The crystal structure of Afc-containing peptides

A systematic structural analysis of Afc (9-amino-fluorene-9-carboxylic acid) containing peptides is here reported. The crystal structures of four fully protected tripeptides containing the Afc residue in position 2: Z-X(1)-Afc(2)-Y(3)-OMe (peptide a: X = Y = Gly; peptide b: X = Aib, C(alpha, alpha)-dimethylglycine, Y = Gly; peptide c: X = Gly, Y = Aib; peptide d: X = Y = Aib) have been solved by x-ray crystallography. All the results suggest that the Afc residue has a high propensity to assume an extended conformation. In fact, the Afc residue adopts an extended conformation in three peptides examined in this paper (peptides a-c). In contrast, Afc was found in a folded conformation, in the 3(10)-helical region, only in the peptide d, in which it is both preceded and followed by the strong helix promoting Aib.     

Synthetic peptide models for the redox-active disulfide loop of glutaredoxin. Conformational studies

Two cyclic peptide disulfides (Sequence: see text). (X = L-Tyr or L-Phe) have been synthesized as models for the 14-membered redox-active disulfide loop of glutaredoxin. 1H NMR studies at 270 MHz in chloroform solutions establish a type I beta-turn conformation for the Pro-X segment in both peptides, stabilized by a 4----1 hydrogen bond between the Cys(1) CO and Cys(4) NH groups. Nuclear Overhauser effects establish that the aromatic ring in the X = Phe peptide is oriented over the central peptide unit. In dimethyl sulfoxide solutions two conformational species are observed in slow exchange on the NMR time scale, for both peptides. These are assigned to type I and type II beta-turn structures with -Pro-Tyr(Phe)- as the corner residues. The structural assignments are based on correlation of NMR parameters with model 14-membered cyclic cystine peptides with Pro-X spacers. Circular dichroism studies based on the -S-S- n-omega* transition suggest a structural change in the disulfide bridge with changing solvent polarity, establishing conformational coupling between the peptide backbone and the disulfide linkage in these systems.     

Kinetics of the interaction of alpha-chymotrypsin with trypsin kallikrein inhibitor (Kunitz) in which the reactive-site peptide bond Lys-15--Ala-16 is split.

Modified trypsin kallikrein inhibitor (I*), with the reactive-site peptide bond Lys-15--Ala-16 split, reacts with alpha-chymotrypsin (E) via an intermediate X to the stable tetrahedral complex C:E + I in equilibrium X leads to C. Formation X constitutes a fast pre-equilibrium (equilibrium constant Kx = 7 X 10(-5) M, association rate constant kx = 4 X 10(3)M-1s-1) to the slow reaction X leads to C (rate constant kc = 2 X 10(-3) s-1), all values at pH 7.5. No intermediate X is observed when alpha-chymotrypsin reacts with I*-OMe in which the carboxyl group of Lys-15 is esterified by methanol. This observation as well as the different pH dependence of the overall association rate constants in the case of I* and I*-OMe indicate tha formation of X precedes formation of the acyl enzyme in the catalytic pathway. The data are compared to the similar results obtained with beta-trypsin and I* or I*-OMe.     

Ring conformation of cyclic octapeptide cyclo (L-Pro-Sar)4 in the crystalline state

A single-crystal X-ray diffraction analysis has been made of the structure of the cyclic octapeptide cyclo(L-Pro-Sar)4. The material [C32H48O8N8 X (21/4) H2O X (1/2) CH3OH, Mr = 799.43] crystallizes in the monoclinic space group C2 with cell dimensions a = 14.544 (3), b = 11.902 (2), c = 14.064 (3), and beta = 122.26 (2) degrees (lambda = 1.54178 A, T = 293 K). The final R value for the 1980 observed reflections is 0.079. The ring conformation has the peptide bond sequences of cis-cis-trans-trans-cis-cis-trans-trans (Pro-Sar-Pro peptide bond linkages are cis-cis- or trans-trans). The pyrrolidine rings in the four proline residues take an envelope form in which the gamma-carbon atom deviates from the plane of the remaining four atoms in the ring.     

Recombinant human cathepsin X is a carboxymonopeptidase only: a comparison with cathepsins B and L

The S1 and S2 subsite specificity of recombinant human cathepsins X was studied using fluorescence resonance energy transfer (FRET) peptides with the general sequences Abz-Phe-Xaa-Lys(Dnp)-OH and Abz-Xaa-Arg-Lys(Dnp)-OH, respectively (Abz=ortho-aminobenzoic acid and Dnp=2,4-dinitrophenyl; Xaa=various amino acids). Cathepsin X cleaved all substrates exclusively as a carboxymonopeptidase and exhibited broad specificity. For comparison, these peptides were also assayed with cathepsins B and L. Cathepsin L hydrolyzed the majority of them with similar or higher catalytic efficiency than cathepsin X, acting as an endopeptidase mimicking a carboxymonopeptidase (pseudo-carboxymonopeptidase). In contrast, cathepsin B exhibited poor catalytic efficiency with these substrates, acting as a carboxydipeptidase or an endopeptidase. The S1' subsite of cathepsin X was mapped with the peptide series Abz-Phe-Arg-Xaa-OH and the enzyme preferentially hydrolyzed substrates with hydrophobic residues in the P1' position.     

Prevention of peptide fibril formation in an aqueous environment by mutation of a single residue to Aib

The behavior of a number of 16 residue polypeptides with a sequence Acetyl-EACARXZAACEAAARQ-amide, where X = V or A and Z = A or Aib, is studied under aqueous conditions. It is shown that the substitution of a single alanine residue by alpha-aminoisobutyric acid (Aib) completely alters both the conformation and the aggregation properties of the peptides. The Ala-Ala (X,Z = A,A) peptide is shown by circular dichroism and FTIR methods to adopt a predominately beta-sheet conformation. Furthermore, the peptide has limited solubility and is shown to form fibrils by electron microscopy and thioflavin T binding assays. In contrast, a single substitution at the center of peptide of alanine to Aib (X,Z = A,Aib) completely abolishes fibril formation and alters the conformation to a mixture of random coil and alpha-helix. The results show that Aib is a strong beta-sheet disrupter that is also able to adopt a helical conformation. This is linked to its role in peptaibol antibiotics. Aib provides an attractive alternative to proline and other substitutions in producing peptide variants with a lower tendency to produce fibril aggregates.     

Binding of monoiodinated neuropeptide Y to hippocampal membranes and human neuroblastoma cell lines

Monoiodinated radioligands of the homologous 36-amino acid peptides, neuropeptide Y (NPY) and peptide YY, were prepared by reverse phase high performance liquid chromatography with isocratic elution. [125I-Tyr1]- and [125I-Tyr36]monoiodoNPY bound equally well to a single class of high affinity binding sites on synaptosomal membranes prepared from porcine hippocampus (Kd = 1.0 X 10(-10) M) whereas iodine substitution in Tyr27, for example, partly interfered with the receptor binding. The receptors on the hippocampal membranes did not distinguish between neuropeptide Y and peptide YY either in their monoiodinated or in their unlabeled forms. Six out of twelve human neuroblastoma cell lines had high affinity binding sites for monoiodinated NPY ranging from 2 to 145 X 10(3) sites per cell. The NPY binding to three of the cell lines, SMS-MSN, SMS-KAN, and CHP-234 was of relatively high affinity (Kd = 1.3 to 6.1 X 10(-10) M), and, as in the hippocampal membranes, the long C-terminal fragment, NPY(13-36)peptide was also a relatively potent ligand for these receptors. Two other neuroblastoma cell lines, MC-IXC and CHP-212, expressed NPY receptors characterized by a lower affinity (Kd = 4.8 and 24.6 X 10(-9) M) and negligible cross-reactivity with the C-terminal fragment. It is concluded that monoiodinated radioligands of the tyrosine-rich neuropeptide Y can be prepared and that receptors for these ligands in two apparently different subtypes are found on a series of human neuroblastoma cell lines.     

Comparative study of various serine alkaline proteinases from microorganisms. Esterase activity against N-acylated peptide ester substrates

Hydrolyses of N-acylated peptide ester substrates by various serine alkaline proteinases from bacterial and mold origin were compared using Ac- or Z-(Ala)_m-X-OMe (m = 0-2 or 0-3; X = phenylalanine, alanine, and lysine) as esterase substrates. The results indicated that the esterase activities of these enzymes were markedly promoted by elongating the peptide chain from P₁ to P₂ or P₃ with alanine, irrespective of the kind of the amino acid residue at the P₁-position (amino acid residues in peptide substrates are numbered according to the system of Schechter and Berger (1)). The effect of the kind of amino acid residue at the P₂-position was further determined using Z-X-Lys-OMe (X = glycine, alanine, leucine, or phenylalanine) as esterase substrates. Alanine was the most efficient residue as X with subtilisins and Streptomyces fradiae Ib enzyme, while leucine or phenylalanine were most efficient with the enzymes from Streptomyces fradiae II, Aspergillus sojae, and Aspergillus melleus. All the serine alkaline proteinases tested in this study were sensitive to Z-Ala-Gly-PheCH₂Cl, the dependence of inhibition on the inhibitor concentration differed among the enzymes.     

Comparison of energy-minimized structures of [Pd(II)(N-methyliminodiacetate)] complexes of X(1)-His-X(3)-His-His peptides as an analysis of steric and specific interactions with synthetic binding tags for IMAC separations

[Pd(II)(mida)(peptide)] complexes for the series of peptides of sequence X(1)-His-X(3)-His-His were studied by molecular mechanics methods using Spartan, MMFF94, and SYBYL programs with X(1) = X(3) = glycine (G), phenylalanine (F), tyrosine (Y), tryptophan (W), and with X(1) = glycine (G) and X(3) = proline (P). For comparison purposes, data were also obtained for the Ser-Pro-His-His-Gly (SPHHG) and the (His)(5) peptides.The latter two peptides and GHPHH are tags in current use for IMAC separations. These provide calibration points as to the binding affinities that have been determined for the entire series. The energies of the complexes, as an average trend found from the composite behavior of the three methods, were found to be SPHHG (205 kcal/mol) (most stable; are values obtained by MMFF94 methods) < HH(#)HH(#)H(#) (222; where # implies the site of attachment to match the other X(1)-His-X(3)-His-His peptides) < YHYHH (249) < GHGHH (265) < WHWHH (284) approximately GHPHH (286) < FHFHH (311) (least stable), implying that FHFHH might be a useful chromatographic tag for IMAC protein separations that would elute more readily than GHPHH from IMAC sites that are of square-planar structure, such as Cu(II)(ida-supported) IMAC columns. Specific H-bonded interactions are observed between the tyrosine X(1) and pendant carboxylates and between X(3) and the N-terminal amine of [Pd(mida)(YHYHH)]. Face-to-pi-face ring stacking occurs between phenylalanine X(1) and X(3) units in [Pd(mida)(FHFHH)], whereas edge C-H to pi H-bonding or pi stacking occurs between the X(1) and X(3) tryptophans of [Pd(mida)(WHWHH)]. Two energy minima were found with tryptophan. The more stable form has the aromatic rings more parallel, similar to the stacked form of phenylalanine, rather than the edge C-H to pi H-bonding, and virtually the same overall energy as for [Pd(mida)(GHPHH)]. The "perpendicular" structure was found as an initial local energy minimum, but additional MMFF94 calculations found the pi -stacked arrangement at energy ca. 39 kcal/mol lower than that of the nearly "perpendicular" arrangement of the tryptophan rings, a composite effect of relaxation of the peptide, together with differences in stabilities imparted by the differing geometries. The use of the terms "pi-stacked " and "perpendicular" forms represent the limiting cases available to the tryptophan side chain groups. A twist of about 15 degrees to 20 degrees in dihedral angle is all that is necessary to change between structures that are nearly described as one form or the other.     

Conformational study of peptides containing dehydrophenylalanine: helical structures without hydrogen bond

The conformational behaviour of deltaZPhe has been investigated in the model dipeptide Ac-deltaZPhe-NHMe and in the model tripeptides Ac-X-deltaZPhe-NHMe with X=Gly,Ala,Val,Leu,Abu,Aib and Phe and is found to be quite different. In the model tripeptides with X=Ala,Val,Leu,Abu,Phe the most stable structure corresponds to phi1=-30 degrees, psi1=120 degrees and phi2=psi2=30 degrees. This structure is stabilized by the hydrogen bond formation between C=O of acetyl group and the NH of the amide group, resulting in the formation of a 10-membered ring but not a 3(10) helical structure. In the peptides Ac-Aib-deltaZPhe-NHMe and Ac-(Aib-deltaZPhe)3-NHMe, the helical conformers with phi = +/-30 degrees, psi = +/-60 degrees for Aib residue and phi=psi= +/-30 degrees for deltaZPhe are predicted to be most stable. The computational studies for the positional preferences of deltaZPhe residue in the peptide containing one deltaZPhe and nine Ala residues reveal the formation of a 3(10) helical structure in all the cases with terminal preferences for deltaZPhe. The conformational behaviour of Ac-(deltaZPhe)n-NHMe with n< or =4 is predicted to be very labile. With n > 4, degenerate conformational states with phi,psi values of 0 degrees +/- 90 degrees adopt helical structures which are stabilized by carbonyl-carbonyl interactions and the N-H-pi interactions between the amino group of every deltaZPhe residue with one C-C edge of its own phenyl ring. The results are in agreement with the experimental finding that screw sense of helix for peptides containing deltaZPhe residues is ambiguous in solution. The helical structures stabilized by hydrogen bond formation are found to be at least 3kCalmol(-1) less stable. Conformational studies have also been carried out for the peptide Ac-(deltaEPhe)6-NHMe and the peptide Ac-deltaAla-(deltaZPhe)6-NHMe containing deltaAla residue at the N-terminal. The N-H-pi interactions are absent in peptide Ac-(deltaEPhe)6-NHMe.     

Manipulation of peptide conformations by fine-tuning of the environment and/or the primary sequence

The widely observed phenomenon that peptides are capable of adopting multiple conformations in different environments suggests that secondary structure formation in a peptide segment is a process involving not only the peptide itself hut also the surrounding solvent media. The influence of the primary sequence and the molecular environment on peptide conformations are now investigated using synthetic peptides of amino acid sequence H₂N-(Ser-Lys)₂-Ala-X-Gly-Ala-X-Gly-Trp-Ala-X-Gly-(Lys-Ser)₃-OH, where X = Ile or Val. These two peptides, namely 3I (X = Ile) and 3V (X = Val), are found to lack defined secondary structure in aqueous buffer. However, discrete conformational states, e.g., α-helices and β-sheets, are readily generated and interconverted for both peptides when the buffer is modulated with the addition of methanol, sodium dodecyl sulfate (SDS) micelles, or phospholipid vesicles. The role of the primary sequence in affecting peptide conformations is manifested in that peptides 3I and 3V, which differ respectively in their content of β-branched Ile or Val residues, differ in their secondary structures at monomeric concentrations in 2 mM SDS and in mixed lipid vesicles of phosphatidic acid and phosphatidylcholine. The overall results suggest that peptide segments can be conformationally flexible entities poised to react to minor modulations in cither the molecular environment or the primary sequence, a circumstance that may he relevant to protein functioning and folding. © 1995 John Wiley & Sons, Inc.     

Main-chain length control of conformation, membrane activity, and antibiotic properties of lipopeptaibol sequential analogues

To investigate the effect of backbone length and amphiphilicity on the 3D structure, membrane permeability, and antibacterial properties of trichogins, a subclass of lipopeptaibols, we prepared, by the segment condensation approach in solution and chemically characterized, a set of N(alpha)-1-octanoylated -X-(GLUG)(n)-I-L- ( X=G or U where U=Aib; n=1-4) sequential peptide esters. In parallel, the 12-mer (UGGL)(3) aneurism peptide, an analogue of the 11-mer sequential peptide (n=2) with an amino acid insertion was also synthesized and studied. By FT-IR absorption technique, we clearly showed that, in CDCl(3) solution, all peptides essentially populate intramolecularly H-bonded, helical conformations. Moreover, CD spectroscopy indicates that all peptides, with the single exception of the shortest oligomer (the heptamer), adopt mixed 3(10)-/alpha-helical structures, to an extent approximately correlating with main-chain length, in MeOH solution and sodium dodecylsulfate (SDS) micelles. Significant membrane permeability properties were found only for the three longest GLUG-based peptides, with the 15-mer oligomer (n=4) resulting the most active. The lack of activity exhibited by the aneurism peptide in this experiment strongly suggests a relevant role for the sequence amphiphilicity. In addition, antibacterial activity and selectivity were highlighted and demonstrated to be dependent on peptide main-chain length and amphiphilicity, in the sense that the two shortest GLUG-based homologues are active against Gram-positive strains, whereas the two longest homologues are able to penetrate the membranes of the Gram-negative strains, and the UGGL-based aneurism peptide is inactive.     

Conformational stability of antamanide and analogs. Crystal structure of perhydrosymmetric antamanide, cyclic (-Val-Pro-Pro-Cha-Cha-Val-Pro-Pro-Cha-Cha-)

The synthetic perhydrogenated symmetric analog of the cyclic decapeptide antamanide is biologically inactive, although the conformation of the molecule and the crystal packing are very similar to that of the active symmetric analog of antamanide. In fact, the same conformation for the molecule has now been found in six polymorphs of uncomplexed antamanide and its analogs. The differences between the active and inactive antamanide analogs are displayed dramatically in the conformations of their metal ion (Na+ or Li+) complexes, thus suggesting strongly that for physiological activity antamanide is not in the conformation assumed by the uncomplexed molecule, but rather in the conformation assumed by the complexed state of natural antamanide. The present structure crystallizes in space group P2(1)2(1)2(1) with a = 20.515(14) A, b = 21.316(16) A, c = 17.128(16) A and four peptide molecules in the unit cell. There are three cocrystallized water molecules at full occupancy intrinsic to the peptide, and several more water molecules or other solvent molecules at partial occupancy. The formula of the peptide is C66H106N10O10.4-H2O.2X.     

Inhibitor of inflammation,peptide fragment (65–76) of monocyte chemotactic protein-1 (MCP-1), inhibits binding of MCP-1 to heparin

Monocyte chemotactic protein-1 (MCP-1, CCL2) is one of the most important chemokines involved in inflammation. MCP-1 stimulates migration of monocytes and certain lymphocyte populations to the affected area, in particular to the sites of atherosclerotic plaque formation. Development of drugs inhibiting MCP-1 is now a topical task. We earlier designed and synthesized a dodecapeptide from C-terminal domain of MCP-1 (65–76, peptide X) that possessed an anti-inflammatory activity. The mechanism of action of chemokines (in particular, of MCP-1) in vivo is based on activation of CCR2 receptor on target cells and binding to glycosaminoglycans (GAGs) on the cell surface of and extracellular matrix. Peptide X did not affect the MCP-1-CCR2 interaction. Thus, we hypothesized that peptide X could impair MCP-1 binding to GAGs. Here we studied the effect of peptide X on the MCP-1 binding to heparin using the label-free biosensing device Picoscope®, enzyme-linked immunoassay (ELISA), and the intrinsic fluorescence method. According to the data obtained, peptide X interfered with the MCP-1-heparin binding, which may be due to the competition of peptide X with MCP-1 for heparin binding sites. Probably, this effect determines the anti-inflammatory activity of peptide X in vivo.     

Demonstration of an Mg2+-induced conformational change by photoaffinity labelling of the high-affinity ATP-binding site of (Na+ + K+)-ATPase with 8-azido-ATP

8-Azido-ATP (8-N3ATP) is a substrate of (Na+ + K+)-ATPase from pork kidney and photoinactivates it by binding to the Mr = 100 000 alpha-subunit. The photoinactivation requires the presence of Mg2+ even though 8-azido-ATP is recognized by the high-affinity ATP binding site (Kd = 3.1 microM). K+ ions protect the enzyme against photoinactivation as does excess ATP. To see whether the Mg2+-requirement of the photoinactivation is due to the action of free Mg2+ or to the existence of an Mg X 8-azido-ATP complex, the action of the stable Mg X ATP complex analogue, chromium X 8-N3ATP (Cr X 8-N3ATP), was studied. Cr X 8-N3ATP photoinactivates (Na+ + K+)-ATPase in the absence of Mg2+, but the photoinactivation is enhanced by Mg2+, indicating that the formation of a Mg X ATP complex is an absolute requirement for photoinactivation. However, the interaction of Mg2+ with a low-affinity site also enhances the photoinactivation. It is therefore concluded that interactions with MgATP and free Mg induce conformational changes in the purine subsite of the high-affinity ATP binding site. Controlled trypsinolysis of the [alpha-32P]8-N3ATP-photolabelled enzyme in the presence of K+ results in the formation of an Mr = 56 000 radioactive peptide, whereas trypsinolysis of a [gamma-32P]Cr X ATP-labelled enzyme under identical conditions forms an Mr = 41 000 radioactive peptide. Extensive trypsinolysis of the [alpha-32P] 8-N3ATP-photolabelled alpha-subunit leads to the formation of a radioactive peptide of Mr = 1800.     

Membrane-spanning peptides induce phospholipid flop: a model for phospholipid translocation across the inner membrane of E. coli

The mechanism by which phospholipids translocate (flop) across the E. coli inner membrane remains to be elucidated. We tested the hypothesis that the membrane-spanning domains of proteins catalyze phospholipid flop by their mere presence in the membrane. As a model, peptides mimicking the transmembrane stretches of proteins, with the amino acid sequence GXXL(AL)(n)XXA (with X = K, H, or W and n = 8 or 12), were incorporated in large unilamellar vesicles composed of E. coli phospholipids. Phospholipid flop was measured by assaying the increase in accessibility to dithionite of a 2,6-(7-nitro-2,1,3-benzoxadiazol-4-yl)aminocaproyl (C(6)NBD)-labeled phospholipid analogue, initially exclusively present in the inner leaflet of the vesicle membrane. Fast flop of C(6)NBD-phosphatidylglycerol (C(6)NBD-PG) was observed in vesicles in which GKKL(AL)(12)KKA was incorporated, with the apparent first-order flop rate constant (K(flop)) linearly increasing with peptide:phospholipid molar ratios, reaching a translocation half-time of approximately 10 min at a 1:250 peptide:phospholipid molar ratio at 25 degrees C. The peptides of the series GXXL(AL)(8)XXA also induced flop of C(6)NBD-PG, supporting the hypothesis that transmembrane parts of proteins mediate phospholipid translocation. In this series, K(flop) decreased in the order X = K > H > W, indicating that peptide-lipid interactions in the interfacial region of the membrane modulate the efficiency of a peptide to cause flop. For the peptides tested, flop of C(6)NBD-phosphatidylethanolamine (C(6)NBD-PE) was substantially slower than that of C(6)NBD-PG. In vesicles without peptide, flop was negligible both for C(6)NBD-PG and for C(6)NBD-PE. A model for peptide-induced flop is proposed, which takes into account the observed peptide and lipid specificity.     

Cytoplasmic domain of natriuretic peptide receptor C constitutes Gi activator sequences that inhibit adenylyl cyclase activity

We have recently demonstrated that a 37-amino acid peptide corresponding to the cytoplasmic domain of the natriuretic peptide receptor C (NPR-C) inhibited adenylyl cyclase activity via pertussis toxin (PT)-sensitive G(i) protein. In the present studies, we have used seven different peptide fragments of the cytoplasmic domain of the NPR-C receptor with complete, partial, or no G(i) activator sequence to examine their effects on adenylyl cyclase activity. The peptides used were KKYRITIERRNH (peptide 1), RRNHQEESNIGK (peptide 2), HRELREDSIRSH (peptide 3), RRNHQEESNIGKHRELR (peptide 4), QEESNIGK (peptide X), ITIERRNH (peptide Y), and ITIYKKRRNHRE (peptide Z). Peptides 1, 3, and 4 have complete G(i) activator sequences, whereas peptides 2 and Y have partial G(i) activator sequences with truncated carboxyl or amino terminus, respectively. Peptide X has no structural specificity, whereas peptide Z is the scrambled peptide control for peptide 1. Peptides 1, 3, and 4 inhibited adenylyl cyclase activity in a concentration-dependent manner with apparent K(i) between 0.1 and 1 nm; however, peptide 2 inhibited adenylyl cyclase activity with a higher K(i) of about 10 nm, and peptides X, Y, and Z were unable to inhibit adenylyl cyclase activity. The maximal inhibitions observed were between 30 and 40%. The inhibition of adenylyl cyclase activity by peptides 1-4 was absolutely dependent on the presence of guanine nucleotides and was completely attenuated by PT treatment. In addition, the stimulatory effects of isoproterenol, glucagon, and forskolin on adenylyl cyclase activity were inhibited to different degrees by these peptides. These results suggest that the small peptide fragments of the cytoplasmic domain of the NPR-C receptor containing 12 or 17 amino acids were sufficient to inhibit adenylyl cyclase activity through a PT-sensitive G(i) protein. The peptides having complete structural specificity of G(i) activator sequences at both amino and carboxyl termini were more potent to inhibit adenylyl cyclase activity as compared with the peptides having a truncated carboxyl terminus, whereas the truncation of the amino-terminal motif completely attenuates adenylyl cyclase inhibition.     

Demonstration of high affinity fibronectin receptors on rat hepatocytes in suspension

A cell-binding peptide (Mr = 85,000) which lacks the gelatin- and heparin-binding domains, was purified from trypsin-digested fibronectin. Preincubation of rat hepatocytes in suspension with the peptide, inhibited initial attachment of the cells to immobilized fibronectin while attachment to immobilized laminin and collagen was unaffected. 125I-labeled 85-kDa peptide bound to the cells in suspension at 4 degrees C in a time-dependent, saturable, and partially reversible reaction. Scatchard analysis of the binding data indicated a single class of receptors with a Kd of 1.7 X 10(-8) M. The number of binding-sites was approximately 2.8 X 10(5)/cell. Unlabeled 85-kDa peptide inhibited the binding of 125I-labeled 85-kDa peptide 30-fold more effectively than intact fibronectin. These results provide direct evidence for the presence of a domain in the fibronectin molecule which has or may acquire a high affinity for receptors involved in adhesion of hepatocytes to immobilized fibronectin.