Factor V and factor Va binding to single bilayer phospholipid vesicles was investigated by light-scattering intensity measurements. This technique allows the measurement of free and phospholipid-bound protein concentrations from which equilibrium constants can be obtained. As controls, the Ca2+-dependent phospholipid binding of prothrombin and factor X were also studied. The average values obtained for the dissociation constants (Kd) and lipid to protein ratio at saturation, moles/mole (n), for prothrombin (Kd = 2.3 X 10(-6) M, n = 104) and factor X (Kd = 2.5 X 10(-6) M, n = 46) binding to vesicles containing 25% Folch fraction III and 75% phosphatidylcholine in the presence of 2 mM Ca2+ were in agreement with those reported in the literature. The average factor V and factor Va values for the dissociation constants and lipid to protein ratio at saturation (moles/mole) were Kd = 7.2 X 10(-8) M and n = 270 for factor V and Kd = 4.4 X 10(-7) M and n = 76 for factor Va. In contrast to prothrombin and factor X, factor V and factor Va demonstrated Ca2+-independent lipid binding. In addition, the number of factor V and factor Va molecules bound per vesicle was found to be dependent both on the phosphatidylserine content of the vesicle and the ionic strength of the buffer.

IgG is a tetrameric protein composed of two copies each of the light and heavy chains. The four-chain structure is maintained by strong noncovalent interactions between the amino-terminal half of pairs of heavy-light chains and between the carboxyl-terminal regions of the two heavy chains. In addition, interchain disulfide bonds link each heavy-light chain and also link the paired heavy chains. An engineered human IgG4 specific for human tumor necrosis factor-cy (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetramers composed of nondisulfide bonded half-IgG4 (heavy chain disulfide bonded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rather than serine, CDP57 I(S229P), or with an IgGl rather than IgG4 hinge region, CDP571(yl), only trace amounts of nondisulfide bonded half-lgG tetramers were observed. Trypsin digest reversephase HPLC peptide mapping studies of CDP57 1 and CDP57 1 (y I ) with on-line electrospray ionization mass spectroscopy supplemented with Edman sequencing identified the chemical factor preventing inter-heavy chain disulfide bond formation between half-IgG molecules: the two cysteines in the IgG4 and IgGl core hinge region KPSCP and GPPCP, respectively) are capable of forming an intrachain disulfide bond. Conformational modeling studies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded energy ranges for the low-energy conformations of 31-33 kcallmol and 40-42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP peptide due to backbone constraints caused by the extra proline. These modeling results suggest a reason why a larger fraction of intrachain bonds are observed in IgG4 rather than IgGl molecules: the serine in the core hinge region of IgG4 allows more hinge region flexibility than the proline of IgGl and thus may permit formation of a stable intrachain disulfide bond more readily.

Circular dichroism experiments indicate that prothrombin fragment 1 undergoes essentially the same secondary structural change whether in the presence of Ca(2+), Mg(2+), or Mn(2+). Titration with any of these metal ions results in a sigmoidal titration curve indicative of cooperative binding. Mg(2+) and Ca(2+) have nearly identical transition midpoints, while that for Mn(2+) is an order of magnitude less. These results correlate well with the results of previous metal ion intrinsic fluorescence quenching experiments. Fragment 1 has previously been shown to undergo a second transition corresponding to dimerization at high calcium concentrations. The present circular dichroism experiments show that this transition does not result in a gross alteration of secondary structure in the fragment 1 molecule. Studies with prothrombin, similar to those with fragment 1, indicate a similar metal ion dependent conformational change but of smaller magnitude. As apparently only the fragment 1 portion of the molecule undergoes the transition, it would appear that the covalently linked fragment 1 is constrained from attaining the same conformation as the purified entity. This suggests that caution must be used in interpreting the results of metal ion binding studies using fragment 1 as an analogue for prothrombin.

Human alpha-thrombin is inhibited by the circulating protease inhibitors alpha1-antitrypsin, antithrombin III, and alpha2-macroglobulin. Kinetic analyses of the inhibitor thrombin interactions were carried out utilizing either fibrinogen or the synthetic substrate Bz-Phe-Val-Arg-p-nitroanilide as substrates to determine residual thrombin activity. These studies demonstrated that the inhibition of thrombin by alpha1-antitrypsin, antithrombin III, and alpha2-macroglobulin followed second-order kinetics. The rate constants for the inhibition of thrombin by alpha1-antitrypsin, antithrombin III, and alpha2-macroglobulin are 6.51 +/- 0.38 x 10(3), 3.36 +/- 0.34 x 10(5), and 2.93 +/- 0.02 x 10(4) M-1 min-1, respectively. Comparison of the second-order rate constants and the normal plasma levels of the three inhibitors demonstrates that, under the in vitro conditions utilized, antithrombin III is five times and alpha2-macroglobulin is one-third as effective as alpha1-antitrypsin in the inhibition of thrombin.

Pituitaryadenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100 -1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and chargescanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.Pituitary adenylate cyclase-activating polypeptide (PACAP), 1 originally isolated from ovine hypothalamus (1) by following pituitary adenylate cyclase activation, belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) family of peptides (2). These peptides are expressed as part of larger proteins that are processed by proteolysis followed by Cterminal amidation to generate the mature amidated peptides (Fig. 1). PACAP exists as a 38-residue form (PACAP38), and as a shorter form corresponding to the N-terminal 27 amino acids of PACAP38 (PACAP27). Both forms of PACAP bind to and activate the G-protein-coupled receptors PAC1, VPAC1, and VPAC2, whereas the related 28-mer peptide VIP only recognizes VPAC1 and VPAC2 (3). Activation of multiple receptors by PACAP or VIP has broad physiological effects on nervous, endocrine, cardiovascular, reproductive, muscular, and immune systems (4). Thus, clinical applications will require selective activation of a particular receptor to minimize potential side effects mediated by the other receptors. For example, we have previously demonstrated that VPAC2 activation induces glucose-dependent insulin secretion without the undesired side effects mediated by VPAC1 such as watery diarrhea (5). Therefore, to provide a potential therapy for type 2 diabetes, VPAC2 selectivity is an absolute requirement.We sought to identify key determinants of VPAC2 selectivity and generate a peptide with the minimum number of mutations. However, structure-function relationship studies on VIP and PACAP (6 -15) have been restricted by the number of peptides that can be tested due to limitations of peptide synth...