A two-step enzymatic synthesis of dipeptides

A simple system is introduced to produce dipeptides continuously by enzyme catalyzed condensation of amino acid esters and amino acid amides. Synthesis of N-terminal free dipeptide-amides is achieved by means of carboxypeptidase Y. The peptide-amide is deamidated utilizing a newly isolated peptide-amide is deamidated utilizing a newly isolated peptide-amidase. Separation of substrates and products is accomplished by anion-exchange chromatography. Modeling of the reactions shows that the two reactions have to be carried out in a cascade of two reactors in order to prevent hydrolysis of the peptide by the carboxypeptidase. Continuous production of Kyotorphin (H-TyrArg-OH) with a space-time yield of 257 g/L . d shows the feasibility of this concept.     

Inositol tetrakisphosphates as second messengers induce Ca(++)-dependent chloride currents in Xenopus laevis oocytes.

Microinjection of inositol 1,3,4,5-tetrakisphosphate or inositol 1,4,5-trisphosphate induced distinct chloride membrane currents in defolliculated Xenopus laevis oocytes. To decide whether these Cl(-)-currents were due to the injected compounds or their metabolic products, [3H]Ins(1,3,4,5)P4 or [3H]Ins(1,4,5)P3 were injected into oocytes and their metabolites were analyzed by HPLC. Our results indicate that Ins(1,3,4,5)P4 itself or its metabolite Ins(1,3,4,6)P4 is able to induce Cl(-)-membrane currents, most likely by increasing the cytosolic Ca(++)-concentration.     

A combined study of aggregation, membrane affinity and pore activity of natural and modified melittin

The pore activity of melittin and several chemically modified derivatives has been investigated using conductance measurements on planar lipid bilayers and marker release from small unilamellar vesicles. The modifications included N-terminal formylation, acetylation, succinylation and modification of the tryptophan residue. All of the compounds showed bilayer permeabilizing properties, though quantitative differences were evident. These comprised changes in the voltage dependence of the conductance, in the single-pore kinetics, in the concentration of aqueous peptide required to induce a given pore activity and in the apparent 'molecularity' reflected by the power law of its concentration dependence. A strong tendency for disrupting bilayers was not always correlated with strong pore activity. For a better understanding of these results, measurements of pore activity were complemented by studying the aggregation behavior in solution and the water-membrane partition equilibrium. Modifications of charged residues gave rise to significant changes in the aggregation properties, had virtually no influence on the partition coefficient. The latter decreased strongly, however, as a result of tryptophan modification. Analysis of the isotherms was consistent with the assumption that the arginine residues in melittin do not contribute very much to charge accumulation at the immediate membrane/water interface.     

Multiple shoot production from seedling explants of slash pine (Pinus elliottii,Engelm.)

Hypocotylary explants obtained from 30- to 40-day-old slash pine (Pinus elliottii, Engelm.) seedlings treated with 6-benzylaminopurine produced multiple buds that eventually elongated into axillary shoots. The explants were pulse treated (45-s dip) with 6-benzylaminopurine (22.2, 111, 222 M) plus a control and cultured on three different basal media containing activated charcoal (0.5% w/v). Hormonal concentration and basal medium were compared for the number and size of axillary shoots induced after 12 and 29 days. The greatest number of axillary shoots was produced by explants that were pulse treated with 111 M 6-benzylaminopurine and cultured on Gresshoff and Doy medium. The axillary shoots were fewer in number per explant than shoots previously reported resulting from hormonally induced advantitious buds of slash pine, but the axillary shoots developed more rapidly.Abbreviations BA 6-benzylaminopurine - DMSO dimethyl sulfoxide - PAR photosynthetically active radiation     

Dielectric probe of the electric-field-sensitive peptide alamethicin

Dielectric constant and loss of alamethicin in solvents of various degrees of lipophilicity (namely mixtures of n-octanol and dioxane) have been measured at frequencies from 5 kHz to 50 MHz. By means of a conventional Cole-Cole approach, dielectric properties were evaluated to obtain information about the structural and dipolar properties of the peptide in view of its function as a voltage-dependent pore former in membranes. The results for a pure octanol solvent (together with an apparent molecular weight determined by ultracentrifugation) indicate the existence of primarily monomeric particles of quite elongated shape and of comparatively high dipole moment. Adding dioxane was found to yield considerable aggregation and a decrease of polarity.     

The α-keto acids of branched-chain amino acids: Simplified derivatization for physiological samples and complete separation as quinoxalinols by packed column gas chromatography

By use of a relatively new mixed stationary phase, complete separation of the branched-chain α-keto acids as O-trimethylsilyl-quinoxalinol derivatives is achieved within 10 min by packed column gas chromatography. Precise quantification of less than 5 nmol of α-keto acids in biological samples is possible. In small aqueous samples the α-keto acids are directly derivatized without prior purification. Plasma need only be deproteinized by perchlorate and neutralized before derivatization. Average relative precision for determination of the three main branched-chain α-keto acids is ± 5.8%.     

Some general aspects regarding the interpretation of binding data by means of a Scatchard plot

The question is quantitatively examined which general information can be obtained from experimental data of ligand binding to macromolecules if represented by a Scatchard plot. In particular, the curvature as well as the intercepts and slopes at both coordinate axes are analyzed assuming rather general conditions including cooperative behavior (with applications to some simplified cases of interest in practice). The results should provide a basis for attempts to elucidate the binding mechanism of a system encountered in experimental work.     

Studies on the interaction of cholesterol with diester- and dietherlecithin

The lamellar repeat distances of aqueous dispersions of rac-1,2-dioctadec-9′-cis-enyl-glycero-3-phosphorylcholine (dietherlecithin) and 1,2-dioctadec-9′-cis-enoyl-sn-glycero-3-phosphorylcholine (diesterlecithin) have been measured by X-ray diffraction as a function of water concentration. The point of maximum hydration was found to be 43% (w/w) and 40% (w/w) for dietherlecithin and diesterlecithin respectively; the corresponding lamellar repeat distances being 62.3 Å and 60.5 A. Incorporation of cholesterol above maximum hydration results in the initial increase in the lamellar repeat distance with a maximum around cholesterol concentrations of 25 and 33 mol % for dietherlecithin and die diesterlecithin respectively.The apparent partial specific volumes of the two lecithins and for lecithin-cholesterol mixtures in sonicated aqueous dispersions were measured. Values of 1.024 cm³ · g^?1 and 0.987 cm³ · g^?1 were obtained for diether- and diesterlecithin, respectively, at 20°C. Diesterlecithin-cholesterol mixtures showed a very small change in partial specific volume while mixtures of dietherlecithin-cholesterol showed a very marked decrease with increasing proportions of cholesterol.From these data a series of structure parameters are derived for the two lecithins and possible implications for the nature of the lecithin-cholesterol interaction are discussed.     

Heterotrophic Activity of Deep-Sea Sediment Bacteria

Sediment samples, containing mixed microbial populations that were decompressed during retrieval from 7,750 and 8,130 m in the Puerto Rican Trench, were recompressed and incubated at the approximate in situ temperature (3 C) and pressure (775 or 815 atm) in the presence of 14C-labeled amino acids. Heterotrophic activity (total uptake, CO2 respiration, and cellular assimilation) and cellular-associated "pool" concentrations were measured. Compared with atmospheric controls held at 3 C, the total uptake at elevated pressure at 3 C was reduced, on an average, 55 times, CO2 respiration was reduced 45 times, and cellular assimilation was reduced 69 times. Rate of total uptake at elevated pressure was found to range from 4.0 X 10(-11) mug/cell per h for leucine to 2.61 X 10(-10) mug/cell per h for an amino acid mixture. Also, the percentage of total uptake at elevated pressures, respired as CO2, increased at the expense of cellular assimilation (ca. 22% increase). Two cellular-associated amino acid pools were detected, a large, loosely bound, outer pool and a small, tightly bound internal pool. The loosely bound outer pool was removed by a change in the pH of the incubation medium. Even though heterotrophic uptake and the outer, cellular-associated pool were markedly reduced at an elevated pressure, the percentage of total uptake calculated for the unincorporated, tightly bound, intracellular pool was 2 to 19 times that obtained for cultures held at 1 atm. The results were interpreted as indicating that bacterial metabolism and biosynthesis in the deep sea are markedly reduced, with a greater proportion of metabolic activity devoted to cellular maintenance.