金属螯合载体定向固定化木瓜蛋白酶的研究

以磁性金属螯合琼脂糖微球为载体,利用金属螯合配体（IDACu²⁺）与蛋白质表面供电子氨基酸相互作用的原理,定向固定了木瓜蛋白酶。固定化最适条件为Cu²⁺1.5×10^-2mol/g载体、固定化时间4h、固定化pH7.0、给酶量30mg/g载体。固定化酶的最适反应温度70℃、最适反应pH8.0,固定化酶的热稳定性明显高于溶液酶,固定化酶活力回收为68.4％,且有较好的操作稳定性,载体重复使用5次后固定化酶酶活为首次固定化酶79.71%。     

Cysteine proteinase activity regulation. A possible role of heparin and heparin-like glycosaminoglycans.

Papain is considered to be the archetype of cysteine proteinases. The interaction of heparin and other glycosaminoglycans with papain may be representative of many mammalian cysteine proteinase-glycosaminoglycan interactions that can regulate the function of this class of proteinases in vivo. The conformational changes in papain structure due to glycosaminoglycan interaction were studied by circular dichroism spectroscopy, and the changes in enzyme behavior were studied by kinetic analysis, monitored with fluorogenic substrate. The presence of heparin significantly increases the alpha-helix content of papain. Heparin binding to papain was demonstrated by affinity chromatography and shown to be mediated by electrostatic interactions. The incubation of papain with heparin promoted a powerful increase in the affinity of the enzyme for the substrate. In order to probe the glycosaminoglycan structure requirements for the papain interaction, the effects of two other glycosaminoglycans were tested. Like heparin, heparan sulfate, to a lesser degree, was able to decrease the papain substrate affinity, and it simultaneously induced alpha-helix structure in papain. On the other hand, dermatan sulfate was not able to decrease the substrate affinity and did not induce alpha-helix structure in papain. Heparin stabilizes the papain structure and thereby its activity at alkaline pH.     

A small hydrophobic domain that localizes human erythrocyte acetylcholinesterase in liposomal membranes is cleaved by papain digestion

A small hydrophobic domain in isolated human erythrocyte acetylcholinesterase is responsible for the interaction of this enzyme with detergent micelles and the aggregation of the enzyme on removal of detergent. Papain has been shown to cleave this hydrophobic domain and to generate a fully active hydrophilic enzyme that shows no tendency to interact with detergents or to aggregate [Dutta-Choudhury, T.A., & Rosenberry, T.L. (1984) J. Biol. Chem. 259, 5653-5660]. We report here that the intact enzyme could be reconstituted into phospholipid liposomes while the papain-disaggregated enzyme showed no capacity for reconstitution. More than 80% of the enzyme reconstituted into small liposomes could be released by papain digestion as the hydrophilic form. Papain was less effective in releasing the enzyme from large liposomes that were probably multilamellar. In a novel application of affinity chromatography on acridinium resin, enzyme reconstituted into small liposomes in the presence of excess phospholipid was purified to a level of 1 enzyme molecule per 4000 phospholipid molecules, a ratio expected if each enzyme molecule was associated with a small, unilamellar liposome. Subunits in the hydrophilic enzyme form released from reconstituted liposomes by papain digestion showed a mass decrease of about 2 kilodaltons relative to the intact subunits according to acrylamide gel electrophoresis in sodium dodecyl sulfate, a difference similar to that observed previously following papain digestion of the soluble enzyme aggregates. The data were consistent with the hypothesis that the same hydrophobic domain in the enzyme is responsible for the interaction of the enzyme with detergent micelles, the aggregation of the enzyme in the absence of detergent, and the incorporation of the enzyme into reconstituted phospholipid membranes.     

Purification of detergent-solubilized form and membrane-binding domain of rat γ-glutamyltransferase by immuno-affinity and hydrophobic chromatography

A new method to purify papain- or detergent-solubilized form (papain or detergent form) of γ-glutamyltransferase from rat hepatomas as well as from rat kidney by immuno-affinity column chromatography is presented. The antibody-column was prepared by coupling the anti-kidney papain form antibody, which had been purified by using a kidney papain form-Sepharose column, to CNBr-activated Sepharose 4B. The enzyme bound to the antibody-column was eluted with 0.04 M NH₄OH. By this method, detergent forms were purified 300 and 1600-fold in approx. 50% yields from rat kidney and rat ascites hepatoma AH 13, respectively, and the papain form was also purified 16 000-fold in a similar yield from primary hepatoma which has a very low activity of this enzyme. Preparations thus obtained apparently did not contain any peptide other than heavy and light subunit peptides of this enzyme on SDS-polyacrylamide gel electrophoresis. The detergent form of kidney enzyme was preferentially adsorbed to a hydrophobic column of aminooctyl-Sepharose, while the papain form was not, suggesting that the detergent form might be adsorbed to the column through hydrophobic interaction of the membrane-binding domain. The domain peptide was also purified by the hydropholic column after release from the detergent form by papain treatment. The molecular weight of the peptide was estimated to be about 16 000 on SDS-polyacrylamide gel electrophoresis. On double immunodiffusion, the domain peptide reacted with anti-detergent form antibody but not with anti-papain form antibody. The domain-specific antibody was also purified from the anti-detergent form antibody.     

Purification of detergent-solubilized form and membrane-binding domain of rat gamma-glutamyltransferase by immuno-affinity and hydrophobic chromatography

A new method to purify papain- or detergent-solubilized form (papain or detergent form) of gamma-glutamyltransferase from rat hepatomas as well as from rat kidney by immuno-affinity column chromatography is presented. The antibody-column was prepared by coupling the anti-kidney papain form antibody, which had been purified by using a kidney papain form-Sepharose column, to CNBr-activated Sepharose 4B. The enzyme bound to the antibody-column was eluted with 0.04 M NH4OH. By this method, detergent forms were purified 300 and 1600-fold in approx. 50% yields from rat kidney and rat ascites hepatoma AH 13, respectively, and the papain form was also purified 16 000-fold in a similar yield from primary hepatoma which has a very low activity of this enzyme. Preparations thus obtained apparently did not contain any peptide other than heavy and light subunit peptides of this enzyme on SDS-polyacrylamide gel electrophoresis. The detergent form of kidney enzyme was preferentially absorbed to a hydrophobic column of aminooctyl-Sepharose, while the papain form was not, suggesting that the detergent form might be adsorbed to the column through hydrophobic interaction of the membrane-binding domain. The domain peptide was also purified by the hydrophobic column after release from the detergent form by papain treatment. The molecular weight of the peptide was estimated to be about 16 000 on SDS-polyacrylamide gel electrophoresis. On double immunodiffusion, the domain peptide reacted with anti-detergent form antibody but not with anti-papain form antibody. The domain-specific antibody was also purified from the anti-detergent form antibody.     

Production and recovery of recombinant propapain with high yield

Papain (EC 3.4.22.2), the archetypal cysteine protease of C1 family, is of considerable commercial significance. In order to obtain substantial quantities of active papain, the DNA coding for propapain, the papain precursor, has been cloned and expressed at a high level in Escherichia coli BL21(DE3) transformed with two T7 promoter based pET expression vectors - pET30 Ek/LIC and pET28a⁺ each containing the propapain gene. In both cases, recombinant propapain was expressed as an insoluble His-tagged fusion protein, which was solubilized, and purified by nickel chelation affinity chromatography under denaturing conditions. By systematic variation of parameters influencing the folding, disulfide bond formation and prevention of aggregate formation, a straightforward refolding procedure, based on dilution method, has been designed. This refolded protein was subjected to size exclusion chromatography to remove impurities and around 400 mg of properly refolded propapain was obtained from 1 L of bacterial culture. The expressed protein was further verified by Western blot analysis by cross-reacting it with a polyclonal anti-papain antibody and the proteolytic activity was confirmed by gelatin SDS-PAGE. This refolded propapain could be converted to mature active papain by autocatalytic processing at low pH and the recombinant papain so obtained has a specific activity closely similar to the native papain. This is a simple and efficient expression and purification procedure to obtain a yield of active papain, which is the highest reported so far for any recombinant plant cysteine protease.     

Active water-insoluble derivatives of papain and other enzymes based on preformed diazonium-type supports.

Papain (EC 3.4.22.2) has been coupled to supports of titanium (IV) oxide and cellulose, which are particulate and pre-coated with diazotised 1,3-diaminobenzene, giving water-insoluble and stable derivatives which possess low proteolytic activity but high esterolytic activity. In addition the reversible binding of zinc (II) at the active site of papain has been exploited to inhibit protectively the enzyme during its linkage to the aforementioned supports, thereby yielding water-insoluble derivatives of papain having superior activity upon reactivation with EDTA. Application of the improved procedure of enzyme coupling to macroporous cellulose particles gave a water-insoluble derivative of papain having further enhanced proteolytic activity. Other properties of the water-insoluble derivatives of papain and of similarly prepared water-insoluble conjugates of urease (EC 3.5.1.5) and cholinesterase (EC 3.1.1.8) with cellulose are also reported.     

Adsorption of papain with Cibacron Blue F3GA carrying chitosan-coated nylon affinity membranes

Covalent coupling of chitosan (CS) to activated nylon membrane was performed after the reaction of the microporous nylon membrane with formaldehyde. Non-specific adsorption on the CS-coated nylon membrane decreased greatly, compared with plain nylon membrane. The dye Cibacron Blue F3GA (CB F3GA) as a ligand was then covalently immobilized on the CS-coated membranes. Physical properties of the composite membrane and its applications in affinity membrane chromatography were examined. The contents of CS and CB F3GA-attached membranes were 89.6 mg/g nylon membrane and 146.1 micromol/g nylon membrane, respectively. These CB F3GA-attached composite membranes were used in the papain adsorption studies. Higher papain adsorption capacity, up to 235.3mg/g affinity membrane, was obtained. The adsorption isotherm fitted the Freundlich model well. Significant amount of the adsorbed papain (about 94.3%) was eluted by 1.0M NaSCN at pH 9.0. Experiments on regeneration and dynamic adsorption were also performed. It appears that CB F3GA-CS nylon membranes can be applied for papain separation without causing any denaturation.     

Interaction of a ferrocenoyl-modified peptide with papain: toward protein-sensitive electrochemical probes

A new ferrocenoyl tetrapeptide, Fc-Gly-Gly-Tyr-Arg-OH, has been synthesized, which acts as an effective competitive inhibitor to papain, with a K(i) of 9 microM at pH 6.2. The electrochemical potential of the ferrocenoyl moiety is influenced by papain binding, resulting in a small cathodic shift of 30 mV.     

The Coupling of an Enzymatic Reaction to Transmembrane Flow of Electric Current in a Synthetic "Active Transport" System

If a chemical reaction is constrained to occur within an asymmetric structure, e.g. by the presence of bound or otherwise trapped enzyme, coupling of the reaction to the flow of one or more solutes, or to the flow of electric current, becomes possible. Such systems can serve as models in which transport is “driven” by chemical reaction. In this respect the processes involved are analogous to active transport, though the molecular mechanisms may be quite different from those in nature. A simple arrangement of this kind has been studied: a composite membrane consisting of two ion exchange membranes of opposite fixed charge, separated by an intermediate layer of solution containing papain. An uncharged substrate of low molecular weight acts as “fuel” for the system, N-acetyl-L-glutamic acid diamide. This material (not previously described) hydrolyzes in the presence of papain to ammonium N-acetyl-L-glutamine. The composite membrane gives rise to an electromotive force, ultimately reaching a stationary state, when clamped between two identical solutions in which the affinity of the reaction has been fixed. Onsager''s reciprocity relation has not hitherto been tested in a case of coupling between chemical reaction and a vectorial flow (here electric current); its validity for this system, in which stationary-state coupling occurs, was established over the experimental range of affinities (up to 3 kcal/mole).     

Inhibition of papain by S-nitrosothiols. Formation of mixed disulfides

S-Nitrosylation of protein thiols is one of the cellular regulatory mechanisms induced by NO. The cysteine protease papain has a critical thiol residue (Cys(25)). It has been demonstrated that NO or NO donors such as sodium nitroprusside and N-nitrosoaniline derivatives can reversibly inhibit this enzyme by S-NO bond formation in its active site. In this study, a different regulated mechanism of inactivation was reported using S-nitrosothiols as the NO donor. Five S-nitroso compounds, S-nitroso-N-acetyl-dl-penicillamine, S-nitrosoglutathione, S-nitrosocaptopril, glucose-S-nitroso-N-acetyl-dl-penicillamine-2, and the S-nitroso tripeptide acetyl-Phe-Gly-S-nitrosopenicillamine, exhibited different inhibitory activities toward the enzyme in a time- and concentration-dependent manner with second-order rate constants (k(i)/K(I)) ranging from 8.9 to 17.2 m(-1) s(-1). The inhibition of papain by S-nitrosothiol was rapidly reversed by dithiothreitol, but not by ascorbate, which could reverse the inhibition of papain by NOBF(4). Incubation of the enzyme with a fluorescent S-nitroso probe (S-nitroso-5-dimethylaminonaphthalene-1-sulfonyl) resulted in the appearance of fluorescence of the protein, indicating the formation of a thiol adduct. Moreover, S-transnitrosylation in the incubation of S-nitroso inactivators with papain was excluded. These results suggest that inactivation of papain by S-nitrosothiols is due to a direct attack of the highly reactive thiolate (Cys(25)) in the enzyme active site on the sulfur of S-nitrosothiols to form a mixed disulfide between the inactivator and papain.     

Expanded bed adsorption on supermacroporous cross-linked cellulose matrix

Rigid spherical macroporous adsorbent beads (CELBEADS) prepared by cross-linking of cellulose were characterised and found eminently suitable for use as expanded bed affinity chromatography matrix. Chromatographic runs were performed on a 10 mm diameter column with three solutes tyrosine, papain and bovine serum albumin under non-retaining conditions on CELBEADS and Streamline^TM DEAE, a commercial agarose based expanded bed matrix. Performance of the runs was measured in terms of height equivalent to theoretical plate, HETP. Variation in HETP with velocity on Streamline^TM DEAE gave flat profiles in packed bed and increasing trend in expanded bed. On CELBEADS, the HETP curves in both packed and expanded bed modes followed profiles typical of macroporous adsorbents i.e. increasing and levelling with velocity. HETP values obtained for papain and bovine serum albumin on CELBEADS were lower than those obtained on Streamline^TM DEAE at all velocities. Lactate dehydrogenase was purified from porcine muscle homogenate using Cibacron blue conjugated to CELBEADS using a protocol reported for supports with surface hydroxyl groups. Elution of the enzyme was investigated both in packed mode as well as in expanded mode at a flow rate of 1 ml min^-1. The purification procedure took about 60 minutes and a purification fold of about 14 was achieved in both cases. The adsorbent could be cleaned in place with 5 M urea and used repeatedly without loss of performance.     

An in vitro assessment of several enzymes for the supplementation of rabbit diets

In order to evaluate the effectiveness of several enzymes for use as feed supplements, the stabilities of these enzymes in relation to rabbit gastrointestinal content and feed processing conditions were investigated. Results showed that in glycine-HCl buffer pH 3.2, cellulase, papain and bromelain were acid tolerant while bacillus -amylase and protease were acid labile. When the buffer pH was 2.0, cellulase and papain still showed some pH resistance while bromelain was completely inactivated. With pepsin added to these buffer systems, similar results were obtained. In the gastric content system of pH 2.0, all the enzymes tested were unstable, but when the pH was 3.2 cellulase was fairly stable. Therefore, some factors in the gastric content other than pepsin might inactivate enzymes such as papain and bromelain. These factors might be the components of mineral premix in feed. On the other hand, preincubation of the enzymes with rabbit intestinal content would not affect the activities of these enzymes significantly. For the thermal stability study, it was found that, except for papain, thermal stabilities of all the enzymes tested could be significantly enhanced by mixing with the feed. Since mineral premix has an inhibitory effect on both bromelain and papain, the enzyme stabilization effect from feed could be caused by components other than mineral premix. Therefore, factors affecting the stabilities of each enzyme vary significantly and care must be taken when using these enzymes as feed supplements.     

小鼠抗天花粉蛋白IgE型单抗的酶解及其Fab的制备

研究了Ｐａｐａｉｎ及Ｔｒｙｐｓｉｎ裂解小鼠抗天花粉蛋白ＩｇＥ单抗的条件及Ｆａｂ的制备。Ｐａｐａｉｎ和Ｔｒｙｐｓｉｎ两者都可产生Ｆ（ａｂ′）_２,分子量在１５０～１６０ｋＤ左右;经Ｐａｐａｉｎ裂解的主要产物中还有Ｆａｂ,分子量７２ｋＤ,可通过凝胶过滤获得纯的Ｆａｂ。而Ｔｒｙｐｓｉｎ裂解物经ＤＴＴ还原、碘乙酰胺烷化虽然也可得到Ｆａｂ′（ｔ）,但不易纯化;可见,要制备Ｆａｂ以采用Ｐａｐａｉｎ裂解为好,而制备Ｆ（ａｂ′）_２则可采用Ｔｒｙｐｓｉｎ裂解。这二个酶的裂解速度是Ｔｒｙｐｓｉｎ大于Ｐａｐａｉｎ。     

Purification and characterization of amine oxidase from pea seedlings

A novel, simple, and rapid procedure for the purification of pea seedling amine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fractionation, was purified in two steps: the first one by anion-exchange chromatography and the second one by affinity chromatography. The first chromatography step was carried out on a diethylaminoethyl-cellulose column. By lowering the amount of protein loaded on the column and the buffer concentration it was possible to obtain an enzyme pure at 95% (sp act 1.2 microkat/mg). To achieve a higher degree of purification various affinity resins were prepared and tested. The resins were obtained by covalent immobilization of polyamines on Sepharose according to three different procedures. The best results were obtained with 6-aminohexyl-Sepharose 2B, prepared using CNBr as coupling agent, and eluting the enzyme by a solution containing 1, 4-diaminocyclohexane. This last compound was found to be a relatively strong competitive inhibitor of the oxidative deamination of cadaverine catalyzed by pea seedling amine oxidase (Ki = 32 microM). According to this procedure an electrophoretically homogeneous enzyme, characterized by a specific activity of 1.63 microkat/mg, was obtained.     

Interaction of chicken cystatin with inactivated papains.

Papain which was inactivated by covalent attachment of small substituents to the active-site cysteine, up to the size of a carbamoylmethyl group, bound with high affinity to chicken cystatin (Kd less than approximately 15 pM), although less tightly than did active papain (Kd approximately 60 fM). However, as the size of the substituent was increased further, the affinity decreased appreciably, generally in proportion to the size of the inactivating group. For instance the dissociation constants for papain inactivated with N-ethylmaleimide and [N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-amido-(4-guanido )butane were 0.17 and approximately 10 microM respectively. The spectroscopic changes accompanying the reaction of all but the most weakly binding (Kd greater than or equal to 2 microM) inactivated papains with cystatin were similar to those induced by the active enzyme. Interactions involving the reactive cysteine residue of papain are thus not crucial for high-affinity binding of the enzyme to cystatin, in accordance with a recently proposed model for the enzyme-inhibitor complex, based on computer docking experiments. In this model there is sufficient space around the reactive cysteine in the complex for a small inactivating group, explaining the tight binding of papains with such substituents. However, larger inactivating groups cannot be accommodated in this space and therefore must displace the inhibitor out of the tight fit with the enzyme, in agreement with the observed decrease in binding affinity with increasing size of bulkier substituents. The kinetics of binding of cystatin to inactivated papains were compatible with simple, reversible, bimolecular reactions, having association rate constants of (7-9) x 10(6) M-1 s-1 at pH 7.4, 25 degrees C, similar to what was shown previously for the binding of cystatin to active papain. The rate of association of the inhibitor with either active or inactivated papain thus appears to be primarily diffusion-controlled. The decreasing affinity of cystatin for papains inactivated with groups of increasing size was shown to be due to progressively higher dissociation rate constants, consistent with the greater impairment of fit between the binding regions of the two molecules.     

Enzymatic Peptide Synthesis in Organic Media. Synthesis of CCK-8 Dipeptide Fragments

The enzymatic synthesis of the seven consecutive dipeptide fragments of the cholecysto kinin C-terminal octapeptide (CCK-8) in organic media is reported. The influence of the reaction medium composition, the protease, and the structure of N-α and C-α protecting groups of both carboxyl and amino components was evaluated. α-Chymotrypsin, papain and thermolysin adsorbed on Celite were used as catalysts, under thermodynamic and kinetic control. The carboxamidomethyl, methyl and allyl ester derivatives of acetyl, benzyloxycarbonyl, tert-butyloxycarbonyl and fluoren-9-ylmethoxycarbonyl amino acids, were assayed as carboxy components. Amino acid amide and ester derivatives were employed as nucleophiles with a preference for the latter, since the dipeptide product obtained could be used directly, without any further chemical modification, as acyl-donor in subsequent coupling steps. All dipeptides selected were successfully synthesized, using the optimal combination of protecting groups, reaction media and enzyme different for each coupling reaction. The information gained with this study should be instrumental in designing an optimal strategy for the total enzymatic synthesis of cholecystokinin C-terminal octapeptide (CCK-8).     

Subsite differences between the active centres of papaya peptidase A and papain as revealed by affinity chromatography. Purification of papaya peptidase A by ionic-strength-dependent affinity adsorption on an immobilized peptide inhibitor of papain.

An affinity column consisting of the specific peptide inhibitor of papain, Gly-Gly (O-benzyl)Tyr-Arg, attached to Sepharose was found to bind the active thiol proteinase papaya peptidase A specifically, but only at an ionic strength significantly higher than the one at which papain is bound. When a mixture of active papaya peptidase A and its irreversibly oxidized contaminant was applied to the column, the active enzyme was bound whereas the inactive material was not. The bound enzyme was released by deionized water and found to contain 1 mol of SH group/mol of protein. The different conditions required for the binding of the two enzymes to the immobilized peptide was shown to reflect different ionic-strength-dependences of the affinity of the two enzymes for the peptide in solution. Whereas the affinity of papain for the inhibitor appears to be insensitive to ionic strength over the range studied, that of papaya peptidase A is ionic-strength-dependent and always lower than that of papain. A rate assay is devised for papaya peptidase A with N-benzyloxycarbonylglycine p-nitrophenyl ester as the substrate at pH 5.5. After calibration against an active-site titration the assay yields the thiol-group concentration without interference from inactive contaminants. For the papaya peptidase A-catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester at pH 5.5 kcat. was found to be 16.7s-1, which is about 3 times the value found for the same reaction catalysed by papain.     

Role of the single cysteine residue, Cys 3, of human and bovine cystatin B (stefin B) in the inhibition of cysteine proteinases

Cystatin B is unique among cysteine proteinase inhibitors of the cystatin superfamily in having a free Cys in the N-terminal segment of the proteinase binding region. The importance of this residue for inhibition of target proteinases was assessed by studies of the affinity and kinetics of interaction of human and bovine wild-type cystatin B and the Cys 3-to-Ser mutants of the inhibitors with papain and cathepsins L, H, and B. The wild-type forms from the two species had about the same affinity for each proteinase, binding tightly to papain and cathepsin L and more weakly to cathepsins H and B. In general, these affinities were appreciably higher than those reported earlier, perhaps because of irreversible oxidation of Cys 3 in previous work. The Cys-to-Ser mutation resulted in weaker binding of cystatin B to all four proteinases examined, the effect varying with both the proteinase and the species variant of the inhibitor. The affinities of the human inhibitor for papain and cathepsin H were decreased by threefold to fourfold and that for cathepsin B by approximately 20-fold, whereas the reductions in the affinities of the bovine inhibitor for papain and cathepsins H and B were approximately 14-fold, approximately 10-fold and approximately 300-fold, respectively. The decreases in affinity for cathepsin L could not be properly quantified but were greater than threefold. Increased dissociation rate constants were responsible for the weaker binding of both mutants to papain. By contrast, the reduced affinities for cathepsins H and B were due to decreased association rate constants. Cys 3 of both human and bovine cystatin B is thus of appreciable importance for inhibition of cysteine proteinases, in particular cathepsin B.     

Role of the tryptophan residue in the interaction of pancreozymin with its receptor

1. Analogues of the C-terminal octapeptide and tetrapeptide of pancreozymin with a modified tryptophan residue have been tested on the rat pancreas adenylate cyclase activity, on the enzyme and fluid secretion of the rat pancreas in vivo and on the amylase release from rabbit pancreatic fragments. 2. Fluorination of the tryptophan residue in position 5 or 6 does not influence the effect of the peptides on any of the measured parameters. 3. Methylation of the nitrogen atom in the indolyl ring, which eliminates hydrogen bond formation, markedly reduces the affinity of the peptides for the adenylate cyclase activity and for the amylase release in rabbit pancreatic fragments. The effects on fluid and enzyme secretion in the rat pancreas in vivo are reduced nearly as much. 4. Tetrafluorination of the tryptophan residue, which reduces its charge donor capacity, causes a still larger reduction in activity and affinity of the octapeptide. 5. The tetrafluorinated tetrapeptide stimulates the adenylate cyclase activity and the enzyme and fluid secretion in vivo more than the unmodified tetrapeptide, which may be due to its increased hydrophobicity. 6. Replacement of the nitrogen atom in the indolyl ring of tryptophan by a sulfur or an oxygen atom, which also reduces the charge donor capacity, leads again to a large reduction in the affinity and activity of both the octapeptide and the tetrapeptide. 7. These findings suggest that the charge donor capacity of the tryptophan residue is of primary importance for the biologic activity of pancreozymin, while hydrogen bond formation and hydrophobicity are of secondary importance.