Crystal Structure Determination of New Antimitotic Agent Bis(p-fluorobenzyl)trisulfide

The purpose of this research was to investigate the physical characteristics and crystalline structure of bis(p-fluorobenzyl)trisulfide, a new anti-tumor agent. Methods used included X-ray single crystal diffraction, X-ray powder diffraction (XRPD), Fourier-transform infrared (FT-IR) spectroscopy, differential scanning calorimetric (DSC) and thermogravimetric (TG) analyses. The findings obtained with X-ray single crystal diffraction showed that a monoclinic unit cell was a = 12.266(1) A, b = 4.7757(4) A, c = 25.510(1) A, beta = 104.25(1) degrees ; cell volume = 1,448.4(2) A(3), Z = 4, and space group C2/c. The XRPD studies of the four crystalline samples, obtained by recrystallization from four different solvents, indicated that they had the same diffraction patterns. The diffraction pattern stimulated from the crystal structure data is in excellent agreement with the experimental results. In addition, the identical FT-IR spectra of the four crystalline samples revealed absorption bands corresponding to S-S and C-S stretching as well as the characteristic aromatic substitution. Five percent weight loss at 163.3 degrees C was observed when TG was used to study the decomposition process in the temperature range of 20-200 degrees C. DSC also allowed for the determination of onset temperatures at 60.4(1)-60.7(3) degrees C and peak temperatures at 62.1(3)-62.4(3) degrees C for the four crystalline samples studied. The results verified that the single crystal structure shared the same crystal form with the four crystalline samples investigated.     

Inactivation of plasminogen activator inhibitor-1 by specific proteolysis with stromelysin-1 (MMP-3)

Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) specifically hydrolyzes the Ser(337)-Ser(338) (P10-P9) and Val(341)-Ile(342) (P6-P5) peptide bonds in human plasminogen activator inhibitor-1 (PAI-1). Cleavage is completely abolished in the presence of the metal chelators EDTA or 1,10-phenanthroline. A stabilized active PAI-1 variant was also cleaved by MMP-3. At an enzyme/substrate ratio of 1/10 at 37 degrees C, PAI-1 protein cleavage occurred with half-lives of 27 or 14 min for active or stable PAI-1 and was associated with rapid loss of inhibitory activity toward tissue-type plasminogen activator with half-lives of 15 or 13 min, respectively. A substrate-like variant of PAI-1, lacking inhibitory activity but with exposed reactive site loop, was cleaved with a half-life of 23 min, whereas latent PAI-1 in which a major part of the reactive site loop is inserted into the molecule, was resistant to cleavage. Biospecific interaction analysis indicated comparable binding of active, stable, and substrate PAI-1 to both proMMP-3 and MMP-3 (K(A) of 12-22 x 10(6) m(-1)), whereas binding of latent PAI-1 occurred with lower affinity (1.7-2.3 x 10(6) m(-1)). Stable PAI-1 bound to vitronectin was cleaved and inactivated by MMP-3 in a manner comparable with that of free PAI-1; however, the cleaved protein did not bind to vitronectin. Cleavage and inactivation of PAI-1 by MMP-3 may thus constitute a mechanism decreasing the antiproteolytic activity of PAI-1 and impairing the potential inhibitory effect of vitronectin-bound PAI-1 on cell adhesion and/or migration.     

Bovine plasminogen activator inhibitor 1: specificity determinations and comparison of the active, latent, and guanidine-activated forms

The plasminogen activator inhibitor 1 (PAI-1) synthesized and released by cultured bovine aortic endothelial cells is present in conditioned medium in a latent form that can be activated by guanidine hydrochloride [Hekman, C. M., & Loskutoff, D. J. (1985) J. Biol. Chem. 260, 11581-11587]. The purified, guanidine-activated PAI-1 was shown to inhibit both plasmin and trypsin in a dose- and time-dependent manner. Second-order rate constants for these interactions were calculated to be 6.6 X 10(5) and 7.0 X 10(6) M-1 s-1 for plasmin and trypsin, respectively. Experiments were conducted to compare the inherently active and the guanidine-activated forms of PAI-1. The two active forms had similar kinetic parameters for interaction with urokinase (Kd, 0.3 pM; kassoc, 1.5 X 10(8) M-1 s-1) and were both inactivated upon treatment with acid or base and by incubation at 37 degrees C. The latent form was relatively stable when incubated under similar conditions. The decrease in PAI-1 activity upon incubation at 37 degrees C was partially restored by a second treatment with guanidine hydrochloride. However, the degree of recovery decreased as a function of incubation time at 37 degrees C. These data suggest that active and guanidine-activated PAI-1 represent a single form of PAI-1. Incubation of this form at 37 degrees C yields two distinct populations of inactive PAI-1, one capable of reactivation and another that appears to be irreversibly inactivated.     

Purification and characterization of recombinant plasminogen activator inhibitor-1 from Escherichia coli

A recombinant form of plasminogen activator inhibitor-1 (rPAI-1) has been purified from lysates of pCE1200, a bacterial expression vector containing the full length PAI-1 gene, by utilizing sequential anion exchange and cation exchange chromatography on Q-Sepharose and S-Sepharose columns. Approximately 140 mg of rPAI-1, estimated at 98% purity on the basis of analytical high performance liquid chromatography, could be obtained from 200 g wet weight of cells. The purified protein exhibited a single Coomassie Blue-stainable band at the region of Mr = 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an NH2-terminal amino acid sequence consistent with the expected translation product of the pCE1200 PAI-1 insert. The rPAI-1 rapidly inhibited single- and two-chain tissue plasminogen activators, as well as urokinase, with apparent second order rate constants in the range of 2-5 x 10(7) M-1 s-1. A specific activity measurement of 250,000 units/mg was calculated for the rPAI-1 based on its ability to inhibit the enzymatic activity of a single-chain tissue plasminogen activator. Stability studies showed that the activity of the rPAI-1 was very stable when stored at temperatures of 25 degrees C or lower, but decayed within hours when stored at 37 degrees C. Sodium dodecyl sulfate treatment, which partially activates the latent form of natural PAI-1, inactivated rPAI-1. These results show that the purified rPAI-1 produced from pCE1200 displays many of the properties associated with the biologically active form of natural PAI-1.     

The importance of helix F in plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1) is the only functionally labile serpin, as it converts spontaneously into a non-reactive 'latent' conformation. Several studies have suggested an important role for helix F in the functional behavior and stability of the serpins, especially for PAI-1. We constructed a mutant of PAI-1 (PAI-1-delhF) in which residues 127-158 (hF-thFs3A) were deleted. Whereas wild-type PAI-1 (wtPAI-1) exhibits inhibitory properties towards t-PA and u-PA to an extent of 60-80% of the theoretical maximum, PAI-1-delhF did not exert any detectable inhibitory properties, but behaved as a stable substrate. Prolonged incubation at 37 degrees C did not change its functional properties in contrast to wtPAI-1 that under those conditions converts to the latent conformation. In contrast to active wtPAI-1 and other substrate-type PAI-1 mutants, PAI-1-delhF showed a 3000-fold decreased binding to vitronectin. The obtained results clearly show the importance of helix F in the inhibitory activity of PAI-1. The absence of helix F apparently leads to an impaired kinetics of insertion of the reactive site loop upon interaction with its target proteinase resulting in the inability to form a stable covalent complex. Moreover, removal of helix F strongly affects the binding of PAI-1 to vitronectin.     

Kinetic analysis of the interaction between type 1 plasminogen activator inhibitor and vitronectin and evidence that the bovine inhibitor binds to a thrombin-derived amino-terminal fragment of bovine vitronectin.

The interaction between guanidine-activated bovine type 1 plasminogen activator inhibitor (PAI-1) and bovine vitronectin was investigated. Activated PAI-1 bound to vitronectin in a dose- and time-dependent manner, and binding was saturable. The dissociation constant (Kd) for this interaction was estimated to be 3.10(-10) mol/l by Scatchard analysis. Complexes of activated PAI-1 and vitronectin were relatively stable at 4 degrees C (T1/2 greater than 24 h), but dissociated with a T1/2 of 4 h at 37 degrees C. The half-life of PAI-1 activity was increased from 2.5 to 4.5 h upon binding to immobilized vitronectin. In order to identify the binding domain(s) in vitronectin for activated PAI-1, the ability of PAI-1 to bind to vitronectin fragments was assessed. Vitronectin was cleaved by thrombin in a dose- and time-dependent manner, generating fragments of Mr 60,000, 54,000 and 38,000. The PAI-1 binding domain(s) were not destroyed by this treatment, since the digested vitronectin competed with immobilized vitronectin for PAI-1 binding to the same extent as uncleaved vitronectin. The thrombin digested vitronectin fragments were fractionated by SDS-PAGE and analyzed by PAI-1 ligand binding. The smallest fragment (Mr 38,000) retained PAI-1 binding function, and sequence analysis demonstrated that this fragment contained the NH2-terminus of bovine vitronectin. These results suggest that the high-affinity binding site for activated PAI-1 is located in the NH2-terminal region of the bovine vitronectin molecule.     

Internalization of the urokinase-plasminogen activator inhibitor type-1 complex is mediated by the urokinase receptor.

The role of the urokinase receptor (uPAR) in the internalization of the urokinase-plasminogen activator inhibitor type-1 (uPA.PAI-1) complex has been investigated. First, exploiting the species specificity of uPA binding, we show that mouse LB6 cells (that express a mouse uPAR) were unable to bind or degrade the human uPA.PAI-1 complex. On the other hand, LB6 clone 19 cells, which express a transfected human uPAR, degraded uPA.PAI-1 complexes with kinetics identical to the human monocytic U937 cells. We also show by immunofluorescence experiments with anti-uPA antibodies that in LB6 clone 19 cells, the uPA.PAI-1 complex is indeed internalized. While at 4 degrees C uPA fluorescence was visible at the cell surface, shift of the temperature to 37 degrees C caused a displacement of the immunoreactivity to the cytoplasmic compartment, with a pattern indicating lysosomal localization. If uPA.PAI-1 internalization/degradation is mediated by uPAR, inhibition of uPA.PAI-1 binding to uPAR should block degradation. Three different treatments, competition with the agonist amino-terminal fragment of uPA, treatment with a monoclonal antibody directed toward the binding domain of uPAR or release of uPAR from the cell surface with phosphatidylinositol-specific phospholipase C completely prevented uPA.PAI-1 degradation. The possibility that a serpin-enzyme complex receptor might be primarily or secondarily involved in the internalization process was excluded since a serpin-enzyme complex peptide failed to inhibit uPA.PAI-1 binding and degradation. Similarly, complexes of PAI-1 with low molecular mass uPA (33 kDa uPA), which lacks the uPAR binding domain, were neither bound nor degraded. Finally we also show that treatment of cells with uPA.PAI-1 complex caused a specific but partial down-regulation of uPAR. A similar result was obtained when PAI-1 was allowed to complex to uPA that had been previously bound to the receptor. The possibility therefore exists that the entire complex uPA.PAI-1-uPAR is internalized. All these data allow us to conclude that internalization of the uPA.PAI-1 complex is mediated by uPAR.     

Prognostic value of plasminogen activator inhibitors in breast cancer

The prognosis of cancer is primarily dependent on its potential to invade and metastasize. Data from both preclinical and clinical studies strongly suggest that serine proteases, as well as their inhibitors and receptor, play a central role in the processes leading to metastasis. We therefore investigated the prognostic value of plasminogen activator inhibitors type 1 (PAI-1) and type 2 (PAI-2) and the combination of both inhibitors in 332 patients with operable breast cancer. PAI-1 and PAI-2 content was measured in the primary tumor cytosols using an enzyme-linked immunosorbent assay. For PAI-1 the median value (3.9 ng/mg protein) was used as cutoff, while the optimized cutoff for PAI-2 (6.5 ng/mg protein) was obtained using the log-rank statistic. After a median follow-up of 46 months 96 (29%) patients relapsed. In univariate analysis patients with a high PAI-1 or a low PAI-2 content had an increased risk of relapse. The difference was statistically significant for PAI-1 (p<0.0001) and almost statistically significant for PAI-2 (p=0.057). Stage, tumor size, differentiation grade, lymph node status and hormone receptor status also showed significant univariate impact on disease-free survival (DFS). In multivariate analysis (Cox model) PAI-1 (p<0.0001, RR=2.78), PAI-2 (p=0.0075, RR=2.17), UICC stage (p=0.0014, RR=2.2), differentiation grade (p=0.0097, RR=1.91) and nodal status (p<0.0001, RR=2.9) retained their significance. When both inhibitors were combined the worst prognosis was observed in patients with simultaneous high PAI-1 and low PAI-2 levels, whereas low PAI-1 in combination with high PAI-2 values indicated a very favorable prognosis. In conclusion, our study showed that both PAI-1 and PAI-2 had independent prognostic value in breast cancer. Combination of both inhibitors further improved the differentiation of patients with respect to prognosis.     

Kinetic analysis of plasminogen activator inhibitor type-2: urokinase complex formation and subsequent internalisation by carcinoma cell lines

The overexpression of urokinase (uPA), which plays a key role in tumour invasion and metastasis, is an established prognostic marker and potential therapeutic target. Plasminogen activator inhibitor type 2 (PAI-2), an efficient and specific inhibitor of uPA, has been shown to selectively deliver potent cytotoxins to tumour cells. However, a direct quantitative analysis of both the inhibition kinetics and subsequent fate of PAI-2 upon interaction with cell-surface uPA has not been previously undertaken. In this study, we analysed specific PAI-2 binding to receptor-bound uPA on human breast and prostate cancer cell lines to directly measure inhibition kinetics. Cell-surface uPA:PAI-2 complex formation, which is reflective of complete uPA inhibition, was found to be very efficient (inactivation constant [K(I)] = 60-80 pM, depending on cell line used) and rapid (inactivation rate constant [k(inact)] = 0.32-0.47 min(-1) at 37 degrees C, depending on cell line used). To directly quantify and visualise cellular internalisation and localisation, we developed a novel assay based on the use of PAI-2 labelled with Alexa(488) fluorochrome and a polyclonal antibody to quench Alexa(488) fluorescence. The efficient and rapid formation of uPA:PAI-2 complexes was thus shown to be associated with specific and rapid internalisation of PAI-2, which could be localised within endosomes and lysosomes. PAI-2 was subsequently degraded, presumably within lysosomes. This study is the first to provide definitive evidence for uPA/uPAR-mediated PAI-2 endocytosis.     

Characterization of crystals of an intact monoclonal antibody for canine lymphoma

A monoclonal antibody of the subclass IgG2a specific for canine lymphoma cells has been crystallized by vapor diffusion from polyethylene glycol 8000. the crystals, which occasionally measure nearly a millimeter on edge, have been examined by X-ray diffraction. The crystals are of triclinic space group P1 with unit cell parameters of a = 66.39 A, b = 77.34 A, c = 101.42 A, alpha = 87.60 degrees, beta = 92.55 degrees, gamma = 97.54 degrees and cell volume of V = 4.84 x 10(5) A3. There is one entire antibody molecule as the asymmetric unit of the crystals. Three-dimensional X-ray diffraction data have been collected to 2.8 A resolution and a self rotation function calculation shows a pronounced peak indicating at least an approximate non-crystallographic dyad axis.     

The conversion of active to latent plasminogen activator inhibitor-1 is an energetically silent event

PAI-1 is a proteinase inhibitor, which plays a key role in the regulation of fibrinolysis. It belongs to the serpins, a family of proteins that behave either as proteinase inhibitors or proteinase substrates, both reactions involving limited proteolysis of the reactive center loop and insertion of part of this loop into beta-sheet A. Titration calorimetry shows that the inhibition of tissue-type plasminogen and pancreatic trypsin are exothermic reactions with DeltaH = -20.3, and -22.5 kcal.mol(-1), respectively. The Pseudomonas aeruginosa elastase-catalyzed reactive center loop cleavage and inactivation of the inhibitor is also exothermic (DeltaH = -38.9 kcal.mol(-1)). The bacterial elastase also hydrolyses peptide-bound PAI-1 in which acetyl-TVASSSTA, the octapeptide corresponding to the P(14)-P(7) sequence of the reactive center loop is inserted into beta-sheet A of the serpin with DeltaH = -4.0 kcal.mol(-1). In contrast, DeltaH = 0 for the spontaneous conversion of the metastable active PAI-1 molecule into its thermodynamically stable inactive (latent) conformer although this conversion also involves loop/sheet insertion. We conclude that the active to latent transition of PAI-1 is an entirely entropy-driven phenomenon.     

Kinetic analysis of the interactions between plasminogen activator inhibitor 1 and both urokinase and tissue plasminogen activator

Plasminogen activator inhibitor 1 (PAI-1) was purified from medium conditioned by cultured bovine aortic endothelial cells by successive chromatography on concanavalin A Sepharose, Sephacryl S-200, Blue B agarose, and Bio-Gel P-60. As shown previously for conditioned media (C. M. Hekman and D. J. Loskutoff (1985) J. Biol. Chem. 260, 11581-11587) the purified PAI-1 preparation contained latent inhibitory activity which could be stimulated 9.4-fold by sodium dodecyl sulfate and 45-fold by guanidine-HCl. The specific activity of the preparation following treatment with 0.1% sodium dodecyl sulfate was 2.5 X 10(3) IU/mg. The reaction between purified, guanidine-activated PAI-1 and both urokinase and tissue plasminogen activator (tPA) was studied. The second-order rate constants (pH 7.2, 35 degrees C) for the interaction between guanidine-activated PAI-1 and urokinase (UK), and one- and two-chain tPA are 1.6 X 10(8), 4.0 X 10(7), and 1.5 X 10(8) M-1 S-1, respectively. The presence of CNBr fibrinogen fragments had no affect on the rate constants of either one- or two-chain tPA. Steady-state kinetic analysis of the effect of PAI-1 on the rate of plasminogen activation revealed that the initial UK/PAI-1 interaction can be competed with plasminogen suggesting that the UK/PAI-1 interaction may involve a competitive type of inhibition. In contrast, the initial tPA/PAI-1 interaction can be competed only partially with plasminogen, suggesting that the tPA/PAI-1 interaction may involve a mixed type of inhibition. The results indicate that PAI-1 interacts more rapidly with UK and tPA than any PAI reported to date and suggest that PAI-1 is the primary physiological inhibitor of single-chain tPA. Moreover, the interaction of PAI-1 with tPA differs from its interaction with UK, and may involve two sites on the tPA molecule.     

Epistatic interactions in genetic regulation of t-PA and PAI-1 levels in a Ghanaian population

The proteins, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1), act in concert to balance thrombus formation and degradation, thereby modulating the development of arterial thrombosis and excessive bleeding. PAI-1 is upregulated by the renin-angiotensin system (RAS), specifically by angiotensin II, the product of angiotensin converting enzyme (ACE) cleavage of angiotensin I, which is produced by the cleavage of angiotensinogen (AGT) by renin (REN). ACE indirectly stimulates the release of t-PA which, in turn, activates the corresponding fibrinolytic system. Single polymorphisms in these pathways have been shown to significantly impact plasma levels of t-PA and PAI-1 differently in Ghanaian males and females. Here we explore the involvement of epistatic interactions between the same polymorphisms in central genes of the RAS and fibrinolytic systems on plasma t-PA and PAI-1 levels within the same population (n = 992). Statistical modeling of pairwise interactions was done using two-way ANOVA between polymorphisms in the ETNK2, RENIN, ACE, PAI-1, t-PA, and AGT genes. The most significant interactions that associated with t-PA levels were between the ETNK2 A6135G and the REN T9435C polymorphisms in females (p = 0.006) and the REN T9435C and the TPA I/D polymorphisms (p = 0.005) in males. The most significant interactions for PAI-1 levels were with REN T9435C and the TPA I/D polymorphisms (p = 0.001) in females, and the association of REN G6567T with the TPA I/D polymorphisms (p = 0.032) in males. Our results provide evidence for multiple genetic effects that may not be detected using single SNP analysis. Because t-PA and PAI-1 have been implicated in cardiovascular disease these results support the idea that the genetic architecture of cardiovascular disease is complex. Therefore, it is necessary to consider the relationship between interacting polymorphisms of pathway specific genes that predict t-PA and PAI-1 levels.     

X-ray diffraction and calorimetric study of N-lignoceryl sphingomyelin membranes.

Differential scanning calorimetry and x-ray diffraction have been used to investigate hydrated multibilayers of N-lignoceryl sphingomyelin (C24:0-SM) in the hydration range 0-75 wt % H2O. Anhydrous C24:0-SM exhibits a single endothermic transition at 81.3 degrees C (delta H = 3.6 kcal/mol). At low hydration (12.1 wt % H2O), three different endothermic transitions are observed: low-temperature transition (T1) at 39.4 degrees C (transition enthalpy (delta H1) = 2.8 kcal/mol), intermediate-temperature transition (T2) at 45.5 degrees C, and high-temperature transition (T3) at 51.3 degrees C (combined transition enthalpy (delta H2 + 3) = 5.03 kcal/mol). On increasing hydration, all three transition temperatures of C24:0-SM decrease slightly to reach limiting values of 36.7 degrees C (T1), 44.4 degrees C (T2), and 48.4 degrees C (T3) at approximately 20 wt % H2O. At 22 degrees C (below T1), x-ray diffraction of C24:0-SM at different hydration levels shows two wide-angle reflections, a sharp one at 1/4.2 A-1 and a more diffuse one at 1/4.0 A-1 together with lamellar reflections corresponding to bilayer periodicities increasing from d = 65.4 A to a limiting value of 71.1 A. Electron density profiles show a constant bilayer thickness dp-p approximately 50 A. In contrast, at 40 degrees C (between T1 and T2) a single sharp wide-angle reflection at approximately 1/4.2 A-1 is observed. The lamellar reflections correspond to a larger bilayer periodicity (increasing from d = 69.3-80.2 A) and there is some increase in dp-p (52-56 A) with hydration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure determination of thymoquinone by high-resolution X-ray powder diffraction

The crystal structure of 2-isopropyl-5-methyl-1,4-benzoquinone (thymoquinone) and its thermal behavior--as necessary physical and chemical properties--were determined in order to enhance the current understanding of thymoquinone chemical action by using high resolution x-ray powder diffraction, Fourier transform infrared spectroscopy (FTIR), and 3 thermo-analytical techniques thermogravimetric analysis (TGA), differential thermal analysis (DTA), and differential scanning calorimetry (DSC). The findings obtained with high-resolution x-ray powder diffraction and molecular location methods based on a simulated annealing algorithm after Rietveld refinement showed that the triclinic unit cell was a = 6.73728(8) A, b = 6.91560(8) A, c = 10.4988(2) A, alpha = 88.864(2) degrees, beta = 82.449(1) degrees, gamma = 77.0299(9) degrees; cell volume = 472.52(1) A3, Z = 2, and space group P1. In addition, FTIR spectrum revealed absorption bands corresponding to the carbonyl and C-H stretching of aliphatic and vinylic groups characteristically observed in such p-benzoquinones. Also, a chemical decomposition process starting at 65 degrees C and ending at 213 degrees C was noted when TGA was used. DSC allowed for the determination of onset at 43.55 degrees C and a melting enthalpy value of DeltaH(m) = 110.6 J/g. The low value obtained for the fusion point displayed a van der Waals pattern for molecular binding, and the thermograms performed evidence that thymoquinone can only be found in crystalline triclinic form, as determined by DRX methods.     

Effect of chain unsaturation on the structure and thermotropic properties of galactocerebrosides.

Differential scanning calorimetry (DSC) and x-ray diffraction have been used to study the effect of increasing chain-unsaturation on the structure and properties of the hydrated cerebrosides N-stearoyl, -oleoyl, and -linoleoyl galactosylsphingosine (NSGS, NOGS, and NLnGS, respectively). DSC of hydrated (70 wt% water) NSGS shows an endothermic transition at 85 degrees C (delta H = 18.0 kcal/mol NSGS) and a broad exothermic transition at 40-60 degrees C, the latter being dependent upon the previous cooling rate. X-Ray diffraction patterns recorded at 21, 61, and 86 degrees C provide evidence for interconversions between metastable and stable crystalline NSGS bilayer phases. The properties of the unsaturated-chain cerebrosides are more complex. Hydrated NOGS shows a single endothermic transition at 44.8 degrees C (delta H = 11.5 kcal/mol NOGS). However, incubation of NOGS at 49 degrees C for 24 h results in a second transition at 55.5 degrees C. By cycling NOGS between 0 and 49 degrees C complete conversion into this higher melting phase (delta H = 12.1 kcal/mol NOGS) is achieved. X-ray diffraction confirms a bilayer phase at all temperatures and delineates the conversions between a crystalline phase at 21 degrees C (bilayer period d = 56.5A), a second crystalline phase at 47 degrees C (d = 69.9A), and a liquid crystalline phase at 59 degrees C (d = 52.0A). The more unsaturated NLnGS shows two transitions, a sharp transition at 28 degrees C (delta H = 8.0 kcal/mol NLGS) and a broad, low-enthalpy transition at 42 degrees C (delta H = 0.4 kcal/mol NLGS). Again, incubation between the two transitions leads to a single transition at 44 degrees C (delta H = 9.3 kcal/mol NLGS). X-ray diffraction demonstrates conversions between two crystalline bilayer phases (d = 55.2A and d = 68.4A), and a liquid crystalline bilayer phase (d = 51.8A). Thus, increased unsaturation in the amide-linked fatty acyl chain of cerebrosides results in decreased chain-melting temperatures (NSGS greater than NOGS greater than NLnGS) and has marked effects on their structural properties.     

Identification and partial characterization by chemical cross-linking of a binding protein for tissue-type plasminogen activator (t-PA) on rat hepatoma cells. A plasminogen activator inhibitor type 1-independent t-PA receptor.

Plasma tissue-type plasminogen activator (t-PA) is cleared rapidly in vivo by the liver. Previous studies with the human hepatoma cell line HepG2 have identified a clearance system for t-PA modulated by plasminogen activator inhibitor type 1 (PAI-1). In the present study, a rat hepatoma cell line MH1C1 is shown to contain a PAI-1-independent t-PA clearance system. At 4 degrees C, binding of 125I-t-PA to MH1C1 cells was rapid, specific, and saturable. Scatchard analysis of the binding data yielded a mean estimate of 105,000 high affinity binding sites per cell (Kd = 4.1 nM). When the bound ligand was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the majority (about 90%) of the specific binding was in the form of uncomplexed 125I-t-PA. This is in contrast to HepG2 cells in which specific binding was mainly in the form of a sodium dodecyl sulfate-stable 125I-t-PA.PAI-1 complex. When availability of matrix-associated PAI-1 was blocked by preincubation with anti-PAI-1 antibody or removed by elastase treatment, specific 125I-t-PA binding to MH1C1 cells was unaffected, whereas most of the specific 125I-t-PA binding to HepG2 cells was abolished. Furthermore, when the active site of t-PA was inactivated with diisopropyl fluorophosphate, the diisopropyl fluorophosphate-t-PA specifically competed for binding of 125I-t-PA to MH1C1 cells, but failed to block specific 125I-t-PA binding to HepG2 cells. At 37 degrees C, PAI-1-independent t-PA binding to MH1C1 cells was followed by ligand uptake and degradation with kinetics similar to that seen in HepG2 cells. Chemical cross-linking of t-PA to MH1C1 cells revealed a specific t-PA binding protein with a molecular mass of about 500,000 daltons. Ligand-receptor complexes generated by chemical cross-linking were immunoprecipitable by anti-t-PA antibody but not by anti-PAI-1 antibody, further supporting the finding that binding of t-PA to MH1C1 cells is PAI-1-independent.     

Crystallization and preliminary X-ray diffraction studies of a toxic phospholipase A2 from the venom of Vipera ammodytes meridionalis complexed to a synthetic inhibitor

A toxic phospholipase A(2) (PLA(2)) is isolated from the neurotoxic complex Vipoxin, the major lethal component of the venom of Vipera ammodytes meridionalis. The enzyme is complexed to the synthetic inhibitor elaidoylamide and crystallized. The crystals belong to the space group P2(1)2(1)2(1), with unit cell dimensions a=46.57 A, b=82.68 A, c=119.47 A and beta=90 degrees. Initial diffraction data to 3.3 A resolution are collected.     

Two crystal forms of Azotobacter ferredoxin.

Two crystal forms of Azotobacter vinelandii (4Fe-4s)2 ferredoxin I (Fd I) have been grown which are suitable for high resolution x-ray diffraction studies. Tetragonal crystals grow as square bipyramids from ammonium sulfate and Tris buffer using a temperature gradient. The space group is P41212 (or P43212) with a = 55.3, c = 95.9 A and 1 molecule/asymmetric unit. Triclinic crystals grow as plates or laths from ammonium sulfate and phosphate buffer at constant temperature. The space group is P1 with a = 46.8, b = 58.7, c = 64.3 A, alpha = = 105 degrees 05 min, beta = 82 degrees 30 min, gamma = 110 degrees 30 min and 4 or 5 molecules/unit cell. Both crystal forms are stable to x-ray irradiation and diffract beyond 3.0 A resolution.