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The human small intestine is the key organ for absorption, metabolism, and excretion of orally administered drugs. To preclinically predict these reactions in drug discovery research, a cell model that can precisely recapitulate the in vivo human intestinal monolayer is desired. In this study, we developed a monolayer platform using human biopsy-derived duodenal organoids for application to pharmacokinetic studies. The human duodenal organoid-derived monolayer was prepared by a simple method in 3–8 days. It consisted of polarized absorptive cells and had tight junctions. It showed much higher cytochrome P450 (CYP)3A4 and carboxylesterase (CES)2 activities than did the existing models (Caco-2 cells). It also showed efflux activity of P-glycoprotein (P-gp) and inducibility of CYP3A4. Finally, its gene expression profile was closer to the adult human duodenum, compared to the profile of Caco-2 cells. Based on these findings, this monolayer assay system using biopsy-derived human intestinal organoids is likely to be widely adopted., Graphical abstract, Mizuguchi and colleagues produced a monolayer from biopsy-derived human duodenal organoids by means of a simple and straightforward method. The monolayer recapitulates the nature of the in vivo small intestine, and therefore this monolayer can be a general platform for a wide range of applications including pharmaceutical research.

Human intestinal organoids are expected to be applied in pharmaceutical research. Various culture media for human intestinal organoids have been developed, but it remains unclear which media are preferable for pharmacokinetic studies. Here, we cultured human intestinal organoids with three major culture media that are already used widely around the world: the medium of Sato et al. (S-medium; reported in 2011), Fujii et al. (F-medium; 2018), and Miyoshi et al. (M-medium; 2013). The growth of human intestinal organoids cultured in S-medium was faster than that in F- or M-medium. The gene expression levels of most pharmacokinetic-related enzymes or transporters in human intestinal organoids cultured in M-medium were higher than those in S- or F-medium, and comparable to those in the adult human small intestine. The level of cytochrome P450 (CYP) 3A4 activity was also highest in human intestinal organoids cultured in M-medium. Collectively, the results underscored the importance of selection and optimization of culture medium for various applications using human intestinal organoids, including pharmacokinetic studies.

Interaction of CC chemokine receptor 5 (CCR5) with the human immunodeficiency virus type 1 (HIV-1) gp120/CD4 complex involves its amino-terminal domain (Nt-CCR5) and requires sulfation of two to four tyrosine residues in Nt-CCR5. The conformation of a 27-residue Nt-CCR5 peptide, sulfated at Y10 and Y14, was studied both in its free form and in a ternary complex with deglycosylated gp120 and a CD4-mimic peptide. NMR experiments revealed a helical conformation at the center of Nt-CCR5(1-27), which is induced upon gp120 binding, as well as a helical propensity for the free peptide. A well-defined structure for the bound peptide was determined for residues 7-23, increasing by 2-fold the length of Nt-CCR5's known structure. Two-dimensional saturation transfer experiments and measurement of relaxation times highlighted Nt-CCR5 residues Y3, V5, P8-T16, E18, I23 and possibly D2 as the main binding determinant. A calculated docking model for Nt-CCR5(1-27) suggests that residues 2-22 of Nt-CCR5 interact with the bases of V3 and C4, while the C-terminal segment of Nt-CCR5(1-27) points toward the target cell membrane, reflecting an Nt-CCR5 orientation that differs by 180° from that of a previous model. A gp120 site that could accommodate (CCR5)Y3 in a sulfated form has been identified. The present model attributes a structural basis for binding interactions to all gp120 residues previously implicated in Nt-CCR5 binding. Moreover, the strong interaction of sulfated (CCR5)Tyr14 with (gp120)Arg440 revealed by the model and the previously found correlation between E322 and R440 mutations shed light on the role of these residues in HIV-1 phenotype conversion, furthering our understanding of CCR5 recognition by HIV-1.

Dendrotoxin I (DTX-I) is a 60-residue peptide from the venom of the black mamba snake Dendroaspis polylepis, which binds to neuronal K+ channels. The structure reported previously for DTX-I was synthesized for the first time by a solution procedure. The synthetic product was confirmed to have the correct primary and disulfide structure determined by peptide mapping, sequence analysis and mass measurements. Comparison of synthetic DTX-I with the natural one by high-performance liquid chromatography and capillary zone electrophoresis, as well as by sequence analysis, revealed that the Asn residue at position 12 in the synthetic peptide was Asp in the natural product. Synthesis of DTX-I with Asp at position 12 gave a peptide identical with the natural product in all aspects. NMR analysis of synthetic [Asn12]- and [Asp12]-DTX-I also supported our findings that the Asn residue at position 12 in the DTX-I molecule should be revised as Asp. [Asn12]- and [Asp12]-DTX-I had very similar binding affinities when tested against radiolabeled dendrotoxin binding to rat brain synaptosomal membranes.

Native chemical ligation (NCL) approaches have been applied extensively to soluble proteins. Fewer successes have been achieved with membrane peptides. In this report, the synthesis and semisynthesis by NCL of peptides corresponding to 1.7 transmembrane domains of the α-factor receptor from Saccharomyces cerevisiae is described. Synthesis was achieved when the ligation point was approximately in the middle of the loop joining the two transmembrane regions. In contrast, little to no ligation was observed when the ligation point was at the putative membrane interface of the sixth transmembrane domain (TM6) and the third extracellular loop (EL3). Ligations of a chemically synthesized 22-residue thioester with a synthetic 29-residue N-Cys peptide and a biosynthetic 73-residue N-Cys peptide were successfully achieved in both trifluoroethanol/guanidinium hydrochloride (TFE/GnHCl) and sodium dodecyl sulfate (SDS) media when mercaptoethanesulfonic acid (MESNA) was used as a catalyst. The resulting 51-residue and 95-residue ligation products were purified by reversed phase HPLC and recovered on a mg scale. Both peptides were >95% pure as determined by HPLC and had the expected molecular weight as judged by mass spectrometry. Segmental labeling of the 95-residue fragment, in which the N-Cys portion was [15N] labeled, resulted in a peptide that gave an NMR spectrum which was comparable to that of the unligated 73-residue peptide alone.

Transmembrane domains (TMDs) of G-protein coupled receptors (GPCRs) have very low water solubility and often aggregate during purification and biophysical investigations. To circumvent this problem many laboratories add oligolysines to the N -a ndC-termini of peptides that correspond to a TMD. To systematically evaluate the effect of the oligolysines on the biophysical properties of a TMD we synthesized 21 peptides corresponding to either the second (TPIFIINQVSLFLIILHSALYFKY) or sixth (SFHILLIMSSQSLLVPSIIFILAYSLK) TMD of Ste2p, a GPCR from Saccharomyces cerevisiae. Added to the termini of these peptides were either Lysn (n = 1,2,3) or the corresponding native loop residues. The biophysical properties of the peptides were investigated by circular dichroism (CD) spectroscopy in trifluoroethanol-water mixtures, sodium dodecyl sulfate (SDS) micelles and dimyristoylphosphocholine (DMPC)-dimyristoylphosphoglycerol (DMPG) vesicles, and by attenuated total reflection Fourier transform infrared (ATR-FTIR) in DMPC/DMPG multilayers. The results show that the conformation assumed depends on the number of lysine residues and the sequence of the TMD. Identical peptides with native or an equal number of lysine residues exhibited different biophysical properties and structural tendencies. Copyright  2006 European Peptide Society and John Wiley &S ons, Ltd.

Segment condensation reaction of sparingly soluble protected peptides proceeded smoothly in CHCl3-phenol mixed solvent without danger of epimerization or of significant ester formation with the carboxyl component when 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was employed in the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt). The optimal conditions for enhancement of peptide coupling mediated by EDC/HOOBt in CHCl3-phenol were determined and successfully applied to the synthesis of amyloid β-peptide (1–42), (1–43) and [Pyr3]-(3–42). These peptides of high homogeneity were used to examine the relation between structure and amyloidogenesis by means of CD spectra and fluorimetric assay.

During HF reaction in the presence of anisole or p -cresol, a side reaction was observed associated with use of the N in -cyclohexyloxycarbonyl (Hoc) group for tryptophan (Trp). The structure of the modified Trp and the HF conditions for suppressing this side reaction are described.

Human pleiotrophin (hPTN), a novel heparin-binding neurotrophic factor consisting of 136 amino acid residues with five intramoleular disulfide bonds, was synthesized by solution procedure in order to demonstrate the utility of our strategy using our newly developed solvent system, a mixture of trifluoroethanol (TFE) and dichloromethane (DCM) or chloroform (CHL). The final protected peptide was synthesized by coupling two larger protected intermediates, Boc-(1-64)-OH and H-(6 5-136)-OBzl, in CHL/TFE (3 : 1; v/v) using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) in the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt). After removal of all protecting groups using the HF procedure followed by treatment with Hg(OAc)2, the fully deprotected peptide was subjected to an oxidative folding reaction. The product was confirmed as having the correct disulfide structure by examining the cystine peptides obtained by enzymatic digestions, and as possessing the same biological activities as those of the natural product. The N- and C-terminal half domains (1-64 and 65-136) were also synthesized, and measurement of their biological activities indicated that the C-terminal half domain displays almost all the activities of the full-length molecule, whereas the N-terminal half domain shows almost no activity. From these results, we were able to confirm that the C-terminal half domain is responsible for the expression of biological activities in the same manner as human midkine (hMK), another heparin-binding neurotrophic growth factor.

The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⋅HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (λ max = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.

The 3-pentyl (Pen) group is a new base-resistant protecting group for tyrosine. It is sufficiently stable in 50% TFA/DCM and completely stable towards 20% piperidine/DMF but is readily cleavable by the standard HF procedure without the formation of any significant amount of alkyltyrosine rearrangement product.

Several new protecting groups were introduced at the Nin-position of tryptophan, and their reactivities were examined under the conditions used for peptide synthesis by Boc-strategy. Among them, the cyclohexyloxycarbonyl (Hoc) group was found to be the most suitable in terms of stability during elongation of the peptide chain and removability at the final HF reaction without resorting to the use of thiols.

Human midkine (hMK), a novel heparin-binding neurotrophic factor consisting of 121 amino acid residues with five intramolecular disulphide bonds, was synthesized by solution procedure in order to demonstrate the usefulness of our newly developed solvent system, a mixture of dichloromethane or chloroform and trifluoroethanol. The final protected 121-residue peptide was assembled from two large fully protected intermediates, Boc-(1-59)-OH and H-(60-121)-OBzl, in CHL/TFE(3:1, v/v) using water-soluble carbodiimide in the presence of HOOBt as coupling reagents. After removal of the protecting groups by HF followed by treatment with Hg(OAc)2 in 50% acetic acid, the fully deprotected peptide was subjected to the oxidative folding reaction. The final product was confirmed to have the correct disulphide structure from its tryptic peptide mapping and to possess the same biological activities as those of the natural product. In order to clarify the active region of the hMK molecule, the N-terminal half domains [(1-59) and (60-121)] were also synthesized by the same procedure used for the hMK synthesis. The C-half domain was confirmed to show the full pattern of bioactivities except for the neuronal cell survival activity, while the N-half one showed much less activity in general.

Chemically synthesized human midkine enhanced plasminogen activator activity and decreased its inhibitor levels in bovine aortric endothelial cells. These activities were preserved in the C-terminal half, but not in the N-terminal half of the midkine molecule. Furthermore, a synthetic peptide of 43 amino acids designated as "C-domain", which formed the compact structure held by two disulfide bonds in the C-terminal half, mimicked intact midkine. Chemically synthesized C-domain of pleiotrophin (43 amino acids), which was 53% identical to midkine C-domain in amino acid sequence, expressed the similar activities. These 43 amino acid peptides are, so far, the shortest peptide able to enhance the fibrinolytic activities of the endothelial cells.

Several new protecting groups were introduced at the Nin-position of tryptophan, and their reactivities were examined under the conditions used for peptide synthesis by Boc-strategy. Among them, the cyclohexyloxycarbonyl (Hoc) group was found to be the most suitable in terms of stability during elongation of the peptide chain and removability at the final HF reaction without resorting to the use of thiols.

The 3-pentyl (Pen) group is a new base-resistant protecting group for tyrosine. It is sufficiently stable in 50% TFA/DCM and completely stable towards 20% piperidine/DMF but is readily cleavable by the standard HF procedure without the formation of any significant amount of alkyltyrosine rearrangement product.

Calcicludine (CaC) and dendrotoxin-I (DTX-I) possess high homology of their primary structures despite their different biological activities acting on calcium and potassium channels, respectively. In order to elucidate the channel specificity displayed by these toxins, their three-dimensional structures were compared by NMR. These analyses revealed that their overall conformations are similar except for the structure at the N-terminus. To demonstrate the significance of this N-terminal, chimeric peptides, CaC(1-30)/DTX-I(31-60) and DTX-I(1-30)/CaC(31-60), were synthesized. The CD spectra and receptor-binding measurements of chimeric peptides indicated that the contribution to the overall conformation and to the affinity of the N-terminal part of molecule seem to be more important than that of the C-terminal one. These results suggest that the N-terminal part may participate in distinguishing between calcium and potassium channels.

Amyloid (1-40, 1-42, 3-42, or 1-43) are sparingly soluble proteolytic fragments present as the major constituent of extracellular proteinaceous deposits known as amyloid plaques. Because of the hydrophobic composition of the C-terminal region of , severe solubility problems arise during the synthesis of these peptides due to aggregation. To prevent this, in stepwise solid-phase synthesis, amide backbone protecting groups (e.g., 2-hydroxy-4-methoxybenzyl) were introduced, but this often led to accumulation of amino acid deletion products which can be extremely difficult to separate from the desired peptide. More promising is the segment condensation method, which can be employed to prepare large quantities of peptides without the formation of amino acid deletion products. This procedure, performed in the solution phase, is extremely efficient for synthesizing sparingly soluble peptides if solvent systems that dissolve the protected intermediates are available during the construction of molecules. In the present study, we developed a new solvent system, a mixture of chloroform and phenol, for the segment condensation of sparingly soluble protected peptides in solution and successfully used it to synthesize and [1].

The total synthesis and structural characterization of the MHCII-associated p41 invariant chain fragment (P41icf) is described. P41icf plays a crucial role in the maturation of MHC class II molecules and antigen processing, acting as a highly selective cathepsin L inhibitor. P41icf synthesis was achieved using a combined solid-phase/solution approach. The entire molecule (65 residues, 7246 Da unprotected) was assembled in solution from fully protected peptides in the size range of 10 residues. After deprotection, oxidative folding in carefully adjusted experimental conditions led to the completely folded and functional P41icf with a disulfide pairing identical to that of native P41icf. CD, NMR, and surface plasmon resonance (SPR) were used for the structural and functional characterization of synthetic P41icf. CD thermal denaturation showed clear cooperative behavior. Tight cathepsin L binding was demonstrated by SPR. (1)H NMR spectroscopy at 800 MHz of unlabeled P41icf was used to solve the three-dimensional structure of the molecule. P41icf behaves as a well-folded protein domain with a topology very close to the crystallographic cathepsin L-bound form.

We have recently demonstrated the utility of a combined solid-phase and solution approach for protein synthesis [1]. The procedure consists of the preparation of protected segments by solid-phase with Boc chemistry and subsequent segment condensation in solution, followed by final deprotection using HF. Each fully protected segment with a free α-carboxyl group is detached from an N-[9-(hydroxymethyl)-2-fluorenyl] succinamic acid (HMFS) linker [2] by treatment with 20% morpholine/DMF. When employing a base-labile linker such as HMFS, the side-chain protecting groups must be completely stable under the basic conditions used to detach the segments from the resin. For this purpose, the cyclohexyloxycarbonyl (Hoc) group was developed as a base-resistant side-chain protecting group for the Trp residue suited to our synthetic strategy. However, side products of MW 108 higher than that of the desired product were observed, when the Hoc group was exposed to HF in the presence of p-cresol, and these side products were identified as two steric trans-isomers of 2-[2’-(p-cresol)]-2,3-dihydrotryptophan (Figure 1) [3]. The side reaction arising from modification of the Trp residue is inherent to the carbamate-type N in-protecting groups when performing HF treatment in the presence a scavenger. In the present study, we investigated the HF conditions applicable to the practical peptide synthesis for suppressing the modification of the Trp residue. Furthermore, application of this side reaction was examined to develop a facile procedure for introducing aromatic ring systems at the 2-position of the indole moiety.

The segment condensation strategy performed using either a solution or solid phase is one of the most advantageous procedures for synthesizing large peptides or proteins. We have recently demonstrated the utility of a combined solid-phase and solution approach for rapid protein synthesis by synthesizing muscarinic toxin 1 (66 AAs) [1] and green fluorescent protein (238 AAs) [2]. The procedure involves performing the segment condensation reaction in solution employing a maximum protection strategy with Boc chemistry, followed by final deprotection using HF. Each segment used in the subsequent segment condensation is prepared by solid-phase assembly on an N-[9-(hydroxymethyl)-2-fluorenyl] succinamic acid (HMFS) linker [3], which is designed to be cleaved by nucleophiles such as morpholine to produce fully protected segments with a free α-carboxyl group. To establish the present strategy for general protein synthesis, we had to resolve the following issues encountered during preparation of the protected segments on the resin as well as the subsequent segment condensation in solution and HF treatment: 1) development of base-resistant side-chain protecting groups for Trp and Tyr, 2) completion of amino acid-coupling in a mixture of chloroform (CHL) and trifluoroethanol (TFE) on the resin, 3) exclusion of deletion products generated at the N-terminus of His(Bom), 4) development of a solvent system for segment condensation of sparingly soluble protected peptides, and 5) suppression of the Trp modification associated with use of an N in-carbamate-type protecting group in HF.

A topochemical model to explain the biological activity of dendrotoxin-I (DTX-I), a potent blocker for potassium channels, was developed by searching common spatial arrangements of functionally important residues between DTX-I, alpha-dendrotoxin, dendrotoxin-K, BgK, ShK, and charybdotoxin. The first three are structurally and functionally related to one another, and specifically target to Kv1 type potassium channels. The last three are structurally unrelated to the first three but have the ability to displace (125)I-labeled dendrotoxins on the same types of potassium channels. In order to obtain the correct electronic surface potential, thought to be crucial for the DTX-I function, we determined the three-dimensional solution structure of DTX-I by nmr spectroscopy using its correct amino acid sequence recently determined by our group. The most interesting characteristic of our model is that DTX-I has two binding sites to potassium channels: one is the cationic domain made up of Lys residues at positions 5 in the 3(10)-helix, 28 and 29 in the beta-turn, and the other is the Lys19/Tyr17/Trp37 triad located in the antiprotease domain. The cationic domain and the triad are located at the opposite sides of the molecular structure and are separated by about 25 A between Lys29 Calpha and Tyr17 Calpha. The functional triad is characterized by three distances, d(1) approximately 7.5 A (Lys19 Calpha-the center of the Tyr17 aromatic ring), d(2) approximately 8.1 A (Lys19 Calpha-the center of the 6-membered ring of the Trp37 indole group), and d(3) approximately 7. 3 A (the center of the Tyr17 aromatic ring-the center of the 6-membered ring of the Trp37 indole group). This model should aid in the pharmaceutical design of peptide and nonpeptide drugs with potassium channel blocking potencies, as well as in understanding of the physiology, pharmacology, biochemistry, and structure-function analysis of potassium channels.

In the synthesis of large peptides or proteins, highly homogeneous segments are indispensable for a convergent strategy either on a solid-phase resin or in solution. Employing Boc/Bzl chemistry to prepare fully protected segments with a free α-carboxyl group from the solid support, base-labile linkers are profitable for practical peptide synthesis since they require no special equipment. For this purpose, an N-[9-(hydroxymethyl)-2-fluorenyl]succinamic acid (HMFS) linker was adopted. Consequently, there must be high compatibility between the protecting groups of the segment and the anchoring group which is cleavable by treatment with morpholine or piperidine in DMF. Instead of using the 2-bromobenzyloxycarbonyl (BrZ) group for the Tyr residue and the formyl (For) group for the Trp residue, both of which are the most susceptible protecting groups under these base-catalysed conditions, the base-resistant 3-pentyl (Pen) and cyclohexyloxycarbonyl (Hoc) groups were introduced to the respective side-chain functional groups. By applying the present strategy, the authors were able to rapidly synthesize homogeneous protected segments for use in the subsequent segment coupling in solution. In the present paper, the utility of the combined solid-phase and solution approach is demonstrated by synthesizing muscarinic toxin 1 (MTX1) which binds to the muscarinic acetylcholine receptors. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.

Midkine (MK) is a 13 kDa heparin-binding polypeptide which enhances neurite outgrowth, neuronal cell survival and plasminogen activator activity. MK is structurally divided into two domains, and most of the biological activities are located on the C-terminal domain. The solution structures of the two domains were determined by NMR. Both domains consist of three antiparallel beta-strands, but the C-terminal domain has a long flexible hairpin loop where a heparin-binding consensus sequence is located. Basic residues on the beta-sheet of the C-terminal domain form another heparin-binding site. Measurement of NMR signals in the presence of a heparin oligosaccharides verified that multiple amino acids in the two sites participated in heparin binding. The MK dimer has been shown to be the active form, giving signals to endothelial cells and probably to neuronal cells. We present a head-to-head dimer model of MK. The model was supported by the results of cross-linking experiments using transglutaminase. The dimer has a fused heparin-binding site at the dimer interface of the C-terminal domain, and the heparin-binding sites on MK fit the sulfate group clusters on heparin. These features are consistent with the proposed stronger heparin-binding activity and biological activity of the dimer.

We studied about the physicochemical stability of a novel heparin-binding growth/differentiation factor, midkine (MK). It was found that synthetic human MK was heat and acid stable. Neither incubation at 80°C for 90 sec nor treatment at low pH affected the elution profile of MK molecule on the high performance liquid chromatography. This physicochemical stability was maintained in the biological activities of MK to enhance plasminogen activator (PA) activity in bovine endothelial cells as well as to promote neurite outgrowth of rat brain cells. A similar stability was observed both with recombinant murine MK and with its homologous protein, recombinant human pleiotrophin. Comparison of physicochemical stability with other several growth factors suggested that MK/pleiotrophin was a unique family of heat and acid stable polypeptides capable of enhancing PA activity and neurite outgrowth.

Endothelin (ET)-related peptides including ET-1 (1–39) were synthesized, and their constricting activity in rat pulmonary artery rings and pressor activity in unanesthetized rat were measured to elucidate their structure-activity relationship. The vasoconstrictor activities of ET-2, ET-3 and sarafotoxin S6b were one-half, one-60th and one-third that of ET-1, respectively. Such differences in biological activities should mainly arise from sequence heterogeneity at the N-terminal portion, especially at position 4 to 7. All of the blocked ETs at the amino or carboxyl termini showed greatly decreased activities. A monocyclic analog, in which Cys3 and Cys11 were replaced by Ala, showed one-third the activity of ET-1; however, its deamino dicarba analog was almost completely inactive. Significant activities were retained even with replacement of amino acids at positions Ser4, Ser5, Leu6, Met7, Lys9, Tyr13, and Trp21 by Ala, Ala, Gly, Met(0), Leu, Phe, and Tyr or Phe, respectively. On the other hand, replacement of Asp8, Glu10 and Phe14 by Asn, Gln and Ala, respectively, resulted in complete loss of the biological activity. These results indicated that two disulfide bonds in ET molecule were not essential for the expression of vasoconstricting activity. Both terminal amino and carboxyl groups, carboxyl groups of Asp8 and Glu10, and the aromatic group of Phe14 seemed to be contributing, more or less, to the expression of the biological activities.

Midkine (MK), a retinoic acid-inducible growth/differentiation factor, serves as a substrate for tissue transglutaminase (Kojima, S. , Muramatsu, H., Amanuma, H., and Muramatsu, T. 1995. J. Biol. Chem. 270, 9590-9596). Upon incubation with transglutaminase MK forms multimers through cross-linkages. Here, we report the following results. 1) Heparin potentiated the multimer formation by MK. 2) The N- and C-terminal half domains each formed a dimer through the action of transglutaminase. 3) Gln42 or Gln44 in the N-terminal half and Gln95 in the C-terminal half served as amine acceptors in the cross-linking reaction, as judged from the incorporation of putrescine into whole MK or each half domain, and the competitive inhibition of the cross-linking by MK-derived peptides containing Gln residue(s). The strongest inhibition was obtained with Ala41-Pro51. 4) This peptide abolished the biological activity of MK to enhance the plasminogen activator activity in bovine aortic endothelial cells. The inhibition was limited against the MK monomer, and not seen against the MK dimer, separated by gel filtration chromatography. These results suggest that dimer formation through transglutaminase-mediated cross-linking is an important step as to the biological activity of MK.