Lipase-catalyzed transamidation of non-activated amides in organic solvent

A novel biocatalytic reaction of transamidation of non-activated amides with amines is reported. Among 45 different lipolytic and proteolytic enzymes tested, only the lipase from Candida antarcticawas able to catalyze this reaction. The reaction proceeded with up to ca. 80% conversion in anhydrous methyl tert-butyl ether and worked with both N-substituted and unsubstituted amides. The biocatalytic transamidation is an equilibrium process and, therefore, higher conversions to the desired amide were achieved by using increased concentrations of the amine nucleophile.     

Biochemical and phenotypical correction of an envelope mutant of Salmonella typhimurium

Salmonella typhimurium DA 361 bears an env D1 mutation with the following abnormal phenotypical and biochemical characteristics: a) it autolyses at stationary phase in nutrient broth; b) it grows in chains of short rods; c) it is a poor maltose fermenter and d) it has a diphosphatidylglycerol (DPG) content twice as high than its isogenic non-lytic pair DA 362 (env D+) and LT2, of which both are derivatives. Growth of DA 361 in the presence of 400 mM ethanol leads on a 50% decrease of DPG level, thereby equalling its PG/DPG ratio with those of the control strain. Consequently, a correction on the other phenotypical and biochemical anomalies are induced since the DA 361 strain decreases its autolytic activity, ferments normally maltose and appear as rods undifferentiated from DA 362.     

Human skin chymotrypsin-like proteinase chymase. Subcellular localization to mast cell granules and interaction with heparin and other glycosaminoglycans

The subcellular localization of human skin chymase to mast cell granules was established by immunoelectron microscopy, and binding of chymase to the area of the dermo-epidermal junction, a basement membrane, was demonstrated immunocytochemically in cryosections incubated with purified proteinase prior to immunolabeling. Because heparin and heparan sulfate proteoglycans are major constituents of mast cell granules and basement membranes, respectively, the ability of chymase to bind to glycosaminoglycans (GAG) was investigated. Among a variety of GAGs, only binding of chymase to heparin and heparan sulfate appears physiologically significant. Binding was ionic strength-dependent, involved amino groups on the proteinase, and correlated with increasing GAG sulfate content, indicating a predominantly electrostatic association. Interaction with heparin was observed in solutions containing up to 0.5 M NaCl, and interaction with heparan sulfate was observed in solutions containing up to 0.3 M NaCl. Binding of heparin did not detectably affect catalysis of peptide substrates, but may reduce accessibility of proteinase to protein substrates. Measurements among a series of serine class proteinases indicated that heparin binding was a more common property of mast cell proteinases than proteinases stored in other secretory granules. Binding of chymase to heparin is likely to have a storage as well as a structural role within the mast cell granule, whereas binding of chymase to heparan sulfate may have physiological significance after degranulation.     

An apparatus to measure in vivo biomechanical behavior of dorsi- and plantarflexors of mouse ankle.

We developed an apparatus to quantify the biomechanical behavior of the dorsi- and plantarflexor muscles of the ankle of an anesthetized mouse. When the dorsi- or plantarflexor muscle group is activated by electrical stimulation of either the peroneal or tibial nerve, the apparatus measures the moment developed about the ankle during isometric, isovelocity shortening, or isovelocity lengthening contractions. Displacements may be performed over the full 105 degrees range of ankle motion with an angular resolution of 0.09 degrees. Bidirectional isovelocity ramps in ankle angle up to 1,100 degrees/s are possible and are equivalent to maximum velocities of 2.3 fiber lengths/s (Lf/s) for the fibers in tibialis anterior muscle and 11.9 Lf/s for those in gastrocnemius muscle. During single contractions of the dorsi- and plantarflexor muscle groups at 37 degrees C and with both knee and ankle joint at 90 degrees neutral position, the isometric tetanic force developed was 1.4 and 3.3 N, power output at 2.2 Lf/s was 3.1 and 5.9 mW, and power absorption at 0.5 Lf/s was 4.9 and 9.0 mW, respectively. These values are in reasonable agreement with data from the same muscle groups tested in situ. We conclude that the apparatus provides valid measurements of force and power of the dorsi- and plantarflexor muscle groups.     

Initiation of the extrinsic pathway of coagulation. Association of factor VIIa with a cell line expressing tissue factor

We have examined initial assembly of the extrinsic pathway of blood coagulation on cell surfaces with radiolabeled human factor VIIa and a human fetal lung cell line possessing abundant functional tissue factor activity. Binding of factor VIIa to these cells was observed and was time- and temperature-dependent. Binding of factor VIIa was quantitatively equivalent at 37 and 6 degrees C, although the kinetics of binding differed. The radiolabeled ligand bound by the cell was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel analysis from the factor VIIa offered. Factor VIIa binding was influenced by calcium ions. The binding appears to involve at least two classes of calcium-dependent binding sites. Optimal binding occurred at 2 mM calcium for both classes of sites, and there was inhibition of binding to the high affinity sites at higher calcium. Association of factor VIIa was specific, saturable, had a Kd of 123 +/- 37 pm, and factor VIIa interacted with about 100,000 binding sites per cell. Once established, specific binding was rapidly reversible. Direct cellular binding of human factor X also was observed and was calcium, time- and temperature-dependent. Factor X binding was specific and saturable with half-maximal binding at 87.6 +/- 27.4 nM to 6.03 +/- 1.03 X 10(6) sites per cell. Specific high affinity binding of factor VIIa correlated with generation of factor Xa. A direct linear relationship was observed at low factor VIIa binding; however, at higher bound factor VIIa, the relationship was nonstoichiometric, i.e. less factor Xa was formed per mole of factor VIIa. Expression of specific binding sites for factors VIIa and X provides further substantiation for the molecular assembly hypothesized to initiate the extrinsic coagulation protease cascade on cells.     

Obligatory amino acids in primitive proteins

Conformational similarity among amino acid residues, a property derived by analysing (φ, ψ)-probability distributions of 20 proteinous amino acids from 38 different globular proteins, is used to arrive at a set of six ‘obligatory’ amino acids of primitive proteins. The amino acids Ser, Val, Leu, Asp, Gly and Pro have been argued to be ‘obligatory’ and to represent, conformationally, the remaining amino acids. The reasons for consideration of these six residues as ‘obligatory’ are discussed. Methods to check the validity of our proposition are suggested.