Characterization and reconstitution of a cell free system for NAD(+)-dependent deoxyhypusine formation on the 18 kDa eIF-4D precursor

Deoxyhypusine formation on the 18 kDa eIF-4D precursor is due to a covalent linkage between a lysine residue of the protein and the aminobutyl moiety derived from spermidine. The deoxyhypusine is then hydroxylated to form hypusine. This post-translational modification represents one of the most specific spermidine-dependent biochemical events in eukaryotic cells. Deoxyhypusine formation can be performed in vitro at pH 9.5 and is greatly stimulated by NAD+. Using the labeling of the 18 kDa protein by [3H]spermidine as an assay for deoxyhypusine formation, we found that (i) significant deoxyhypusine formation can be demonstrated in vitro at pH 7.2 only if NAD+ is present, (ii) deoxyhypusine formation was sensitive to buffer composition; buffers made of basic amino acids and Tris were inhibitory, (iii) sulfhydryl reagents and metal ions such as Cu2+ and Fe3+ were potent inhibitors of deoxyhypusine formation and (iv) the 18 kDa protein substrate was heat-stable. The in vitro activity of deoxyhypusine formation, which depends on the presence of both enzyme and protein substrate, can be separated from the product, eIF-4D, by a one-step Cibacron blue dye affinity column. Taking advantage of this finding, we have developed a simple procedure, based on the use of Cibacron blue dye, for partially purifying both the deoxyhypusine-forming enzyme and the 18 kDa protein substrate. When the partially purified enzyme and protein substrate were mixed in the presence of 1 mM NAD+ and [3H]spermidine, the 18 kDa protein was radiolabeled, no labeling could be detected if any one component was absent. Using partially purified enzyme, we have also determined the half-life of the protein substrate in alpha-difluoromethyl ornithine (DFMO)-treated NB-15 cells and found it to be longer than 10 h.     

The identification of an eukaryotic initiation factor 4D precursor in spermidine-depleted Chinese hamster ovary cells

When Chinese hamster ovary cells are incubated with [terminal methylenes-3H]spermidine, radioactivity is incorporated into a single cellular protein, eukaryotic initiation factor 4D (eIF-4D), through posttranslational synthesis of the amino acid hypusine (N epsilon-(4-amino-2-hydroxybuyly)lysine). The effect of spermidine depletion on this protein modification reaction was studied by high resolution two-dimensional gel electrophoresis. Factor eIF-4D containing both [3H]lysine and [3H]hypusine was detected as one of the major labeled cellular proteins on the fluorographic map of the proteins from Chinese hamster ovary cells that had been incubated with [3H]lysine. When these cells were depleted of spermidine by the use of DL-alpha-difluoromethylornithine before addition of [3H]lysine, no radiolabeling of this mature eIF-4D (hypusine form, Mr approximately 18,000; pI approximately 5.3) occurred. Instead, a new radiolabeled protein (Mr 18,000; pI 5.1) that contained [3H]lysine but no [3H]hypusine or [3H]deoxyhypusine was seen. This protein was identified as an eIF-4D precursor by comparison of the two-dimensional map of its tryptic peptides with that of the tryptic peptides from [3H]lysine-labeled eIF-4D. Further comparisons also suggest that additional post-translational modification processes are involved in the biogenesis of eIF-4D.     

Synthesis and content of polyamines in bloodstream Trypanosma brucei

The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [3H]ornithine, [14C]arginine and [14C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [4C]methionine. Putrescine and sperimidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous 14C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.     

Polyamines in vaccinia virions and polypeptides released from viral cores by acid extraction.

Vaccinia virions propagated in the presence of [3H]ornithine were found to contain two labeled polyamines, spermine and spermidine. In complete virions the ratio of radioactively labeled spermine to spermidine was about 1:10, whereas in viral cores the ratio was 2:5. This suggests that some spermidine was preferentially lost during the conversion of virions to cores or that spermidine was present in the virions both inside and outside the core structure. Addition of [3H]ornithine to vaccinia virus-infected cells as late as 6 h postinfection demonstrated that, although the conversion of this precursor to polyamines was reduced by 50% or more as compared to mock-infected cells, complete inhibition of polyamine synthesis did not occur. Two percent or less of the total radioactivity associated with virions grown in the presence of [3H]ornithine was found to be acid soluble. Polyacrylamide gel electrophoretic analysis showed that all the structural polypeptides were labeled when virions were propagated in the presence of [3H]ornithine. When cores labeled with a mixture of 14C-labeled amino acids were extracted with 0.25 N H2SO4, 12 to 15% of the labeled core polypeptides were released and could be precipitated with acetone. About 40% of [3H]arginine-labeled polypeptides associated with cores were extracted with acid. Four polypeptides or groups of polypeptides were resolved after polyacrylamide gel electrophoresis analysis of the acid-soluble fraction of cores with molecular weights of about 58,000, 34,000, 24,000 and 10,000 to 12,000. About 40% of the [3H]arginine radioactivity extracted from cores coelectrophoresed with the 10,000 to 12,000-molecular weight polypeptide, indicating that this may represent an arginine-rich, histone-like structural polypeptide of the virion.     

Isolation and characterization of an 18 kDa hypusine-containing protein from cultured NB-15 mouse neuroblastoma cells

An 18 kDa protein can be metabolically labeled by [3H]putrescine or [3H]spermidine in various mammalian cells. The labeling is due to a post-translational modification of one lysine residue to hypusine using the aminobutyl moiety derived from spermidine. In view of the lack of knowledge of the function of this spermidine-modified protein, we decided to use the radioactivity associated with the [3H]spermidine-labeled 18 kDa protein as a tracer to develop a simple procedure for purifying this protein from cultured cells. We first screened more than 15 different affinity adsorbents for their ability to bind the labeled 18 kDa protein. This approach enabled us to develop a four-step procedure to purify the labeled 18 kDa protein from NB-15 mouse neuroblastoma cells. The procedure, including a Cibacron Blue column, an omega-aminooctyl-agarose, a Sepharose G-50, and a Mono Q column, resulted in an 800-fold purification of the labeled 18 kDa protein. Two-dimensional gel analysis of fractions enriched in the labeled 18 kDa protein revealed (i) the presence of isoforms of hypusine-containing 18 kDa protein, with pI values ranging from 4.7 to 5.2, and (ii) the presence of an additional labeled protein with an apparent molecular mass of 22 kDa and a pI value of 5.0. The labeling intensity of the 22 kDa protein, however, was less than 5% of that of the 18 kDa protein. Peptide map analysis, using the V-8 proteinase digestion method, indicated that the 18 kDa hypusine-containing protein obtained from NB-15 cells was similar to eukaryotic initiation factor 4D isolated from rabbit reticulocytes.     

Age dependency of the metabolic conversion of polyamines into amino acids in IMR-90 human embryonic lung diploid fibroblasts

When radioactive polyamines (putrescine or spermidine) were incubated with mammalian cells in tissue culture, the radioactivity was incorporated into cellular proteins via two different metabolic pathways; one is metabolic labeling of an 18,000-dalton protein via hypusine formation, and the other is general protein synthesis employing radioactive amino acids derived from biodegradation of polyamines via GABA shunt and Krebs cycle. Aminoguanidine, a potent inhibitor of diamine oxidase, blocked the metabolic conversion of polyamines to amino acids but had no effect on the metabolic labeling of the 18,000-dalton protein. We have investigated these two polyamine-associated biochemical events in IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL). We found that (1) the metabolic labeling of the 18,000-dalton protein was about two-fold greater in young cells (PDL = 22) than that in old cells (PDL = 48), and (2) the metabolic labeling of other cellular proteins, employing amino acids derived from putrescine via polyamine catabolic pathway, was more than six-fold greater in the old cells (PDL = 48) than in the young cells (PDL = 22). Since the rate of protein synthesis was about 1.4-fold higher in the young cells as compared to the old cells, our data indicated that the activity of catabolic conversion of putrescine (or spermidine) to amino acids in old IMR-90 cells was about eight-fold greater than that in young cells. This remarkable increase of polyamine catabolism and the slight decrease of metabolic labeling of the 18,000-dalton protein were also observed in cell strains derived from patients with premature aging disease.     

The multiple nicotinamide nucleotide-binding subunits of bovine heart mitochondrial NADH:ubiquinone oxidoreductase (complex I).

Direct photoaffinity labeling of purified bovine heart NADH:ubiquinone oxidoreductase (complex I) with 32P-labeled NAD(H), NADP(H) and ADP has shown that five polypeptides become labeled, with molecular masses of 51, 42, 39, 30, and 18-20 kDa. The 51 and the 30-kDa polypeptides were labeled with either [32P]NAD(H), [32P]NADP(H) or [beta-32P]ADP. The 42-kDa polypeptide was labeled with [32P]NAD(H) and to a small extent with [beta-32P]ADP. It was not labeled with [32P]NADP(H). The 39-kDa polypeptide was labeled with [32P]NADPH and to a small extent with [beta-32P]ADP. Our previous studies had shown that this subunit also binds NADP, but not NAD(H) [Yamaguchi, M., Belogrudov, G.I. & Hatefi, Y. (1998) J. Biol. Chem. 273, 8094-8098]. The 18-20-kDa polypeptide was labeled only with [32P]NADPH. Among these polypeptides, the 51-kDa subunit is known to contain FMN and a [4Fe-4S] cluster, and is the NAD(P)H-binding subunit of the primary dehydrogenase domain of complex I. The possible roles of the other nucleotide-binding subunits of complex I have been discussed.     

On the turnover of polyamines spermidine and spermine in mouse brain and other organs

The apparent biological half-lives of spermidine and spermine in mouse brain and other organs were determined by measurement of the specific radioactivities of these compounds over long periods of time. The endogenous polyamine pools were labeled by repeated intraperitoneal injections of [1,4-¹⁴C]putrescine·2HCl, [2-¹⁴C]d,l-methionine, [2-³H]l-methionine, andS-adenosyl-[2-³H]l-methionine. Repeated injections were given to ensure labeling of both fast and slow polyamine pools. It was shown that the two parts of the polyamine molecules which derive from ornithine and methionine have significantly different life spans, especially in the brain. Actual turnover rates of polyamines could not be determined because of the active interconversion between spermine and spermidine, and between spermidine and putrescine. The observed reutilization of putrescine originating from spermidine degradation for spermidine biosynthesis, and the analogous reutilization of spermidine in spermine biosynthesis is discussed with respect to its physiological significance and its relationship to cellular organization.     

Covalent incorporation of polyamines as γ-glutamyl derivatives into CHO cell protein

The possible role of polyamines in the covalent modification of proteins in CHO cells was investigated by metabolic labeling with [³H]putrescine. A single radiolabeled protein band with an apparent relative molecular mass of 18 000 Da was observed by SDS-polyacrylamide gel electrophoresis. Almost all the radioactivity covalently linked to this protein was recovered as hypusine. The labeling of this protein was increased several-fold when cells were cultured with α-difluoromethylornithine (DFMO) or with this drug plus methylglyoxal bis(guanylhydrazone) (MGBG), as a result of increase in specific radioactivity of the hypusine immediate precursor, spermidine. Also labeled under the latter condition were other cellular proteins. These were aggregates on the top both of the stacking gel and of the running gel, and protein-like materials with relative molecular masses of 36 and 8 kDa. The radioactivity covalently associated with these proteins was recovered after acid hydrolofsis as polyamines. The identification of γ-glutamylputrescine and γ-glutamylspermidines in proteolytic digests of the acid-insoluble fraction of treated cells indicates that polyamines are covalently linked to these cellular protein. Several possible cellular functions of γ-glutamylpolyamine protein components are discussed.     

An 18000-dalton protein metabolically labeled by polyamines in various mammalian cell lines

The possible role of polyamines in the covalent modification of cellular protein(s) was investigated by studying the metabolic labeling of NB-15 mouse neuroblastoma cells by [14C]putrescine in fresh Dulbecco's medium followed by separation of cellular proteins through sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under such incubation conditions, a single protein band with an apparent molecular weight of 18000 was radioactively labeled. [14C]Spermidine also specifically labeled this protein. The majority of the radioactivity covalently linked to the 18-kDa protein was recovered as hypusine. The radioactive labeling of this protein was stimulated 1.3-fold by 1 mM dibutyryl cAMP and 2.8-fold by 4% fetal calf serum. Fetal calf serum also stimulated the labeling of many other cellular proteins. This may be due to the conversion of putrescine to amino acids via the formation of gamma-aminobutyric acid. Aminoguanidine, a potent inhibitor of diamine oxidase, completely inhibited the fetal calf serum-stimulated labeling of these cellular proteins but had no effect on the labeling of the 18-kDa protein. The specific labeling of the 18-kDa protein by [14C]putrescine occurred in various mammalian cells examined including the N-18 mouse neuroblastoma cells, 3T3-L1 murine preadipocytes, and H-35 rat hepatoma cells. The specificity of labeling of the apparently ubiquitous 18-kDa protein and the stimulation of this labeling by fetal calf serum suggest that this protein may be important in mediating some of the actions of polyamines in cell growth regulation.     

An 18 000-dalton protein metabolically labeled by polyamines in various mammalian cell lines

The possible role of polyamines in the covalent modification of cellular protein(s) was investigated by studying the metabolic labeling of NB-15 mouse neuroblastoma cells by [¹⁴C]putrescine in fresh Dulbecco's medium followed by separation of cellular proteins through sodium dodecyl sulfate-polyacrylamide gel electrophoreses. Under such incubation conditions, a single protein band with an apparent molecular weight of 18 000 was radioactively labeled. [¹⁴C]Spermidine also specifically labeled this protein. The majority of the radioactivity covalently linked to the 18-kDa protein was recovered as hypusine. The radioactive labeling of this protein was stimulated 1.3-fold by 1 mM dibutyryl cAMP and 2.8-fold by 4% fetal calf serum. Fetal calf serum also stimulated the labeling of many other cellular proteins. This may be due to the conversion of putrescine to amino acids via the formation of γ-aminobutyric acid. Aminoguanidine, a potent inhibitor of diamine oxidase, completely inhibited the fetal calf serum-stimulated labeling of these cellular proteins but had no effect on the labeling of the 18-kDa protein. The specific labeling of the 18-kDa protein by [¹⁴C]putrescine occurred in various mammalian cells examined including the N-18 mouse neuroblastoma cells, 3T3-L1 murine preadipocytes, and H-35 rat hepatoma cells. The specificity of labeling of the apparently ubiquitous 18-kDa protein and the stimulation of this labeling by fetal calf serum suggest that this protein may be important in mediating some of the actions of polyamines in cell growth regulation.     

Polyamine-stimulated alteration of the ornithine decarboxylase molecule in Physarum polycephalum.

The molecular mechanism for polyamine-stimulated feedback modification of ornithine decarboxylase isolated from Physarum polycephalum was investigated by using two-dimensional polyacrylamide-gel electrophoresis. Partially purified A-form enzyme was converted into the B-form enzyme by isolated fractions of the Physarum A-B-converting protein, and the substrates and products were subsequently labelled by covalent addition of alpha-difluoro[14C]methylornithine, an enzyme-activated irreversible inhibitor. The active (A-form) and inactive (B-form) states of this enzyme were found to have the same Mr value, 52 000, yet they differed noticeably in their pI values, 5.45 and 5.65 respectively. In further experiments, the use of high-specific-radioactivity [3H]spermidine to stimulate this enzyme modification was shown not to result in the covalent attachment of this polyamine to ornithine decarboxylase. These results demonstrate that the polyamine-induced modification of ornithine decarboxylase in Physarum is not due to any of the mechanisms previously suggested for ornithine decarboxylase inactivation in this and other eukaryotes, namely phosphorylation, covalent polyamine addition or the non-covalent association of a specific low-Mr protein.     

Purification of a benzo[a]pyrene binding protein by affinity chromatography and photoaffinity labeling

Binding proteins for the polycyclic aromatic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) have been purified from C57B1/6J mouse liver. Following affinity chromatography on aminopyrene-Sepharose, a single polypeptide of 29,000 daltons was isolated. The photolabile compound 1-azidopyrene was developed as a photoaffinity labeling agent to identify the protein during its purification. 1-Azidopyrene was found to be a competitive inhibitor of [3H]B[a]P binding. Affinity labeling studies with [3H]-1-azidopyrene in unfractionated cytosol, and in purified preparations, yielded a single covalently labeled protein of 29,000 daltons. The formation of this labeled species was blocked by preincubation with excess unlabeled B[a]P. A native molecular weight of 30,000 was estimated by gel filtration chromatography of [3H]B[a]P- and [3H]-1-azidopyrene-labeled cytosol proteins. An equilibrium dissociation constant of 2.69 +/- 0.66 nM and a maximum number of binding sites of 2.07 +/- 0.10 nmol of [3H]B[a]P bound/mg of protein were estimated for the pure protein. Two-dimensional gel electrophoresis further resolved the purified 29,000-dalton protein into three major isoelectric variants, each of which was specifically labeled by [3H]-1-azidopyrene.     

Synthesis and Content of Polyamines in Bloodstream Trypanosoma brucei*

SYNOPSIS. The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [³H]ornithine, [¹⁴C]arginine and [¹⁴C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [¹⁴C]methionine. Putrescine and spermidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous ¹⁴C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.     

Localization and biosynthesis of polyamines in insulin-producing cells.

Two recently developed fluorescence cytochemical methods, specific for spermidine and spermine, were used to localize polyamines in the endocrine pancreas. The polyamines were restricted to the insulin-producing beta-cells and were mainly associated with the secretory granules. Chemical polyamine determinations carried out on isolated rat and mouse pancreatic islets revealed large amounts of polyamines. Compared with extracts of whole pancreas, the islets contained very high concentrations of spermine relative to spermidine. Biosynthesis of polyamines from [3H]ornithine or from [3H]putrescine in isolated islets was significantly stimulated at high glucose concentrations. Moreover, significant incorporation of label from [3H]putrescine was also detected in gamma-aminobutyric acid. This incorporation, however, was not stimulated by high glucose. Possible roles for polyamines associated with the secretory granules in insulin-producing cells are discussed.     

Polyamines as physiological substrates for transglutaminases

When normal human blood lymphocytes are treated with mitogen in the presence of [3H]putrescine, label is incorporated into a few cellular proteins. Labeled N-(gamma-glutamyl) putrescine, N1-(gamma-glutamyl)spermidine, and N8-(gamma-glutamyl)spermidine were identified in exhaustive proteolytic digests of the cellular protein fraction. The enzyme-mediated clotting of rat seminal plasma to which 14C-labeled spermidine and spermine are added is accompanied by incorporation of the polyamines into a number of seminal plasma proteins. Proteolytic digests of the protein fraction from this clotted seminal plasma contain labeled N1-(gamma-glutamyl)spermidine, N8-(gamma-glutamyl)spermidine, N1,N8-bis(gamma-glutamyl)spermidine, N1-(gamma-glutamyl)spermine, and N1,N12-bis(gamma-glutamyl)spermine. These findings support a proposal that polyamines serve as substrates for transglutaminases both in cells and in an extracellular fluid. They show differences in cellular and extracellular substrate properties of the polyamines and indicate cross-linking through these amines in the extracellular system, but provide no evidence for such cross-linking in the cells.     

A novel approach to detect toxin-catalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin

Certain microbial toxins are ADP-ribosyltransferases, acting on specific substrate proteins. Although these toxins have been of great utility in studies of cellular regulatory processes, a simple procedure to directly study toxin-catalyzed ADP-ribosylation in intact cells has not been described. Our approach was to use [2-3H]adenine to metabolically label the cellular NAD+ pool. Labeled proteins were then denatured with SDS, resolved by PAGE, and detected by flurography. In this manner, we show that pertussis toxin, after a dose-dependent lag period, [3H]-labeled a 40-kD protein intact cells. Furthermore, incubation of the gel with trichloroacetic acid at 95 degrees C before fluorography caused the release of label from bands other than the pertussis toxin substrate, thus, allowing its selective visualization. The modification of the 40-kD protein was ascribed to ADP-ribosylation of a cysteine residue on the basis of inhibition of labeling by nicotinamide and the release of [3H]ADP-ribose from the labeled protein by mercuric acetate. Cholera toxin catalyzed the [3H]-labeling of a 46-kD protein in the [2-3H]adenine-labeled cells. Pretreatment of the cells with pertussis toxin before the labeling of NAD+ with [2-3H]adenine blocked [2-3H]ADP-ribosylation catalyzed by pertussis toxin, but not that by cholera toxin. Thus, labeling with [2-3H]adenine permits the study of toxin-catalyzed ADP-ribosylation in intact cells. Pasteurella multocida toxin has recently been described as a novel and potent mitogen for Swiss 3T3 cell and acts to stimulate the phospholipase C-mediated hydrolysis of polyphosphoinositides. The basis of the action of the toxin is not known. Using the methodology described here, P. multocida toxin was not found to act by ADP-ribosylation.     

Polyamine depletion induces enhanced synthesis and accumulation of cadaverine in cultured Ehrlich ascites carcinoma cells

An exposure of cultured Ehrlich ascites carcinoma cells to DL-α-difluoromethyl ornithine, an irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17), rapidly depleted the tumor cells of putrescine and spermidine. The decrease in the cellular concentrations of these two natural polyamines, however, was accompanied by a striking appearance of two new major amines: cadaverine and a compound tentatively identified as N-3-aminopropyl-1,5-diaminopentane (aminopropylcadaverine). When the cultures were grown in the presence of uniformly labeled [¹⁴C]lysine, tumor cells exposed to difluoromethyl ornithine converted lysine to cadaverine and aminopropyl cadaverine at strikingly enhanced rate. The difluoromethyl ornithine-induced accumulation and synthesis of cadaverine and aminopropylcadaverine were totally prevented by the presence of micromolar concentrations of spermidine (or spermine) in the culture media.     

Effect of portal branch ligation on polyamine metabolism in rat liver

The activity of ornithine decarboxylase and the intracellular concentrations of putrescine and spermidine in the non-ligated lobes of the liver increased after portal branch ligation. These changes were followed by increased [3H]thymidine uptake into the acid-insoluble fraction of the liver. The induction of ornithine decarboxylase and changes in intracellular polyamines are important biochemical events in liver regeneration, so our results suggest that portal branch ligation causes formation of some stimuli that trigger liver regeneration. Changes were less with ligation than with partial hepatectomy.     

Mitochondrial nicotinamide nucleotide transhydrogenase: active site modification by 5'-[p-(fluorosulfonyl)benzoyl]adenosine

Membrane-bound and purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) was inhibited by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), which is an analogue of TH substrates and their competitive inhibitors, namely, 5'-, 2'-, or 3'-AMP. NAD(H) and analogues, NADP, 5'-AMP, 5'-ADP, and 2'-AMP/3'-AMP mixed isomers protected TH against inhibition by FSBA, but NADPH accelerated the inhibition rate. In the absence of protective ligands or in the presence of NADP, FSBA appeared to modify the NAD(H) binding site of TH, because, unlike unmodified TH, the enzyme modified by FSBA under these conditions did not bind to an NAD-affinity column (NAD-agarose). However, when the NAD(H) binding site of TH was protected in the presence of 5'-AMP or NAD, then FSBA modification resulted in an inhibited enzyme that did bind to NAD-agarose, suggesting FSBA modification of the NADP(H) binding site or an essential residue outside the active site. [3H]FSBA was covalently bound to TH, and complete inhibition corresponded to the binding of about 0.5 mol of [3H]FSBA/mol of TH. Since purified TH is known to be dimeric in the isolated state, this binding stoichiometry suggests half-of-the-sites reactivity. A similar binding stoichiometry was found earlier for complete inhibition of TH by [14C]DCCD [Phelps, D.C., & Hatefi, Y. (1984) Biochemistry 23, 4475-4480]. The active site directed labeling of TH by radioactive FSBA should allow isolation of appropriate peptides for sequence analysis of the NAD(H) and possibly the NADP(H) binding domains.