Delbert D. Mueller

Tyrosine metabolism for cuticle tanning in the tobacco hornworm, Manduca sexta (L.) and other lepidoptera: Identification of β-d-glucopyranosyl-O-l-tyrosine and other metabolites

Abstract When [ 14 C ]tyrosine and [ 14 C ]glucose were fed or injected into feeding fifth-instar larvae of the tobacco hornworm, Manduca sexta (L.), they were incorporated into a conjugate identified in hemolymph and carcass extracts as β- d -glucopyranosyl-O- l -tyrosine. In wandering larvae and pupae, the conjugate was hydrolyzed, and tyrosine was hydroxylated and decarboxylated to dihydroxyphenylalanine and 2-(dihydroxyphenyl)ethylamine. None of these metabolites were formed in fourth-instar larvae or in adults. [ 14 C ]Phenylalanine was hydroxylated to tyrosine in all stages of insect development. β- d -Glucopyranosyl-O- l -tyrosine was also detected in 18 other species of Lepidoptera but not in species from other insect orders. This conjugate appears to be the major tyrosine storage metabolite for production of tanning diphenol substrates in Lepidoptera.

β-d-Glucopyranosyl-O-l-tyrosine: Synthesis, properties and titre during insect development

β-d-Glucopyranosyl-O-l-tyrosine (tyrosine glucoside) was synthesized by acid-catalyzed fusion of β-glucopyranose pentaacetate with N-carbobenzoxy-l-tyrosine methyl ester and subsequent deblocking, transesterification and saponification. The [13C]-NMR spectrum was identical to the natural product isolated from the tobacco hornworm, Manduca sexta L. This conjugate was detected by HPLC in whole body extracts of the Indian meal moth, Plodia interpunctella (Hubner), almond moth, Ephestia cautella (Walker) and bagworm, Thyridopteryx ephemeraeformis (Haworth). Highest levels were found in mature larvae and pharate pupae. Tyrosine glucoside serves as a primary source of tyrosine and glucose for cuticle formation in Lepidoptera undergoing metamorphosis.

Comparisons of diisopropylfluorophosphate derivatives of chymotrypsin and chymotrypsinogen by phosphorus-31 nuclear magnetic resonance.

Abstract The nature of the differences in the active sites of α-chymotrypsin and chymotrypsinogen has been investigated by phosphorus-31 NMR studies of their diisopropylfluorophosphate derivatives. The phosphorus-31 resonance of the modified zymogen occurs 2 ppm upfield from that for the enzyme. An even greater separation is seen between diisopropylphosphoryl-neo-chymotrypsinogen and -α-chymotrypsin. A plausible interpretation of the chemical shift differences is based on the known structures for α-chymotrypsin, chymotrypsinogen and diisopropylphosphoryl-trypsin.

Spin lattice relaxation in an AM{Xn} system: Application to 13C relaxation in enriched molecules

In 13C‐enriched molecules, 13C–13C dipolar interactions can contribute significantly to the relaxation of 13C nuclei which are not directly bonded to protons. Comparison of the relaxation behavior of the 13C–13C multiplet lines with that of the singlet for which there are no 13C–13C dipolar interactions allows a quantitative analysis of this interaction. However, since 13C–13C dipolar interactions are necessarily weak, substantial contributions to the relaxation of nonprotonated carbon atoms are made by protons attached to adjacent atoms. The general features of the spin lattice relaxation behavior of an AM{X} system in which {X} represents the decoupled protons, A, the nonprotonated carbon, and M, the adjacent protonated carbon, have been derived. In general, the A relaxation will be nonexponential and dependent on the initial state of the Mz magnetization. A particularly useful application of these measurements on 13C‐enriched systems is found in their sensitivity to internal and/or anisotropic motions....

Defensive chemistry of the flour beetleTribolium brevicornis (LeC): : Presence of known and potential prostaglandin synthetase inhibitors.

The defensive secretion ofTribolium brevicornis contains 12 organic components, including quinones, hydroquinones, hydrocarbons, aromatic ketones, and aromatic esters. The two ketones, 2′-hydroxy-4′-methoxyacetophenone and 2′-hydroxy-4′-methoxypropiophenone, and the two esters, methyl 2,5-dihydroxy-6-methylbenzoate and methyl 2,5-dihydroxy-6-ethylbenzoate, represent ca. 0.25% of the biomass of the beetles. Mass spectral and NMR analyses were used to elucidate the structures of all components. The ketones are potent prostaglandin synthetase inhibitors (PSI), and the esters are suspected to be PSI.

Effects of net charge on the hydrogen-deuterium exchange parameters of lysozyme☆☆☆

Abstract Possible effects of changes in net charge on protein hydrogen exchange rates were investigated by desalting hen egg-white lysozyme, which allowed its net charge to increase with decreasing pH in the acid region. Chloride ion-binding ratios, expressed as ratios of free to total Cl−, were measured with a chloride-specific electrode at pH 5 on a 2.4% solution of a five-time-desalted product. This ratio was used to show a 97% reduction of the 11% Cl− present in a commercial lysozyme preparation upon three passes of the enzyme through a column of ion-retardation resin. Net charges on the purified product were assigned from a combination of electrophoretic mobility and proton titration data gathered under minimal ionic strength conditions. The net charge on the desalted product increased by 1.64 units between pH 5.0 and 3.0. Hydrogendeuterium exchange studies on the purified lysozyme in D2O were obtained using the near-infrared region of a Cary 14R spectrophotometer. The rate-pD profile for k2, the rate constant for the intermediate class of exchanging hydrogens, showed a decrease in the apparent pD of minimum exchange rate of 0.3 units, when compared to that obtained earlier in 0.2 m added NaCl. However, the rate of exchange at pD minimum and the number of hydrogens in the class remained largely unaffected. A similar shift was observed for the rate-pD profile of the class 1 hydrogens. Thus, the effect of an increase in net positive charge is to shift the rate-pD profile to a lower pD. Moreover, the effect extended to the interior peptide hydrogens of this globular protein. Consequently, the exchange rates of all the observable hydrogens are altered by the net charge changes, and the effect appeared uniform. The shift can be accounted for quantitatively by applying electrostatic interaction terms to the acid and base catalytic constants characterizing the exchange process. The calculated electrostatic interaction factors in minimal salt and 0.2 m added NaCl were found to be 29 and 18% lower, respectively, than those obtained theoretically. Therefore, under conditions where changes in net charge may occur for a globular protein, the effect on hydrogen exchange rates can be estimated fairly well theoretically, especially at moderate ionic strengths.

Identification of catecholamine β-glucosides in the hemolymph of the tobacco hornworm, Manduca sexta (L.), during development

Abstract Catecholamine conjugates were isolated from the hemolymph of larval, pupal and adult stages of the tobacco hornworm, Manduca sexta, and their identities were determined by synthesis from 14C-labeled precursors, HPLC analysis, 1H-NMR and FAB-mass spectrometry, and GLC analysis of the sugar moieties. β-Glucosides of N-acetyldopamine (NADA) and N-β-alanyldopamine (NBAD) were the primary conjugates identified and their relative abundance depended on the stage of development. NADA glucosides were the predominant conjugates in hemolymph of pharate or newly ecdysed fifth stadium larvae with 86% as the 3-O-glucoside and 14% as the 4-O-glucoside. Low concentrations of dopamine (DA) 3-O-glucoside and NBAD 3-O-glucoside also were found. In pharate or newly ecdysed pupal hemolymph, NBAD 3-O-glucoside was the major conjugate (98%) along with small amounts of NBAD 4-O-glucoside, DA 3-O-glucoside and NADA 3-O-glucoside. In hemolymph of pharate or newly ecdysed adults, NADA 4-O-glucoside (67%) was more abundant than NADA 3-O-glucoside. Only traces of NBAD 3-O-glucoside were found. Therefore, a preponderance of catecholamine 3-O-glucoside occurred in larval and pupal hemolymph as storage precursors for cuticle sclerotization agents, whereas the 4-O-glucoside of NADA predominated in adult hemolymph. The role of catecholamine glucosides in hemolymph in relation to cuticle sclerotization is discussed.

Phosphorus-31 nuclear magnetic resonance of insect hemolymph sera.

Abstract Sera from larval and pupal stages of the tobacco hornworn, Manduca sexta, have been investigated using phosphorus-31 pulsed Fourier transform nuclear magnetic resonance. Spectra of larval and pupal sera containing 5 m m EDTA were characterized by four major peaks and one or more minor resonances. A phosphorus-31 spectrum of dialyzed larval serum showed several weak signals which indicated the presence of some higher-molecular-weight phosphorylated compounds as well. None of those signals, however, corresponded to any of the ones seen with undialyzed sera. Three of the four prominent peaks and one minor peak in the whole larval serum had the same chemical shifts as those in the pupal samples. The pupal sera, in addition, displayed an extra peak well upfield from those of the larval stage. All of the low-molecular-weight resonances detectable in the hemolymphs have been identified and included four compounds not previously reported; trehalose-6-phosphate, phosphoarginine, phosphatidylcholine, and phosphatidylethanol-amine. The phosphometabolites found at millimolar or higher concentrations in larval hemolymph were α-glycerolphosphate, phosphorylcholine, phosphorylethanolamine, inorganic phosphate, trehalose-6-phosphate, phosphatidylcholine, and phosphatidylethanolamine. All of the above compounds were found in pupal sera as well except for the addition of phosphoarginine and the deletion of phosphorylethanolamine. The levels of the phosphometabolites in common between the two stages of development, however, were quite different as were their stabilities after extraction. While the intensities of the larval phosphates remained virtually constant in the presence of EDTA at pH 7.8, those of the pupal sera changed rapidly. This was especially true for arginine phosphate which disappeared quickly.

Ribulose-1, 5-bisphosphate carboxylase from comfrey: Isolation and characterization

Abstract Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified to electrophoretic homogeneity from comfrey, Symphytum spp. Sodium dodecyl sulfate polyacrylamide and polyacrylamide gel electrophoresis studies on the purified product showed no extraneous proteins. Comparisons of the electrophoretic mobilities of the subunits to those of standard proteins indicated a large subunit MW of 50 000 and a small subunit of 12 700, which for an octameric structure of each subunit indicates a native MW of 502 000. Specific activities of the comfrey enzyme ranged from 1.2 to nearly 2 μmol 14 CO 2 fixed/min.mg of protein over several preparations and were maintained for months when stored from the sucrose gradient at − 70°. The specific activities depended critically on the amounts of enzyme used in the assay even under saturating conditions of substrates and cofactors. The effective pH dependence for carboxylase catalysis peaked near 7.4, which apparently is the lowest elective optimum yet reported for this enzyme from any source. However, on a constant carbon dioxide basis the pH dependence profile was reversed with a maximum near pH 8.6 which was 0.4 units higher than the value for the spinach enzyme. The K m s for carbon dioxide and ribulose-1,5-bisphosphate at pH 7.5 were 130 μM and 30 μM, respectively, which are comparable to the accepted values for the carboxylase from spinach at pH 7.2.

Hydrogen-deuterium exchange of a primary amide as a model for asparagine and glutamine exchange in proteins

Hydrogen-deuterium exchange of the primary amide, isobutyramide, was investigated as a model for asparagine and glutamine-NH2 exchange in a protein. A simple amide was chosen since the structures of several well-characterized proteins show most of these residues to be exposed to solvent. Isobutyramide-exchange data were obtained in 1:1 D2O:dioxane solutions using a near-infrared method. The rate data were strictly pseudo-first order and yielded an average of 95% exchange of the primary amide hydrogens. In analogy with secondary amides, the pD dependence of the rate constants was characteristic of specific acid and base catalysis. In addition, analysis of the rate-pD profile for isobutyramide indicated a significant uncatalyzed exchange reaction. Temperature-dependence studies of the first-order rate constants at a fixed pD yielded an apparent activation energy of 19.3 kcal/mole. Predicted half-life times for the exposed primary amide hydrogens in proteins, based on these exchange parameters, indicate that asparagine and glutamine side chains generally would contribute to the overall rate data only below 15°C and then only for approximately 1 pD unit around the point of minimum reaction velocity.