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Roles for glycerophospholipids in exocytosis have been proposed, but remain controversial. Phospholipases are stimulated following the activation of the high-affinity receptor for immunoglobulin E (IgE) in mast cells. To study the biochemical sequelae that lead to degranulation, broken cell systems were employed. We demonstrate that the addition of three distinct types of exogenous phospholipases (i.e., bcPLC, scPLD, and tfPLA(2)), all of which hydrolyze phosphatidylcholine (PC), trigger degranulation in permeabilized RBL-2H3 cells, a mucosal mast cell line. Production of bioactive lipids by these phospholipases promotes release of granule contents through the plasma membrane and acts downstream of PKC, PIP(2), and Rho subfamily GTPases in regulated secretion. These exogenous phospholipase-induced degranulation pathways circumvent specific factors activated following stimulation of the IgE receptor as well as in ATP- and GTP-dependent intracellular pathways. Taken together, these results suggest that regulated secretion may be achieved in vitro in the absence of cytosolic factors via phospholipase activation and that products of PC hydrolysis can promote exocytosis in mast cells.

Hen egg white lysozyme has been prepared in which the C epsilon position of the single histidine residue is substituted by a deuterium atom as a nondisturbing stable isotope probe. The deuterium nuclear magnetic resonance (2H NMR) spectrum in H2O shows a broad resonance (500--1000 Hz) due to the histidine deuteron and a sharp signal from residual HOD. The line width of the deuterium signal increases with pH, reflecting the self-association of lysozyme which is known to involve this histidine [shindo, H., Cohen, J.S., & Rupley, J. A. (1977) Biochemistry 16, 3879]. Correlation times calculated from spin-spin relaxation times (T2) derived from the 2H widths indicate that His-15 is restricted in motion and that lysozyme is predominantly dimerized at pH 7.5. Controls carried out with [epsilon-2H]imidazole showed a small pH dependence of the spin-lattice relaxation time (T1), which parallels the 2H chemical shift change upon ionization of the imidazole. Similar results cannot generally be observed by proton nuclear magnetic resonance (1H NMR) because of paramagnetic relaxation due to trace metal ion impurities. The pH dependence of the 2H T1 values indicates a change in the 2H quadrupole coupling constant upon protonation of the imidazole ring.

A polysaccharide, antigenically related to group C meningococcus, has been isolated from Escherichia coli strain Bos-12 (016; K92; NM). Like groups B and C meningococcal polysaccharide, the Bos-12 antigen is a pure polymer of sialic acid. 13C NMR studies on the meningococcal group B and C polysaccharides have indicated that the former consists of sialic acid units linked 2 leads to 8- alpha, whereas the latter contains the sialic acid residues linked 2 leads to 9-alpha (Bhattacharjee, A.K., Jennings, H.J., Kenny, C.P., Martin, A., and Smith, I.C.P. (1975), J. Biol. Chem. 250, 1926). Comparison of natural abundance 13C NMR spectra of the Bos-12 polysaccharide with group B and C meningococcal polysaccharides established that Bos-12 was either (a) an equimolar mixture of 2 leads to 8-alpha linked sialic acid homopolymers or (b) a 2 leads to 8-alpha/2 leads to 9-alpha heteropolymer. These possibilities were distinguished in the following manner. The fact that Bos-12 polysaccharide precipitated with anti-group C serum but not with anti-group B serum would seem to exclude a. Further, chemical studies (periodate oxidation followed by tritiated NaBH4 reduction) gave saccharides with a radioactive-labeling pattern expected for alternating 2 leads to 8-alpha/2 leads to 9-alpha sialic acid linkages. Bos-12 is thus an 2 leads to 8/2 lead to 9-alpha heteropolymer.

The B to Z transition of poly(dG-m5dC) . poly(dG-m5dC) has been monitored by 31P nuclear magnetic resonance (NMR) spectroscopy as a function of concentration of several salts. In 5 mM Tris- [tris(hydroxymethyl)amino-methane] HCl buffer two peaks of equal area (0.27 ppm separation) are observed at -4.2 ppm (upfield from trimethyl phosphate), indicating an alternating (right-handed) B conformation. Upon addition of NaCl, CsF, MgCl2, or Co(NH3)6Cl3, a signal arises at -3 ppm that is characteristic of the left-handed Z form. This signal is half of a doublet (1.3-2.1 ppm separation) and is tentatively assigned to the tg+ phosphodiester conformation in the Z form. The midpoints of the cooperative B to Z transitions with these salts are in good agreement with the values reported from circular dichroism (CD) studies for this copolymer. The 13C NMR spectra at natural abundance of poly(dG-m5dC) . poly(dG-m5dC) in the alternating B conformation in low salt show a broad resonance for the C2' and no clear resonance for the C3'. This is in contrast to the alternating B form of poly(dA-dT) . poly(dA-dT) under similar conditions, which exhibits a well-resolved doublet for both C2' and C3' that is presumed to derive from alternating C2'-endo and C3'-endo deoxyribose conformations. In the 13C NMR spectrum of the Z form of poly(dG-m5dC) . poly(dG-m5dC), two major peaks are observed for the C2', which may also indicate an alternating sugar conformation.

Medium length (500-200 bp) alternating purine-pyrimidine DNAs were prepared by sonication of synthetic polymers at low temperature. The products, and the hairpin structures derived from them after melting, were sufficiently small for high-resolution 31P NMR studies. Of the five sequences studied, two DNAs, poly(dG-dC).poly(dG-dC) and poly(dA-dU).poly(dA-dU), gave singlet 31P resonances, while three others, poly(dA-dT).poly(dA-dT), poly(dA-br5U).poly(dA-br5U), and poly(dI-dC).poly(dI-dC), exhibited two resolved signals of equal area. This indicates the existence of two distinct alternating phosphodiester backbone conformations for these latter three B-DNAs in solution. Controls of homopolymers, which were also prepared by sonication, showed only singlet 31P resonances. Of the alternating sequences DNAs, only sonicated poly(dG-dC).poly(dG-dC) exhibited a conformational transition to a high salt (greater than 2.5 M) form which exhibited two well-resolved 31P resonances of equal area. This indicates that the high salt form of poly(dG-dC).poly(dG-dC) also has an alternating backbone structure, and it is presumed to be a Z-DNA. These results indicate a general response of the DNA backbone conformation to alternating purine-pyrimidine base sequences but with a degree of sequence and environmental specificity which might have functional genetic significance.

The complex of ribonuclease A (RNase A) with uridine vanadate (U-V), a transition-state analogue, has been studied with 51V and proton NMR spectroscopy in solution and by neutron diffraction in the crystalline state. Upon the addition of aliquots of U-V at pH 6.6, the C epsilon-H resonances of the two active-site histidine residues 119 and 12 decrease in intensity while four new resonances appear. Above pH 8 and below pH 5, these four resonances decrease in intensity as the complex dissociates. These four resonances are assigned to His-119 and His-12 in protonated and unprotonated forms in the RNase-U-V complex. These resonances do not titrate or change in relative area in the pH range 5-8, indicating a slow protonation process, and the extent of protonation remains constant with ca. 58% of His-12 and ca. 26% of His-119 being protonated. The results of diffraction studies show that both His-12 and His-119 occupy well-defined positions in the RNase-U-V complex and that both are protonated. However, while the classic interpretation of the mechanism of action of RNase based on the proposal of Findlay et al. [Findlay, D., Herries, D. G., Mathias, A. P., Rabin, B. R., & Ross, C. A. (1962) Biochem. J. 85, 152-153] requires both His-12 and His-119 to be in axial positions relative to the pentacoordinate transition state, in the diffraction structure His-12 is found to be in an equatorial position, while Lys-41 is close to an axial position. Hydrogen exchange data show that the mobility and accessibility of amides in the RNase-U-V complex do not significantly differ from what was observed in the native enzyme. The results of both proton NMR in solution and neutron diffraction in the crystal are compared and interpreted in terms of the mechanism of action of RNase.

We have studied the tridecadeoxynucleotide CGCGAATTACGCG (I), which contains an additional A at position 9 compared to the dodecanucleotide of which the crystal structure has been determined. Sequence I exhibits no distinct melting curve and also has a concentration-dependent pattern of peaks on reverse-phase chromatography. This behavior is explained by a slow equilibration between loop and duplex forms in solution. We have characterized this equilibrium by proton NMR spectroscopy and shown that it is fully reversible by monitoring the two thymine methyl resonances, each of which occurs in two environments. Lower temperature and higher concentration favor the duplex; the midpoint of the transition is such that the loop predominates at room temperature. We have measured the van't Hoff enthalpy of formation of the duplex and the activation energy by temperature-jump and saturation-transfer experiments. The results are compared with those for the 17-mer sequence CGCGCGAATTACGCGCG (II), which contains two additional base pairs in the stem of the loop. The thermodynamic parameters and the effect of increasing salt concentration on the rate of conversion of the loop and duplex forms lead us to presume that the mechanism of interconversion involves complete strand separation and re-formation rather than cruciform formation and branch migration.

Poly(d2NH2A-dT) forms a structure in high salt that is clearly distinct from the B form present in low salt. Two-dimensional nuclear Overhauser effect (2D NOE) NMR spectra establish that the conformation of the high-salt form is not Z. Correlations of observed cross peaks in the 2D NOE spectra and estimated interproton distances of the common DNA conformations are consistent only with an A form or closely related structure. This interpretation is also consistent with the negative circular dichroic band observed in the high-salt form of poly(d2NH2A-dT) and in A-form ribohomopolymer helices containing 2NH2A.

Proton NMR titration curves of the histidine Cepsilon-H resonances of the deoxy and oxy forms of human, horse, and sperm whale myoglobins (Mb) were determined and compared with the results for the met and azide forms. One extra titrating resonance (H-8) was observed for each deoxy-Mb compared with the corresponding met-Mb, and a further extra resonance (H-9) was observed for the oxy-Mb form. These resonances correspond to the two additional resonances previously described for azide-Mb [Hayes, M., Hagenmaier, H., & Cohen, J. S. (1975) J. Biol. Chem. 250, 7461--7472]. This new evidence prompts us to reassign these resonances to the near-heme histidine residues.

13C spin-lattice (T1) relaxation times determined at four frequencies (25, 68, 100, and 125 MHz) have been used to probe the molecular dynamics of ribonuclease S' complexes prepared from synthetic amino-terminal peptides containing 13C enrichment (ca. 90%) at selected sites [Niu, C., Matsuura, S., Shindo, H., & Cohen, J. S. (1979) J. Biol. Chem. 254, 3788]. It was found that the motion of the C alpha-H bond of Ala-5 could not be determined by isotropic reorientation alone. The time scale and spatial restriction on the internal motion of this residue were determined by the model-free approach of Lipari and Sazbo [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. It was found that the C alpha-H bond, in addition to an overall correlation time of 20 ns, underwent internal motion with a correlation time of 0.5 ns and a generalized order parameter S corresponding to a cone semiangle of 23 degrees C. The C beta-H bond had a correlation time of 37 ps, reflecting the fast rotation of the methyl group, and had an S value close to that expected if the C alpha-C beta and C alpha-H bonds have the same degree of spatial restriction.

Proton one- and two-dimensional nuclear Overhauser enhancement (1D and 2D NOE) spectroscopy has been used to demonstrate that poly(d2NH2A-d5IU) and poly(d2NH2A-d5BrU) are converted from the B to the A conformation in high salt, as found previously for poly(d2NH2A-dT) [Borah, B., Cohen, J. S., Howard, F. B., & Miles, H. T. (1985) Biochemistry 24, 7456-7462]. The 2D NOE and 1D NOE spectra exhibit strong base proton (H8,H6)-H3' cross relaxation, suggesting short interproton distances. These results are indicative of a C3'-endo sugar pucker for both purine and pyrimidine residues in an A or closely related structure. The circular dichroism and UV spectra are consistent with the interpretation of an A conformation in high salt.

Primer recognition by purified HIV reverse transcriptase has been investigated. Earlier we found that the reaction pathway for DNA synthesis is ordered, with template-primer and free enzyme combining to form the first complex in the reaction sequence (Majumdar et al., 1988). We now find that d(C)28 is a linear competitive inhibitor of DNA synthesis against poly[r(A)].oligo[d(T)] as template.primer, indicating that d(C)28 and the template.primer combine with the same form of the enzyme in the reaction scheme, i.e., the free enzyme. The phosphorothioate oligodeoxynucleotide Sd(C)28 also is a linear competitive inhibitor against template.primer. However, the Ki for inhibition (approximately 2.8 nM) is approximately 200-fold lower than the Ki for inhibition by d(C)28. Since the inhibition is linear competitive, the dissociation constant is equal to the Ki for inhibition. Filter binding assays confirmed high-affinity binding between Sd(C)28 and the enzyme and yielded a KD similar to the Ki for inhibition. Substrate kinetic studies of DNA synthesis using Sd(C)28 as primer, and poly[r(I)] as template, revealed that the Km for Sd(C)28 is 24 nM. The Km for this primer is, therefore, 8-fold higher than the KD for enzyme-primer binding (2.8 nM). These results enable calculation of real time rate values for the enzyme-primer association (kon = 5.7 x 10(8) M-1 s-1) and dissociation (koff = 1.6 s-1).