Purification and properties of multiple forms of mouse trypsinogen

Three trypsinogens and one chymotrypsinogen were found in and purified from the pancreas of a mouse strain (CFO). The molecular weights of the trypsinogens and the chymotrypsinogen were all estimated as 25 000. The enzyme properties of the three trypsinogens were studied and they showed very low K_m values (3.2?6.5 μM) for the substrates, BzArgOEt and TosArgOMe, and the dame pH optimum profile between pH 8.0–10.0. However, the ratios of catalytic rate constants, k_cat (s^?1), with BzArgOEt as substrates compared to that with TosArgOMe were very different. The values of Try-III were similar with the two substrates, Try-I was slightly higher value with TosArgOMe than with BzArgOEt, and the values of Try-II were much higher with TosArgOMe than with BzArgOEt. Also, the trypsinogens and the chymotrypsinogen were purified from pancreas of Mol-A strain mice. When the enzyme properties of the three trypsinogens were examined, one form of trypsinogen (Try-I) was shown to have different properties in k_cat (s^?1) for the two substrates, compared to the trypsinogen of CFO mice.     

NMR Investigations of the interaction of water with lecithin in benzene solutions

NMR investigations of ¹H (chemical shifts, line widths) and of ³¹P (relaxation times, T₁) performed on the three-component system of lecithin-benzene-water show that there is an interaction of water with the phosphate group in two regions of different mobility and structure. A fast exchange of the water molecules takes place between both regions. The region of strong interaction involves about 2 and that of the weaker interaction about 5 water molecules per lecithin molecule. When the water concentration is increased a third region is formed which is assigned to the water molecules that are located beyond the two regions of interaction with the phosphate group, but within the micelle. This water has a different structure from that of the second region of interaction with the phosphate group and may also have a different mobility.Addition of water increases the motion of the head groups of the lecithin molecules. This is due to a loosening of the packing of lecithin molecules.     

High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. I. The hydrogen bonded protons of the "charge relay" system

High resolution proton nuclear magnetic resonance has been used to observe protons at the active site of chymotrypsin A_δ and at the same region of chymotrypsinogen A. A single resonance with the intensity of one proton is located in the low field region of the nuclear magnetic resonance spectrum. This resonance is observed in H₂O solutions but not in ²H₂O. On going from low to high pH the resonance titrates upfield 3 parts per million in both proteins and has a pK of 7.5. The titration can be prevented by alkylating His57 with either of two active site directed chloromethyl ketones. Using these data the proton resonance has been assigned to a proton in a hydrogen bond between His57 and Asp102. Further confirmation of this assignment lies in the observation of a similar resonance in this same low field region of the nuclear magnetic resonance spectrum of trypsin, trypsinogen, subtilisin BPN′ and α-lytic protease all of which have the Asp-His-Ser triad at their active sites.This proton resonance in chymotrypsin A_δ was used as a probe to monitor the charge state of the active site upon formation of a stable acyl-enzyme analogue N²(N-acetylalanyl)-N¹benzoylcarbazoyl-chymotrypsin A_δ. In this derivative the His-Asp proton resonance titrates from the same low pH end point as in the native enzyme, ?18 parts per million, to a new high pH end point of ?14.4 parts per million (versus ?15.0 parts per million in the native enzyme). The difference of 0.6 parts per million in the high pH end points between the native and acyl enzyme is interpreted as supporting the suggestion that a hydrogen bond exists between Ser195 and His57 in the native enzyme and zymogen.We conclude from these studies that the charge relay system from Asp102 across His57 to Ser195 is intact in chymotrypsin A_δ and chymotrypsinogen A, and that, in the native enzyme, it slightly polarizes Ser195.     

A Cytochemical Study on the Pancreas of the Guinea Pig : V. In vivo Incorporation of Leucine-1-C14 into the Chymotrypsinogen of Various Cell Fractions

Chymotrypsinogen synthesis in the exocrine cell of the guinea pig pancreas was studied under the following conditions: Animals fed after a fast of ∼48 hours received ∼1 hour after feeding an intravenous injection of DL-leucine-1-C¹⁴. At various time intervals (1 to 45 minutes) after the injection, the glands were removed and fractionated into a series of cell fractions of known cytological significance. Ten to twelve animals were used for each time point. From each cell fraction, the chymotrypsinogen was isolated by acid extraction and purified by (NH₄)₂SO₄ fractionation, isoelectric precipitation, and chromatography. Because of the minuteness of the quantities involved, chymotrypsinogen amounts were calculated from enzymatic activity figures, and a carrier method was used to precipitate and count the enzyme. The chymotrypsinogen isolated from the attached ribonucleoprotein particles of the microsomal fraction had the highest specific radioactivity at the early time points (1 to 3 minutes). After long intervals (at 15 to 45 minutes), the specific radioactivity of the enzyme increased in the microsomal contents and finally in the zymogen granules. The results are compatible with the view that the chymotrypsinogen is synthesized in or on the attached RNP particles and subsequently transported to other cell compartments.     

Host-guest properties of the trinuclear arene-ruthenium cluster cation [H3Ru3(C6H6)(C6Me6)2(O)]

The trinuclear arene-ruthenium cluster cation [H₃Ru₃(C₆H₆)(C₆Me₆)₂(O)]⁺, containing a μ₃-oxo cap and three arene ligands that span a hydrophobic pocket above the metal skeleton, has been crystallised as tetrafluoroborate salt in the presence of various guest molecules. The host-guest complexes have been characterised by single-crystal X-ray structure analysis. With chloroform as the guest molecule, a CHCl₃ molecule sits perfectly in the hydrophobic pocket, the hydrogen atom being encapsulated inside the cavity. When dioxane is added during the crystallisation process, the cluster forms infinite chains which are connected by a complex network of hydrogen bonds involving the μ₃-oxo ligand, water and dioxane molecules. Interestingly, in the presence of phenol, a water molecule is hydrogen-bonded between the μ₃-oxo ligand and the phenol molecule, forming a one-dimensional μ₃-O ? H₂O ? HO hydrogen-bonded chain. Finally, with benzoic acid, a head-to-tail host-guest chain is obtained, the phenyl ring being incorporated in the hydrophobic pocket, while the acid group is hydrogen-bonded to the μ₃-oxo ligand.     

Trypsin cleavage of the α-subunit of beef heart F1-ATPase abolishes ATP synthesis and ATP-driven energy-transduction capabilities

Previous work has shown that mild trypsin treatment eliminates energy-transduction capability and tight (non-exchangeable) nucleotide binding in beef heart mitochondrial F₁-ATPase (Leimgruber, R.M. and Senior, A.E. (1976) J. Biol. Chem. 251, 7103–7109). The structural change brought about by trypsin was, however, too subtle to be identified by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, and was not defined. In this work we have applied two-dimensional electrophoresis (isoelectric focussing then sodium dodecyl sulfate polyacrylamide gradient electrophoresis) to the problem, and have determined that the α-subunit of F₁ is altered by the mild trypsin treatment, whereas no change was detected in β-, γ-, δ- or ?-subunits. Binding of ADP to the trypsin-treated F₁ was compared to binding to control enzyme over a range of 0–40 μM ADP in a 30 min incubation period. There was no difference between the two enzymes, K^ADP_d in Mg²⁺-containing buffer was about 2 μM in each. Since the tight (nonexchangeable) sites are abolished in trypsin-treated F₁, this shows that tight exchangeable ADP-binding sites are different from the tight nonexchangeable ADP-binding sites. There was no effect of trypsin cleavage of the α-subunit on β-subunit conformation as judged by aurovertin fluorescence studies. The cleavage of the α-subunit which occurred was judged to occur very close to the C- or N-terminus of the subunit and constitutes therefore a small and specific chemical modification which abolishes overall function in F₁ but leaves partial functions intact.     

Pocket analysis of the full-length cholix toxin. An assessment of the structure–dynamics of the apo catalytic domain

Cholix toxin from Vibrio cholerae is the third member of the diphtheria toxin (DT) group of mono-ADP-ribosyltransferase (mART) bacterial toxins. It shares structural and functional properties with Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae DT. Cholix toxin is an important model for the development of antivirulence approaches and therapeutics against these toxins from pathogenic bacteria. Herein, we have used the high-resolution X-ray structure of full-length cholix complexed with NAD⁺ to describe the properties of the NAD⁺-binding pocket at the residue level, including the role of crystallographic water molecules in the NAD⁺ substrate interaction. The full-length apo cholix structure is used to describe the putative NAD⁺-binding site(s) and to correlate biochemical with crystallographic data to study the stoichiometry and orientation of bound NAD⁺ molecules. We quantitatively describe the NAD⁺ substrate interactions on a residue basis for the main 22 pocket residues in cholix_f, a glycerol and 5 contact water molecules as part of the recognition surface by the substrate according to the conditions of crystallization. In addition, the dynamic properties of an in silico version of the catalytic domain were investigated in order to understand the lack of electronic density for one of the main flexible loops (R-loop) in the pocket of X-ray complexes. Implications for a rational drug design approach for mART toxins are derived.     

Crystal and molecular structure of the amylose–DMSO complex

The crystal and molecular structure of the complex of amylose with dimethyl sulfoxide has been studied by a combination of stereochemical analysis, potential energy, and X-ray diffraction methods. The complex crystallizes in a pseudotetragonal unit cell with a = b = 19.17 Å and c (fiber axis) = 24.39 Å, with two antiparallel chains per unit cell and space group P2₁2₁2₁. The amylose chain is a left-handed 6₁(1.355) helix with three turns per crystallographic repeat. The O(6) rotational position is approximately gt. Dimethyl sulfoxide is located inside the helix with one DMSO molecule for every three glucose residues. An additional four DMSO molecules and eight water molecules each are located in the large interstices between chains, and it is the interaction of these molecules with the helix that results in the pseudotetragonal chain packing. The interstitial DMSO is the source of the previously reported additional layer lines, which are not consistent with the 8.13-Å amylose repeat distance. The final R factor for the layers with amylose contribution to the structure factors was 0.29, while the overall R factor was 0.35. The stereochemical packing analysis provided suitable phasing models for the subsequent X-ray refinement.     

How dry are anhydrous enzymes? Measurement of residual and buried 18O-labeled water molecules using mass spectrometry

There is continual debate over the central role of water in protein folding, structure, stability, and dynamics. Catalytic activity has been demonstrated in organic media with, apparently “anhydrous” enzymes. Hence there is considerable discussion over whether there are a few residual water molecules or if the enzymes are demonstrating activity in the complete absence of water. Here we present measurements designed to test this hypothesis based on the detection of ¹⁸O-labeled water by mass spectrometry. This extremely sensitive technique avoids many of the potential errors associated with published methods for measuring water content such as gravimetry or Karl Fischer titrations. We have also explored the mass spectrometric detection of ²H-enriched water and found that lyophilization of deuteron-labeled protein can lead to extensive loss of the isotopic label during the drying process. “Anhydrous” protein was produced by extended drying, over P₂O₅, of lyophilized powders hydrated through the vapor phase with ¹⁸O-labeled water. Redissolution in standard water released the remaining protein-bound ¹⁸O-labeled water molecules, and the isotopic enrichment of the water was used to calculate the number of bound molecules per mole of protein. In the cases of lysozyme and subtilisin Carlsberg, 4 ± 2 and 15 ± 2 waters per mole were found, respectively. Comparisons with crystal structures showed these values correspond closely to the expected number of buried water molecules in these proteins. This is consistent with the idea that water physically entrapped within the rigid protein structure is retained but all the other more accessible surface-bound hydration molecules can be removed by the drying process. Such anhydrous subtilisin Carlsberg preparations have been found to be weakly catalytic and therefore it appears that additional water molecules on the surface of the enzyme are not essential for this level of enzyme activity. © 1997 John Wiley & Sons, Inc.     

The distribution and interconversion of ammonium and nitrate in the Skallingen salt marsh (Denmark) and their exchange with the adjacent coastal water

The potential nitrogen sources for the primary production in the intertidal area are nitrogen compounds obtained from mineralization in the sediment and the water column, nitrogen fixation, outflow from rivers and groundwater seeping from the mainland. The available inorganic nitrogen in the adjacent coastal waters decreases from 50–80 μmol NO₃ ^-/l and 6–15 μmol NH₄ ⁺/l in early spring to ca one tenth during the growing season. In the sediment of the tidal flats available ammonia and nitrate vary between 50 and 100 μmol/1 pw. In the salt marsh available ammonia increases from 200–300 nmol NH₄ ⁺/g fwt to approximately double the amount, and the available nitrate varies from 100–300 nmol NO₃ ^-/g fwt (250–750 μmol NO₃ ^-/l pw) to ca one third during the growing season. The exchange of NH₄ ⁺, NO₂ ^- and NO₃ ^- across the sediment water interface has been estimated during tidal cycles under light and dark conditions on the tidal flats. The flux of nitrogen was dependent on the flora and fauna as well as the time of the year. The tidal activity, frequency and length of inundation are considered the driving force in a two-way process between salt marshes and adjacent coastal waters. The role of marsh sediment, tidal water and sediments of the tidal flats as sites of accumulation, consumption and remineralization of organic matter is emphasized. The possible exchange of ammonia and nitrate between the salt marsh and the different compartments of the tidal water is discussed.     

Critical roles of the CuB site in efficient proton pumping as revealed by crystal structures of mammalian cytochrome c oxidase catalytic intermediates

Mammalian cytochrome c oxidase (CcO) reduces O₂ to water in a bimetallic site including Fe_a3 and Cu_B giving intermediate molecules, termed A-, P-, F-, O-, E-, and R-forms. From the P-form on, each reaction step is driven by single-electron donations from cytochrome c coupled with the pumping of a single proton through the H-pathway, a proton-conducting pathway composed of a hydrogen-bond network and a water channel. The proton-gradient formed is utilized for ATP production by F-ATPase. For elucidation of the proton pumping mechanism, crystal structural determination of these intermediate forms is necessary. Here we report X-ray crystallographic analysis at ∼1.8 Å resolution of fully reduced CcO crystals treated with O₂ for three different time periods. Our disentanglement of intermediate forms from crystals that were composed of multiple forms determined that these three crystallographic data sets contained ∼45% of the O-form structure, ∼45% of the E-form structure, and ∼20% of an oxymyoglobin-type structure consistent with the A-form, respectively. The O- and E-forms exhibit an unusually long Cu_B²⁺-OH⁻ distance and Cu_B¹⁺-H₂O structure keeping Fe_a3³⁺-OH⁻ state, respectively, suggesting that the O- and E-forms have high electron affinities that cause the O→E and E→R transitions to be essentially irreversible and thus enable tightly coupled proton pumping. The water channel of the H-pathway is closed in the O- and E-forms and partially open in the R-form. These structures, together with those of the recently reported P- and F-forms, indicate that closure of the H-pathway water channel avoids back-leaking of protons for facilitating the effective proton pumping.     

Possible processes releasing nonexchangeable potassium from the rhizosphere of maize

The source and the releasing processes of nonexchangeable K from the rhizosphere were evaluated by using a 0.01 M HCl sequential extraction that enables the detection of subtle depletion of nonexchangeable K in the rhizosphere. Rhizobox experiments were conducted in which maize (Zea mays L.) plants were grown on different K sources (non-allophanic Andosol, Fluvisol, biotite and orthoclase) for 17 days. Nonexchangeable K decreased significantly in the rhizosphere of Andosol, Fluvisol and biotite, but not of orthoclase. The width of depletion was about 0–1 mm from the root-accumulating compartment regardless of the K sources, and was much less than that of exchangeable (5–10 mm) and water-soluble (50 mm) K. Rhizosphere pHs were above 4.5 in any treatment. These results suggested that the main source of nonexchangeable K for maize was interlayer K in 2:1 type phyllosilicate, and that the releasing process involved was cation exchange of the K rather than mineral dissolution by protons. For the exchange of interlayer K, a decrease of solution K⁺ below a certain threshold is known as a prerequisite. But the concentration of solution K⁺ at the root compartment of Andosol or Fluvisol, estimated to be more than 100 M, was relatively higher than the known thresholds. Moreover, the significant release of nonexchangeable K from biotite occurred only at the root compartment where marked depletion of solution K⁺ was not observed. We therefore suggest that the release of interlayer K from the rhizosphere can occur even without a marked depletion of solution K⁺ through the following processes; (1) accumulation of cations such as Ca²⁺, Mg²⁺ or Na⁺ in the rhizosphere, (2) their adsorption on 2:1 type clay minerals near the edge of interlayer but inaccessible and nonexchangeable by NH₄ ⁺, (3) concomitant removal of the NH₄ ⁺-nonexchangable K even above the known thresholds of solution K⁺, which is followed by the expansion of the interlayer space, and (4) further removal of the deeper K by repetition of the above processes.     

On the kinetic distinction of ordered and random bireactant enzyme systems.

The molecular weights of alkylated small subunits of ribulose-1,5-bisphosphate carboxylase of pea and spinach were determined from gel filtration data in the presence of 6M guanidinium chloride as 12,800 and 13,500, respectively. In the presence of 0.1 M sodium phosphate (pH 12.0) these molecules chromatograph at the same position as chymotrypsinogen (M=25,700) on Sephadex G-75. The intrinsic viscosity of the small subunit of spinach ribulose bisphosphate carboxylase, measured in this solvent, was [η] = 30 cm³/gm, while the intrinsic viscosity of chymotrypsinogen measured in this solvent was [η] = 2.4 cm³/gm. These data rule out a globular dimeric model for the sructure of the small subunit in 0.1 M sodium phosphate (pH 12.0).     

Raman scattering of chymotrypsinogen A, ribonuclease, and ovalbumin in the aqueous solution and solid state

The Raman spectra of three globular proteins, beef pancreas chymotrypsinogen A, beef pancreas ribonuclease, and hen egg white ovalbumin have been obtained in the solid state and aqueous solution. X-ray diffraction and circular dichroism evidence have indicated that these proteins have a low α-helical content and a large fraction of the residues in the unordered and β-sheet conformation. The frequencies and intensities of the amide I and amide III lines are consistent with assignments based on the Raman spectra of polypeptides. The intense amide III lines observed in all the spectra would be expected for proteins with a low fraction of the residues in the α-helical conformation. Several spectra changes occur upon dissolution of the proteins in water and may be associated with further hydration of the proteins. The spectrum of thermally denatured chymotrypsinogen is presented. A 3 cm^–1 decrease in the frequency of the amide I line of the protein dissolved in D₂O upon heating was observed. This observation is consistent with a denaturation mechanism allowing only slight changes in the secondary structure but an increase in solvent penetration upon going from the native to the reversibly denatured state.     

Effect of hydration upon the thermal stability of tropocollagen and its dependence on the presence of neutral salts

The endotherm enthalpy changes ΔH_D and temperatures T_D of thermal denaturation of tropocollagen fibers were measured by DSC calorimetry as functions of water content. The denaturation temperatures decrease with increasing water content. The enthalpy change values increase sharply in the range 0–28% of water content, where a maximum of 14.3 cal g^?1 is reached. The effect of water uptake on the enthalpy term is explained by water bridge formation within the collagen triple helix. Evidence is given for the existence of approximately three intercatenary water bridges per triplet at the enthalpy maximum, their H-bond energy amounting to approximately 4000 kcal/mol of protein. In the 30–60% range of water content, ΔH_D decreases by 2 cal^?1 probably due to interactions between secondary water structures and the stabilizing intrahelical water bonds. The influence of two neutral potassium salts, with a structure-stabilizing and a structure-breaking anion (F^? and I^?), on the hydration dependence of ΔH_D and T_D was also studied. It was shown that the primary hydration is not influenced by these ions, but that T_D and ΔH_D are altered in an ion specific way in the presence of interface and bulk water. Hydrophobic interactions do not explain the experimental results. A reaction mechanism of the effects of ions upon the structural stability of collagen is proposed and discussed in terms of interactions of the medium water molecules with the intrahelical water bonds, and in terms of proton-donor/proton-acceptor equilibria between peptide groups, hydrated ions, and intrahelical water molecules.     

Interrelations between sulfate-reducing and methane-producing bacteria in bottom deposits of a fresh-water lake. III. Experiments with 14C-labeled substrates

An ecological substrate relationship between sulfate-reducing and methane-producing bacteria in mud of Lake Vechten has been studied in experiments using ¹⁴C-labeled acetate and lactate as substrates. Fluoroacetate strongly inhibited the formation of ¹⁴CO₂ from [U-¹⁴C]-acetate and β-fluorolactate gave an inhibition of similar magnitude of the breakdown of [U-¹⁴C]-l-lactate to ¹⁴CO₂ thus confirming earlier results on the specific action of these inhibitors. The turnover-rate constant of l-lactate was 2.37 hr^-1 and the average l-lactate pool size was 12.2 μg per gram of wet mud, giving a turnover rate of 28.9 μg of lactate/gram of mud per hr. The turnover-rate constant of acetate was 0.35 hr^-1 and the average pool size was 5.7 μg per gram of wet mud, giving a rate of disappearance of 1.99 μg of acetate/gram of mud per hr. Estimations of the acetate turnover rate based upon the formation of ¹⁴CO₂ from [U-¹⁴C]-acetate or [1-¹⁴C]-acetate yielded figures of the same magnitude (range 0.45 to 1.74). These and other results suggest that only a portion of the lactate dissimilated is turned over through the acetate pool. The ratio of ¹⁴CO₂/¹⁴CH₄ produced from [U-¹⁴C]-acetate by mud was 1.32; indicating that 0.862 moles of CH₄ and 1.138 moles of CO₂ are formed per mole of acetate. From the rate of disappearance of acetate (0.027 μmoles/gram wet mud per hr) and the rate of methane production (0.034 μmoles/gram wet mud per hr), it may be concluded that acetate is an important precursor of methanogenesis in mud (approximately 70%). A substrate relationship between the two groups of bacteria is likely since ¹⁴CH₄ was formed from [U-¹⁴C]-l-lactate.     

Aerial and Aquatic Respiration in the River Crab Potamonautes Warreni Calman with Notes on Gill Structure

Summary

The oxygen consumption rate (?O₂) for Potamonauteus warreni Calman (= Potamon warreni (Calman) kept in 25 °C water was 34,4 μmol 1^?1 O₂ kg^?1 and after 72 hours in 98% R.H. air the rate was 31,9 μmol 1^?1 O₂ kg^?1 min^?1. The ?O₂ values for each of the two groups are not significantly different (P > 0,05). The partial oxygen tension of pre-branchial (v = venous) haemolymph (P_vCO₂) is 15,3 mm Hg in water and 13,0 mm Hg in air); partial carbon dioxide tension of pre-branchial (v) haemolymph (P_vCO₂) is 13,2 mm Hg in water and 13,0 mm Hg in air); the total carbon dioxide concentration in pre-branchial (v) haemolymph (C_vCO₂) tot. is 12,3 mmol 1^?1 in air and 13,9 mmol 1^?1 in water) are not significantly different for the two groups (P > 0,05). The haemolymph pH and the lactate concentration for crabs in water was found to be 7,51 and 0,38 mmol 1^?1 respectively. No significant differences were found in pre-branchial haemolymph oxygen tension, carbon dioxide tension, total carbon dioxide content, haemolymph pH, lactate level, chloride concentration, P₅₀ and haemocyanin-oxygen cooperativity in control crabs kept in water, and experimental crabs held in air for 72 hours. The chloride concentration, (327,0 mmol 1^?1) for crabs kept in water does not differ from that of crabs held in air for 72 hours but is at least 15% higher than the sodium concentration (255 mmol 1^?1) for crabs kept in water. The gill surface area is 520 mm² g^?1 wet body mass; on average 9,2 gill platelets (lamellae) can be found on a gill length of one millimetre. Each lamella is spaced 60–70 μm apart, each with a thickness of 30–40 μm. It is concluded that P. warreni may be described as a truly amphibious fresh-water crab.     

Specific platinum chelation by the guanines of the deoxyhexanucleotide d(T-G-G-C-C-A) upon reaction with cis-[Pt(NH3)2(H2O)2](NO3)2

The stoichiometric reaction between d-TpGpGpCpCpA (d(T-G-G-C-C-A)) and $\text{cis}$-[Pt(NH₃)₂(H₂O)₂](NO₃)₂ (8.4 × 10^?6 to 1.3 × 10^?4M in water at pH 5.5–6) gives a single complex. High pressure gel permeation chromatography and pH-dependent ¹H NMR analyses of the nonexchangeable base protons, show that it is a platinum chelate with the $\text{cis}$-Pt^II(NH₃)₂ moiety bound to the two N7 atoms of the adjacent guanines. A 3 × 10^?3M reaction gives the same platinum chelate, via the formation of intermediate complexes, together with unsoluble adducts.     

An NMR Study of the Conformations of N-terminal Substance P Fragments and Antagonists

Abstract

Three N-terminal fragments of the neurotransmitter Substance P as well as two antagonist heptapeptides containing D-amino-acid residues were studied using different ID and 2D NMR techniques. Total nonexchangeable ¹H-NMR assignments were carried out in D₂O and the NH protons were assigned in H₂O by means of COSY experiments. The spectral data indicates that there are no preferred conformations for the backbone. The N-terminal tetrapeptide SP_1–4-OH exists as a mixture of cis/trans isomers and this effect was studied as a function of pH.     

An X-ray diffraction study of poly-L-arginine hydrochloride

An x-ray study has been made of polyarginine hydrochloride to investigate whether, like polylysine hydrochloride, it can undergo conformational changes merely from variations in the degree of hydration. X-ray powder and fiber photographs of specimens containing up to about five molecules of water per arginine residue show features characteristic of α-helical structures including a 5.4-Å layer line and a meridional 1.5-Å reflection. Increasing the water content from ¹/₂ to 6¹/₂ molecules per residue causes the a axis of the hexagonal unit cell to increase from 14.4 Å to 15.8 Å, with no appreciable change in the 27.0 Å c axis. Removal of the last half molecule of water results in a very diffuse α pattern, but on rehydration the sharp pattern reappears. Specimens containing five to twenty water molecules per residue show quite a different pattern, the spacing of which do not vary appreciably with hydration. This pattern includes a meridional 3.4-Å reflection, a feature commonly shown by β structures, and indeed all the reflections can be satisfactorily indexed in terms of a monoclinic unit cell with a = 9.26 Å, b = 22.05 Å, c = 6.76 Å, and γ = 108.9°. These dimensions are shown by models to be compatible with a β pleated-sheet structure.