Microsequence analysis of peptides and proteins: trimethylsilylisothiocyanate as a reagent for COOH-terminal sequence analysis

A reinvestigation of the isothiocyanate-based chemistry for cyclic degradations of peptides and proteins revealed that the reagent trimethylsilylisothiocyanate (TMS-ITC) gives superior results in terms of coupling efficiency and lack of complicating side reactions. Acetic anhydride (10 min at various temperatures) was used to activate the carboxyl terminus, and 6 N HCl (30 min at room temperature) was used for cleavage as originally described by G.R. Stark (Biochemistry 8, 4735, 1968). Reaction conditions for efficient coupling were explored using subtractive chemistry on bradykinin, a nonapeptide, and separation of the reaction products by reverse-phase high-performance liquid chromatography. The products were analyzed by fast atom bombardment-mass spectrometry and shown to be the N-acetylated starting material and the N-acetylated des-Arg9 derivative of bradykinin. The pseudo-first-order rate constants measured at 50, 70, and 90 degrees C were 5.6 X 10(-5), 5.1 X 10(-4), and 8.6 X 10(-4) s-1, respectively. In order to obtain complete couplings within 30-40 min at 50 degrees C, the effect of pyridine catalysis was studied. The addition of 0.225 M pyridine resulted in roughly doubling the rates at 50 and 70 degrees C. In the case of bradykinin, the reaction with TMS-ITC in the presence of the pyridine catalyst at 50 degrees C was complete in 15 min. In order to apply this methodology to the analysis of proteins, the thiohydantoin derivatives of amino acids were synthesized and separated by reverse-phase HPLC. The derivatives were also characterized by mass spectrometry. The above reaction conditions were tested on 3 nmol of sperm whale apomyoglobin for three cycles of degradation. The sample was first coupled to p-phenylene diisothiocyanate-derivatized aminopropyl glass with a 90% yield. The approximate initial yield of glycine at cycle one was 30%. The first three cycles corresponded exactly to the predicted carboxy-terminal sequence of myoglobin. These results demonstrate the feasibility of a new Stark reagent for automated carboxy-terminal chemistry.     

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

The use of micropreparative electrophoresis of protein/peptide isolations for primary structure determinations

High-performance electrophoresis chromatography (HPEC) is a recent development that features continuous-elution gel electrophoresis for isolating proteins or peptides in range of 1 to 300 microgram quantities. Column gel electrophoresis is conducted under thermostated conditions, and the field voltage can be varied within a run with a programmable power supply. Applications of this apparatus in protein purification are presented to demonstrate the utility of the (Model 230A) HPEC. These examples include on-line detection with direct analyte recovery of highly purified sample, which mimics high-performance liquid chromatography, for subsequent structure-function characterization. A method to remove salts from sodium dodecyl sulfate electrophoresed samples for subsequent sequencing or amino acid analysis is described. This desalting procedure recovers from 90%-95% of the sample and employs a low molecular weight cut-off membrane during sample centrifugation onto a polyvinylidene difluoride membrane. Subsequent washings are performed to efficiently remove salts, free amino acids and detergents that are known to interfere with sequence analysis. Sequence information such as initial recovery, repetitive yields and chromatogram comparisons are presented to demonstrate the utility of this procedure when used following isolation of sample with HPEC.     

Isolation and primary structure of the califins, three biologically active egg-laying hormone-like peptides from the atrial gland of Aplysia californica

The atrial gland of the marine mollusk Aplysia californica contains several biologically active peptides that are thought to be important in reproductive function. In the present study, three novel peptides, which we named califin A, B, and C, were purified from extracts of atrial glands by high performance liquid chromatography, and their primary structures were determined. Each consists of a 36-residue subunit bound by a single disulfide bond to an 18-residue subunit. The large subunits differ from each other by one or two residues, whereas the small subunits are identical. The large subunits are 78-83% homologous to egg-laying hormone (ELH), a 36-residue peptide synthesized by the neuroendocrine bag cells of Aplysia. Like ELH, the califins excite LB and LC cells of the abdominal ganglion and cause egg laying when injected into sexually mature animals. Based on previously described DNA sequence data, each califin is likely to be derived from one of several precursor proteins that are encoded by members of the ELH gene family. Califin A is encoded on the peptide A precursor, and califin B may be encoded on the peptide B precursor. No gene encoding califin C has been sequenced. Because peptides A and B are also biologically active, the precursors encoding them and califins A and B are polyproteins. The possible role of atrial gland peptides as pheromones is discussed.     

Microsequence analysis of peptides and proteins. V. Design and performance of a novel gas-liquid-solid phase instrument

We describe the construction and performance of a novel, automated, Edman chemistry-based microsequencer. The reagent and solvent delivery system, the reaction cartridge for coupling and cleavage, and the conversion flask are all constructed from chemically inert perfluoroelastomers. The delivery valves are of a new design incorporating the use of electromagnetically actuated solenoids and zero-dead-volume construction, and may be connected in a modular fashion resulting in multiple inputs with a single output line which can be flushed with inert gas. The bottle closures are of a new design based on an all-Teflon compression fitting. The reaction cartridge and conversion flask are thermostated by solid-state heaters in an aluminum block. The overall size of the instrument is 25 X 34 X 14 in. The chemistry utilizes 2% aqueous triethylamine as the coupling base which is delivered to the reaction cartridge via a stream of nitrogen. The "gas-phase" delivery of the coupling base and the cleavage acid (trifluoroacetic acid) is modeled after the method described by R. M. Hewick et al. (J. Biol. Chem. 256, 7990-7997,1981). The instrument has performed well over a period of 3 years in terms of low background peaks, sensitivity in the picomole range, and reliability of operation. The use of economical components, ease of construction and operation, and sensitive analytical capability make this instrument a useful tool for microsequence analysis of peptides and proteins.     

The role of pore structures in the selective permeability of antennal sensilla of the desert burrowing cockroach, Arenivaga sp

To obtain information about the chemical composition of pore structures in antennal sensilla, the antennae were exposed to lipid solvents, or they were prepared to show negative-contrast images in electron micrographs. A heavy-metal tracer, lanthanum nitrate, was also used to indicate the permeability of the receptors to water. The grooves of the large grooved peg open into tubular cavities containing electronopaque material, through which stimulatory molecules must pass to reach the sensory dendrites at the center of the sensillum. The material in these cavities was removed by chloroform or acetone, suggesting a lipid composition. Lanthanum penetrated this receptor only after it had been exposed to acetone or chloroform. Strands at pores of the thin-walled pegs were also removed by the lipid solvents, and the water-soluble tracer failed to penetrate these receptors unless they had been previously exposed to chloroform or acetone. The pore structures appear to be hydrophobic, allowing entry of lipid-soluble substances, while preventing passage of water. The differential action of the solvents on the various types of sensilla suggests that receptor discrimination among different classes of chemical stimuli may be partially determined by the chemical properties of structures at the sensillar pores.     

Isolation and characterization of rat vasoactive intestinal peptide

We have used gel filtration, ion exchange chromatography, affinity chromatography and reversed-phase HPLC to isolate vasoactive intestinal peptide from rat intestine. Microsequence analysis of 1 nmole peptide indicated that the sequence was identical to the porcine octacosapeptide VIP. In radioimmunoassay with four antisera and in the turkey pancreas bioassay, rat VIP was equipotent with highly purified preparations of porcine, human and canine VIP. A less basic rat VIP-variant was also isolated and the N-terminal decapeptide region that was sequenced was identical with that of porcine VIP.     

Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.     

Desaturation of Oleic and Linoleic Acids by Leaves of Dark- and Light-grown Maize Seedlings

Oleate and linoleate desaturation in leaves of maize seedlings was largely independent of previous light treatment of the seedlings; there was no evidence of light-induced desaturase activities. These results are in sharp contrast to those observed with developing cucumber cotyledons in which pronounced increase in desaturation occurs after exposure of tissue to light. The rates of desaturation of oleate were about four times those of linoleate in both etiolated and 16-hour greened maize leaves. In both etiolated and greened tissues, about two-thirds of the label from oleate was esterified after 4 hours, half of which was in phosphatidylcholine. Phosphatidylcholine and diglyceride contained large proportions of [¹⁴C]linoleate formed from [¹⁴C]oleate but not [¹⁴C]linolenate. In monogalactolipid, about two-thirds of the labeled fatty acids were linolenate. In vivo desaturase activity was present in tissue of widely different levels of differentiation and chlorophyll content obtained from light-grown maize seedlings.