Evidence of two separate mechanisms for the decrease in aryl sulfotransferase activity in rat liver during early stages of 2-acetylaminofluorene-induced hepatocarcinogenesis.

Enzymatic and immunohistochemical experiments were conducted to evaluate the mechanistic basis for the downregulation of the important detoxication/bioactivation enzyme aryl sulfotransferase IV (AST IV) during 2-acetylaminofluorene (2AAF)-induced hepatocarcinogenesis. To distinguish between possible genotoxic and cytotoxic actions of 2AAF, three different dietary protocols were used in these experiments: group 1 received 2AAF for 12 wk, group 2 received 2AAF for 3 or 6 wk and then a control diet lacking xenobiotics for 3 or 6 wk, and group 3 received 2AAF for 3 or 6 wk and then phenobarbital for 3 or 6 wk. When hepatic AST IV activity was assessed, N-hydroxy-2AAF sulfotransferase activity was found to decrease 80-90% in response to 2AAF feeding, but activity recovered to essentially normal levels in the livers of rats subsequently placed on either control diets or diets with phenobarbital, suggesting a reversible cytotoxic mechanism for loss of AST IV activity. However, when liver sections from the rats were evaluated immunohistochemically, two distinct patterns were detected for the downregulation of AST IV activity. In the livers of rats administered only 2AAF (group 1), a general pattern of overall downregulation of AST IV expression was observed throughout the liver and among most but not all newly developed nodules. In tissue sections from rats initially fed 2AAF and then placed on a control diet (group 2) or a diet with phenobarbital (group 3), the nodules continued to show low levels of AST IV expression, while expression in the areas surrounding nodules returned to the normal, high levels. In addition, among those rats fed 2AAF for just 3 wk and then control diet or diet containing phenobarbital for 6 wk, only rats fed phenobarbital developed altered foci that stained weakly for AST IV expression. These results show that there were two kinds of 2AAF-mediated decrease in hepatic AST IV activity: a general overall loss of AST IV expression dependent on administration of 2AAF and reversible upon removal of 2AAF from the diet and a loss of AST IV expression among newly developed liver foci and nodules that persisted in the absence of 2AAF administration and appeared to be a property of 2AAF-induced subpopulations of cells. These patterns may correspond, respectively, to cytotoxic and genotoxic mechanisms of 2AAF action.

Characterization of a complementary DNA for rat liver aryl sulfotransferase IV and use in evaluating the hepatic gene transcript levels of rats at various stages of 2-acetylaminofluorene-induced hepatocarcinogenesis.

A complementary DNA (cDNA) for rat hepatic aryl sulfotransferase IV (AST IV) was isolated, characterized, and used as a hybridization probe to evaluate the molecular basis for the differential expression of AST IV during 2-acetylaminofluorine (2AAF)-induced hepatocarcinogensis. The AST IV cDNA clone was obtained by immunochemical screening of a male Sprague-Dawley rat liver cDNA library. The AST IV cDNA was found to be 1.3 kilobases long and to encode a fusion protein which was reactive with an antibody to AST IV and enzymatically able to generate the sulfuric acid ester of N-hydroxy-2AAF. Sequence analysis of the AST IV cDNA showed it to be 1127 residues in length and to have essentially complete homology with PST-I cDNA, a previously reported (S. Ozawa, et al., Nucleic Acids Res., 18: 4001, 1990), 1028-base cDNA for an uncharacterized rat liver aryl sulfotransferase. Comparison of the PST-I/AST IV cDNA-deduced amino acid sequence with data from a partial (51%) amino acid sequence analysis of purified AST IV showed complete amino acid homology, confirming the identity of the cDNA and establishing that AST IV was an N-blocked, 291-amino acid protein with a molecular mass of 33,909 daltons. The AST IV cDNA sequence differed from the PST-I cDNA in two principal ways: the 5' end lacked 18 coding bases, and the 3' end contained a 190-base extention in the untranslated region, including a consensus sequence for signalling polyadenylation. Studies of AST IV gene transcript levels showed that the livers of rats fed 2AAF for 3 wk (early stage hepatocarcinogenesis) and hyperplastic nodules from the livers of rats fed 2AAF for 19 wk (intermediate stage hepatocarcinogenesis) displayed transcript levels similar to those of livers from normal rats. This contrasted with the 60 to 70% lower than normal capacity of the mRNA fractions to express AST IV observed during in vitro translation. These results indicated that modulation of AST IV expression at early and intermediate stages of hepatocarcinogenesis involved regulatory mechanisms at the translational level. In contrast, mRNA fractions isolated from some 2AAF-induced liver tumors or from known chemical carcinogen-derived rat hepatoma cell lines showed losses of both AST IV transcript level and in vitro translation capacity, suggesting that regulation at the transcriptional level may become important at late stages of 2AAF-induced hepatocarcinogenesis. These results indicated that the molecular mechanisms for the 2AAF-mediated down regulation of AST IV expression during 2AAF-induced hepatocarcinogenesis involved alterations in regulation at both translational and transcriptional levels.

Reaction product inactivation of aryl sulfotransferase IV following electrophilic substitution by the sulfuric acid ester of N-hydroxy-2-acetylaminofluorene.

Rat liver N-hydroxy-2-acetylaminofluorene (N-OH-2AAF) sulfotransferase activity is mediated by aryl sulfotransferase IV (AST IV) and causes the bioactivation of N-OH-2AAF to a highly reactive sulfuric acid ester form putatively capable of inducing liver cancer. Dietary administration of 2-acetylaminofluorene (2AAF) to induce hepatocarcinogenesis in rats has been shown to cause a rapid loss in N-OH-2AAF sulfotransferase activity. A possible mechanism for the in vivo loss in sulfotransferase activity may be the PAPS-dependent, sulfotransferase-catalyzed, reaction product inactivation of the enzyme by covalent reaction with the N-OH-2AAF sulfuric acid ester. In vitro studies to evaluate this possibility utilized a highly purified form of AST IV and measured the extent of PAPS-dependent interaction between the enzyme and N-OH-2[9-14C]AAF. The results showed the presence of a adenosine-3'-phospho-5'-phosphosulfate (PAPS)-dependent 14C-labeling of AST IV. The labeling could be blocked if the sulfotransferase inhibitor pentachlorophenol was present. Analysis of 14C-labeled AST IV following alkaline digestion and chromatography of digestion products indicated that AST IV cysteine and methionine residues were primary sites of 2[9-14C]AAF adduction. Studies involving the pretreatment of AST IV with PAPS and N-OH-2AAF prior to the measurement of N-OH-2AAF sulfotransferase activity showed a close parallel between formation of the AST IV cysteine-2AAF adduct and loss of activity. Similar studies showed that enzyme inactivation and cysteine-2AAF adduct formation could be blocked when excessive amounts of a competing nucleophile, methionine, were present during the pretreatment step, suggesting that inactivation does not proceed by a mechanism-based process. Finally, experiments involving prior reaction of AST IV with the thiol-blocking agent, N-ethylmaleimide, before measurement of enzyme activity showed essentially full loss of sulfotransferase activity and suggested that formation of AST IV cysteine-2AAF adducts could be a mechanism for enzyme inactivation. These results indicate that the in vitro inactivation of AST IV by the reactive N-OH-2AAF sulfuric acid ester is accompanied by covalent binding to AST IV, possibly through the formation of cysteine-2AAF adducts, and suggests that this mechanism merits further consideration as a basis for the loss of N-OH-2AAF sulfotransferase activity in vivo.

Modulation of hepatic mRNA translation activity and specific expression of arylsulfotransferase IV during acetylaminofluorene-induced rat hepatocarcinogenesis.

Enzymatic sulfation of N-hydroxylated arylamines by mammalian hepatic cytosol sulfotransferases (AST; EC 2.8.2.1) is an important metabolic step which generates ultimate carcinogens. The metabolic activity of AST IV, the putative isozymic form of AST primarily responsible for catalyzing N-hydroxy-2-acetylaminofluorene sulfation, is modulated during 2-acetylaminofluorene (AAF)-induced rat hepatocarcinogenesis. To characterize the molecular mechanisms regulating the differential expression of AST IV, we have assessed polyadenylated mRNA derived from the livers of Sprague-Dawley rats undergoing different stages of AAF hepatocarcinogenesis for general in vitro translation capacity and specific expression of AST IV and albumin. Following 1 and 3 cycles of a cyclical feeding regimen (3 weeks 0.05% AAF, then 1 week basal diet), the mRNA capacity for translation was lowered and the expression of AST IV and albumin was down-regulated about 2-fold each but recovered to normal levels when treated rats were subsequently placed on basal diet for 3 continuous weeks. Cytosolic albumin levels were determined by Western blot analysis to be lowered about 1.5-2-fold. In contrast, however, mRNA from rats on basal diets for 3 weeks subsequent to cycle 5 of the feeding regimen recovered only about 50% of the capacity for AST IV expression, although overall translation capacity and albumin expression returned to normal levels. This pattern of reversible expression, followed by irreversible expression of AST IV at early and late stages of AAF hepatocarcinogenesis, respectively, provides the first evidence correlating the modulation of hepatic mRNA capacity for AST IV expression with differential cytosolic AST IV activity in the AAF hepatocarcinogenesis model. The results further suggest that during early stages in hepatocarcinogenesis modulation of mRNA protein synthesis functions may be a critical factor in AAF-mediated lowering of AST IV expression, while other persistent genetic lesions are likely playing a more significant role at the late stages of the carcinogenic process leading to neoplastic transformation of initiated hepatocytes.

2-Acetylaminofluorene-mediated alteration in the level of liver arylsulfotransferase IV during rat hepatocarcinogenesis.

Rat liver cytosolic sulfotransferase activity forms the highly reactive sulfuric acid ester of N-hydroxy-2-acetylaminofluorene (N-OH-2AAF), an ultimate carcinogen in 2-acetylaminofluorene (2AAF) hepatocarcinogenesis. A previous report demonstrated that 2AAF-induced liver hyperplastic nodules displayed a persistent loss of cytosolic N-OH-2AAF sulfotransferase activity following a hepatocarcinogenesis-producing regimen of 2AAF administration. As an initial step in examining the mechanism responsible for lowering N-OH-2AAF sulfotransferase activity, a monospecific polyclonal antibody to aryl sulfotransferase IV (AST IV) was produced and used in the assessment of AST IV as a candidate enzyme for liver cytosolic N-OH-2AAF sulfotransferase activity. Studies comparing the levels of N-OH-2AAF sulfotransferase activity of highly purified AST IV and rat liver cytosols with corresponding immunochemical analysis of AST IV contents demonstrated that there was sufficient AST IV activity in liver cytosols to indicate that it was the primary enzyme catalyzing cytosolic N-OH-2AAF sulfation. A subsequent immunochemical survey of nine extrahepatic tissues showed no detectable AST IV content and indicated that AST IV expression may be tissue specific. An immunochemical comparison of AST IV levels in control liver cytosols (high in sulfotransferase activity) with cytosols from 2AAF-derived hyperplastic nodules (low in sulfotransferase activity) or liver tumors (no sulfotransferase activity) showed low or no detectable levels, respectively, of AST IV. In addition, an immunochemical analysis of four rat hepatoma cell lines showed they contained no detectable levels of AST IV. These results suggested a strong correlation existed between a decrease in AST IV expression and tumor development. When the liver cytosols of rats taken from early, intermediate, and late stages of 2AAF carcinogenesis were analyzed for the development of a persistent loss of N-OH-2AAF sulfotransferase activity, a parallel loss of cytosolic N-OH-2AAF sulfotransferase activity and AST IV content was observed in rats which had proceeded from a stage of low risk to high risk for liver cancer. These findings indicated that (a) AST IV, a liver-specific enzyme, was the principle enzyme comprising cytosolic N-OH-2AAF sulfotransferase activity and (b) the decrease in sulfotransferase activity in nodules and tumors resulted from a decrease in the level of AST IV expression. Furthermore, it is suggested that a persistent decrease in AST IV expression may reflect a role for AST IV as part of a resistance phenotype in which transforming liver cells are able to escape the cytotoxic effects of highly reactive 2AAF metabolites and progress to cancer.

Changes in rat liver N-hydroxy-2-acetylaminofluorene aryl sulfotransferase activity at early and late stages of hepatocarcinogenesis resulting from dietary administration of 2-acetylaminofluorene.

The ability of 2-acetylaminofluorene (AAF) to mediate a loss in N-hydroxy-AAF (N-OH-AAF) aryl sulfotransferase activity when fed to male Sprague-Dawley rats was examined at early and late stages of hepatocarcinogenesis. Administration of 0.05% AAF in the diet for 1 week caused liver N-OH-AAF aryl sulfotransferase activity to decrease to 15 +/- 5% of that for liver from non-carcinogen-fed rats, and the activity remained low throughout 19 weeks of AAF feeding. When rats were fed AAF diet for 3 weeks, then placed on a control diet, liver N-OH-AAF aryl sulfotransferase activity returned to normal levels within 3 weeks. In contrast, when rats were fed AAF for 19 weeks, then placed on control diet for an additional 10 weeks, little or no recovery of N-OH-AAF aryl sulfotransferase activity was observed in cytosols from whole livers or isolated hyperplastic nodules, respectively. These findings suggest two types of AAF-mediated decreases in sulfotransferase activity: (a) a decrease observed early in the initial stages of AAF feeding which returns to normal levels when AAF is removed from diet, and (b) a persistent decrease in activity following long term AAF administration.

Further characterization of the ability of hepatocarcinogens to lower rat liver aryl sulfotransferase activity.

Aryl sulfotransferase (AST) activity in rat liver is thought to be a primary pathway in the bio-activation of various hepatocarcinogens to forms which act as ultimate carcinogens in chemical hepatocarcinogenesis. In an effort to understand the significance of rapid and sustained decreases in liver AST that accompany dietary administration of hepatocarcinogens and to further assess its relationship to carcinogenic processes, we determined the abilities of various xenobiotics known to be hepatocarcinogens or non-hepatic carcinogens to lower AST activity. We also determined whether the co-administration of the AST enzyme inhibitor, pentachlorophenol, with hepatocarcinogens will abrogate the lowering of AST activity caused by hepatocarcinogens which do not utilize AST for bio-activation versus hepatocarcinogens which can utilize AST. Among carcinogens tested thus far, we have found the AST activity of liver cytosols to be lowered by the hepatocarcinogens 2-acetylaminofluorene, ethionine, 3'-methyl-4-dimethylaminoazobenzene, thioacetamide, aflatoxin B1, diethylnitrosamine and benzidine, but not by the non-hepatic carcinogens 2-acetylaminophenanthrene or 3-methylcholanthrene. Pentachlorophenol reversed activity losses when co-administered with all carcinogens which lowers AST activity with the exception of ethionine and thioacetamide. We suggest that AST activity lowering is relatively specific for liver carcinogens and involves two different mechanisms.

Assignment of aromatic resonances in the 1H nuclear magnetic resonance spectra of cardioactive polypeptides from sea anemones.

High-resolution 1H NMR spectroscopy at 300 MHz has been used to investigate the aromatic residues of a series of homologous polypeptides from sea anemones: anthopleurin-A from Anthopleura xanthogrammica and toxins I and II from Anemonia sulcata. Using two-dimensional NMR techniques, specific assignments to individual protons have been made for all aromatic resonances in the spectra of these molecules. In all three polypeptides the resonances from the two conserved Trp residues, 23 and 33, are shifted significantly from their random coil values, and the indole NH resonance of Trp-23 is not observed. These shift perturbations are due in part to a mutual interaction of the two indole rings, which is also indicated by the observation of nuclear Overhauser enhancements between protons of the two rings. Several other nonpolar side chains also interact with these two Trp residues, forming a hydrophobic region, the overall structure of which is conserved throughout the series. The other aromatic residues in these polypeptides appear not to participate in this structural region.

Amino acid sequence of the Anthopleura xanthogrammica heart stimulant, anthopleurin-B.

Anthopleurin-B, the most potent peptide heart stimulant from the sea anemone Anthopleura xanthogrammica, was shown to exist as a single polypeptide chain consisting of 49 amino acid residues. The sequence of the peptide was shown to be: Gly-Val-Pro-Cys-Leu-Cys-Asp-Ser-Asp-Gly- Pro-Arg-Pro-Arg-Gly-Asn-Thr-Leu-Ser-Gly-Ile-Leu-Trp-Phe-Tyr-Pro-Ser- Gly-Cys-Pro-Ser-Gly-Trp-His-Asn-Cys-Lys-Ala-His-Gly-Pro-Asn-Ile-Gly- Trp-Cys-Cys-Lys-Lys. The carboxymethylcysteine derivative, tryptic and chymotryptic peptides (obtained from the derivative and separated by high performance liquid chromatography) were sequenced by manual Edman degradation. Although six carboxymethylcysteine residues were formed by reduction and alkylation of the polypeptide, no cysteine residues were detectable in the native protein, indicating that there are three cystine residues in anthopleurin-B. The amino acid sequence differs in 7 places from anthopleurin-A: at residues 3 (Pro for Ser), 12 (Arg for Ser), 13 (Pro for Val), 21 (Ile for Thr), 24 (Phe for Leu), 42 (Asn for Thr), and 49 (Lys for Gln). These differences are important since anthopleurin-B is about a 12.5-fold better heart stimulant than anthopleurin-A from A. xanthogrammica, anthopleurin-C from Anthopleura elegantissima, and toxin II from Anemonia sulcata.

Suppression of natural killer and lymphocyte functions associated with carcinogen-induced premalignant hyperplastic nodules in rat liver.

The effects of chemical carcinogenesis to produce premalignant hyperplastic nodules in rat liver on concomitant immune function were studied. Induction of hyperplastic nodules in Fischer rats was accomplished using a combined regimen of diethylnitrosamine, 2-acetylaminofluorene, and partial hepatectomy. Hyperplastic nodules were detected in carcinogen-treated rats from 5 to 23 weeks as confirmed by gross pathology, histopathology, and significantly elevated liver gamma-glutamyltransferase activity. Suppression of natural killer activity of either peritoneal or peripheral blood lymphoid, but not splenic, cells for YAC-1 target cells occurred during 5 to 20 weeks in carcinogen-treated rats. Spleen and blood lymphocyte mitogenic responses to concanavalin A and pokeweed mitogen were also suppressed at most intervals from 8 through 20 weeks. Control groups given individual carcinogen or partial hepatectomy alone or in dual combination were not suppressed in their immune function and failed to develop hyperplastic foci or changes in liver gamma-glutamyltransferase. Our findings indicate that immunosuppression of natural killer and lymphocyte mitogenic functions occurs for a protracted period concurrently with the development of the premalignant hyperplastic state in rat liver. The data suggest a potential role for immune competency during the onset of malignant neoplasia.

Excitatory effect of a new polypeptide (anthopleurin-B) from sea anemone on the guinea-pig vas deferens.

1 Anthopleurin-B (AP-B, greater than 3 x 10(-9) M), a newly isolated polypeptide from sea anemone (Anthopleura xanthogrammica), caused powerful rhythmic contractions in the guinea-pig isolated vas deferens. The other polypeptides anthopleurin-A from A. xanthogrammica and anthopleurin-C from A. elegantissima, elicited similar effects but in higher concentrations ( less than 5 x 10(-8) M). Toxin II (10(-6) M) isolated from the sea anemone, Anemonia sulcata, had no effect. 2 The rhythmic contractions induced by AP-B were inhibited by phentolamine, bretylium, guanethidine, reserpine, 6-hydroxydopamine, tetrodotoxin (TTX) and verapamil. Mecamylamine, atropine, methysergide, chlorpheniramine, and indomethacin had no effect. 3 AP-B (10(-8) M approximately 10(-5) M) caused a dose-dependent increase in the amount of endogenous noradrenaline (NA) released from the vas deferens. AP-B (10-5M) increased the amount of NA released to approximately 310 times (12 micrograms/g tissue) that of untreated tissues. 4 The AP-B-induced release of NA was inhibited or abolished by TTX, verapamil, or incubation in Ca-free medium. 5 These results suggest that the AP-B-induced rhythmic contraction of the vas deferens is mediated through the release of NA from adrenergic nerve endings; AP-B is one of the most potent substances in stimulating NA release from the vas deferens.

Cardiotonic polypeptides from Anthopleura xanthogrammica (Brandt) and A. elegantissima (Brandt).

While studying certain coelenterates for antitumor activity, crude aqueous-ethanolic extracts were found to be very highly toxic to mice, and they exhibited potent cardiotoxicity to rats. When tested at sublethal concentrations, extracts of the sea anemones Anthopleura xanthogrammica (Brandt) and A. elegantissima (Brandt) showed a powerful positive inotropic effect with no chronotopic effect. Further pharmacological studies indicated that the adrenergic system was not involved in the mechanism of action, Na+,K+-ATPase was not inhibited, and vascular tissue was not affected by the extracts. Two cardiotonic polypeptides were then isolated from A. xanthogrammica and named anthopleurin-A (AP-A) and anthopleurin-B (AP-B). A third cardiotonic polypeptide, anthopleurin-C (AP-C), was isolated from A elegantissima. AP-A and AP-C have been sequenced and AP-B partially sequenced. Studies of carbon-13 nuclear magnetic resonance, circular dichroism, laser-Raman and fluorescence spectroscopy have given some clues as to tertiary structure. Chemical amino acid blocking studies also have provided some information as to which functional groups are required for activity. AP-A has been thoroughly studied pharmacologically, but the exact mechanism of action is still not known.

Electrical and mechanical effects of anthopleurin-A, a polypeptide from a sea anemone, on isolated rabbit ventricular muscle under conditions of hypoxia and glucose-free medium.

The effects of anthopleurin-A (AP-A, 1 x 10(-8) M) on the membrane action potential and contraction of isolated rabbit ventricular muscle were compared with those of ouabain (5 x 10(-7) M). Under control conditions, AP-A and ouabain showed submaximal (about 80% of maximal) positive inotropic effects without any toxic manifestations. AP-A caused a marked prolongation of action potential duration (APD) without affecting any other parameters of the action potential, whereas ouabain caused a shortening of APD and a slight decrease in maximum diastolic potential (MDP), overshoot (OS), and upstroke velocity (dV/dtmax) of the action potential. The positive inotropic effect of AP-A was relatively well maintained even under hypoxia or in glucose-free medium. Under these abnormal experimental conditions, AP-A caused a prolongation of APD only at the late stage of repolarization (APD80), whereas APD at the early stage of repolarization (APD30) was further shortened. Other parameters of action potential and resting tension (RT) were not influenced by AP-A. In contrast, under similar experimental conditions, ouabain caused no apparent positive inotropic action, but resulted in a marked increase in RT (contracture). In addition, after exposure to ouabain, a progressive shortening of APD and marked decreases in MDP, OS, and dV/dtmax were observed. These results indicate the AP-A has pharmacological properties quite different from those of ouabain and suggest possible advantages of this newly developed cardiotonic agent over cardiac glycosides when acting on the energy-depleted myocardium.

Antineoplastic evaluation of Pacific basin marine algae.

Extracts of 107 marine alga specimens from Pacific islands were tested for P-388 lymphocytic leukemia and Ehrlich ascites tumor in mice. Several specimens showed high antitumor activity in both systems, with some featuring a notable lack of toxicity.

Conformational studies of peptide heart stimulant anthopleurin A. Laser Raman, circular dichroism, fluorescence spectral studies, and Chou-Fasman calculations.

Sea anemone contain a number of closely related peptide heart stimulants. In the present investigation, the conformation of anthropleurin A from Anthopleura xanthogrammica was investigated by laser Raman, circular dichroism, and fluorescence spectral methods and by the Chou-Fasman method using sequence data. The recent 13C NMR data of the peptide (Norton, R.S., and Norton, T.R. (1979) J. Biol. Chem., in press) provided useful information for the interpretation of the above-mentioned spectral data. The results from these spectral methods suggested that anthropleurin A and the related sea anemone peptides are roughly spherical in shape due to the presence of some beta-bends, possibly due to a beta-pleated sheet region and due to the 3 cystine residues in the peptide which exist in the gauche-gauche-gauche configuration. The sole tyrosine residue is exposed to the solvent, a finding which has now been confirmed by 13C NMR. The laser Raman and fluorescence spectral procedures showed that one or more of the tryptophan residues are buried. Interestingly, the reduction of the native protein with dithioerythritol did not change the spherical shape even in the presence of 5 M guanidine HCl and the carboxymethylcysteine derivative of the peptide was folded even in the presence of the denaturing agent, guanidine HCl.

Natural abundance carbon-13 nuclear magnetic resonance study of anthopleurin-A, a cardiac stimulant from the sea anemone Anthopleura xanthogrammica.

Natural-abundance 13C NMR spectra (at 15.04 MHz) of the polypeptide cardiac stimulant Anthopleurin-A are presented. The spectra contain many resolved one- and two-carbon resonances from carbonyl and aromatic carbons and a few resolved resonances from aliphatic carbons. Most of these have been assigned to individual carbons in the protein. The effect of pH on the 13C spectrum has been investigated. In conjunction with the resonance assignments, this yields estimates for the pK alpha values of the COOH-terminal and NH2-terminal residues, the side chain carboxylate of 1 of the 2 aspartic acid residues, and the imidazolium groups of the 2 histidine residues. The effects of the lanthanides La3+ and Gd3+ on the spectrum have also been studied. The results suggest that there are at least two binding sites, and further studies will be required to characterize these before they can be utilized as an aid in structural mapping. Finally, the results are discussed in relation to a postulated model for the mode of action of Anthopleurin-A.