not the corresponding 2',3'-acyclic nucleotides (ATPDO, ADPDO, AMPDO, APSDO), produced rapid and irreversible inactivation of phenol sulfotransferase. These ribonucleotide dialdehydes appear to modify the active site of the enzyme, since inclusion of the sulfate donor, PAPS, or the product, adenosine 3',5'-bisphosphate (PAP), in the incubation mixture prevented loss of enzyme activity. In contrast, the sulfate acceptor, p-nitrophenol, did not show similar protective effects. Kinetic studies indicated that the ribonucleotide dialdehydes inactivated the enzyme via a unimolecular reaction within a dissociable enzyme-inhibitor complex rather than via a nonspecific bimolecular process. Radioactively labeled ribonucleotide dialdehydes (e.g.,[2, 8-3H]ATP) were incorporated into protein concomitant with loss of enzyme activity. The incorporated ligand could be removed by dialysis in phosphate or Tris buffer. The protein-ligand complex could be stabilized to dialysis by pretreatment with sodium borohydride. The results of these studies suggest that ribonucleotide dialdehydes are affinity labeling reagents for phenol sulfotransferase, causing enzyme inactivation by the possible formation of a Schiff base adduct with an active-site lysine residue.