Comparison of soluble and immobilized trypsin kinetics: Implications for peptide synthesis

The protease trypsin was immobilized to porous glass in both the presence and absence of acetylated soybean trypsin inhibitor (STI) to determine whether immobilization could alter enzyme activity in favor of aminolysis over hydrolysis. Actiive-site titration with 4-methylumbelliferylguanidinobenzoate (MUGB) showed that only about 10% of immobilized trypsin had catalytic activity. Immobilization in the presence of STI produced a higher yield of active enzyme accessible to the inhibitor but did not increase the total yield of MUGB-active immobilized enzyme. Thus, enzyme inactivation upon immobilization could not be attributed to an inaccessible enzyme orientation, nor did STI prevent inactivation by stabilizing the active-site conformation. Kinetic parameters were determined for soluble and immobilized trypsin for two esters, N-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-arginine ethyl ester (BAEE), and two amides, N-benzoyl-L-arginine p-nitroanilide (BAPNA) and N-t-boc-leucylglycylarginine p-nitroanilide (LGRNA). In all cases, immobilization caused a greater decrease in k(cat) for amidase activity than for esterase activity. The ratio [k(cat)/ K(m) (ester)]/[k(cat)/K(m) (amide)] increased slightly or stayed the same (for I.GRNA) or decreased sharply (for BAPNA). Including STI during immobilization had little effect on the active enzyme's intrinsic kinetics. A direct comparison of energy diagrams and free energies of activation for BAEE and BAPNA indicates that immobilization raises the free energy barriers for both amide and ester hydrolysis and lowers the energy barrier for aminolysis. In practice, these effects should lower the amidase activity and increase the aminolysis-hydrolysis ratio, rendering the immobilized enzyme a more efficient catalyst for peptide synthesis. (c) 1993 John Wiley & Sons, Inc.     

Use of winter wheat x Triticum tauschii backcross populations for germplasm evaluation

The wild diploid goatgrass, Triticum tauschii (Coss.) Schmal., is an important source of genes for resistance to both diseases and insects in common wheat (Triticum aestivum L.) We have evaluated grain yield, kernel weight, protein concentration, and kernel hardness of 641 BC₂ F₁-derived families from direct crosses involving four T. aestivum cultivars and 13 T. tauschii accessions over 2 years and at two Kansas, USA, locations. On average, T. tauschii germplasm depressed grain yield and increased protein concentration, whereas kernel weight was affected either positively or negatively, depending on the T. tauschii parent. Three T. tauschii parents produced a large proportion of families with very soft endosperm. Some variation among progeny of different T. tauschii parents resulted from the segregation of genes for resistance to leaf rust (caused by Puccinia recondita Rob. ex Desm.). This study confirmed that random BC₂-derived families can be used to evaluate the effects of T. tauschii genes in the field. This methodology, although laborious, can provide useful information which is not obtainable by the screening of T. tauschii accessions themselves.Joint contribution of USDA-ARS, the Kansas Agricultural Experiment Station, and the Wheat Genetics Resource Center. Contribution no. 94-242-J. Mention of a proprietary name in the article does not imply approval to the exclusion of other suitable products     

DNA sequence of the ribosomal protein genes rp12 and rps19 from a plant-pathogenic mycoplasma-like organism

A segment of a ribosomal protein operon from a plant-pathogenic mycoplasma-like organism (MLO) was cloned and sequenced, to provide supplemental molecular data pertinent to the question of MLO phylogeny. Comparisons of the deduced amino acid sequences indicate an ancient divergence of the MLOs from the animal-pathogenic mycoplasmas. Furthermore, although both the plant and animal pathogens have A-T rich genomes, a fundamental difference was apparent in their usage of the UGA codon.     

Regression and replacement of hydranths in thecate hydroids,and the structure of hydrothecae

In 1953 the author described a regression and replacement cycle of hydranths in Laomedea flexuosa and proposed that this is a common feature of thecate hydroids. Here examples are presented of replacement of hydranths in species from 5 families of thecates, but it has not been demonstrated that this is as common as had been thought. More continuous studies are needed. A brief consideration is given to the origin of hydrothecae and their development.     

Monosomic analysis of tissue culture response in wheat (Triticum aestivum L.)

Summary The ability of immature embryos of wheat (Triticum aestivum L.) to respond in cell culture was examined in crosses between the Wichita monosomic series and a highly regenerable line, ND7532. Segregation in disomic controls and 13 monosomic families showed a good fit to a monogenic ratio indicating a qualitative mode of inheritance. Segregation in the cross involving monosomic 2D showed a high frequency of regeneration (93.6%) and high callus growth rate (1.87 g/90 days) indicating that 2D is a critical chromosome. Modifying genes may be located on other chromosomes. Substitution of chromosomes from a low regenerable cultivar Vona further indicated that the group 2 chromosomes, in particular chromosome 2D, possess genetic factors promoting callus growth and regeneration.     

Order and fluidity in the terminal methyl region of dipalmitoyl phosphatidylcholine multibilayers: A comparison of raman and H-NMR spectroscopy

Deuterium magnetic resonance (²H-NMR) and Raman spectroscopy are used to investigate order and fluidity at the terminal methyl position in 16-d₃, 16′-d₃ dipalmitoylphosphatidylcholine (16-d₆ DPPC) multibilayers. These methods reveal substantial motion and disorder in the gel phase, 5–10°C below the gel-liquid crystal phase transition temperature (T_m). The phase transition is sensed in the ²H-NMR spectrum as a reduction in the quadrupole splitting from 14 kHz to 3 kHz. In contrast, the Raman parameter used to characterize the CD₃ vibrations is quite insensitive to the melting process, although an analogous parameter does sense disordering at T_m at the 10 and 10′ position in 10-d₂, 10′-d₂ DPPC. The difference in the response of the NMR and Raman parameters may arise because the vibrational spectrum of the CD₃ group is inhomogenously broadened and is therefore quite sensitive to alterations in the local environment around the methyl group. In contrast, the NMR quadrupole splitting is sensitive to both local motion of the methyl group and, near Tm, to motions of the CD₂ group induced by transgauche isomerizations further up the chain. The difficulties that arise when results from different spectroscopic techniques are compared are demonstrated.     

Cell surface distribution and intracellular fate of asialoglycoproteins: a morphological and biochemical study of isolated rat hepatocytes and monolayer cultures

A combination of biochemistry and morphology was used to demonstrate that more than 95 percent of the isolated rat hepatocytes prepared by collagenase dissociation of rat livers retained the pathway for receptor-mediated endocytosis of asialoglycoproteins (ASGPs). Maximal specific binding of (125)I-asialoorosomucoid ((125)I-ASOR) to dissociated hepatocytes at 5 degrees C (at which temperature no internalization occurred) averaged 100,000-400,000 molecules per cell. Binding, uptake, and degredation of (125)I- ASOR at 37 degrees C occurred at a rate of 1 x 10(6) molecules per cell over 2 h. Light and electron microscopic autoradiography (LM- and EM-ARG) of (125)I-ASOR were used to visualize the surface binding sites at 5 degrees C and the intracellular pathway at 37 degrees C. In the EM-ARG experiments, ARG grains corresponding to (125)I-ASOR were distributed randomly over the cell surface at 5 degrees C but over time at 37 degrees C were concentrated in the lysosome region. Cytochemical detection of an ASOR-horseradish peroxidase conjugate (ASOR-HRP) at the ultrastructural level revealed that at 5 degrees C this specific ASGP tracer was concentrated in pits at the cell surface as well as diffusely distributed along the rest of the plasma membrane. Such a result indicates that redistribution of ASGP surface receptors had occurred. Because the number of surface binding sites of (125)I-ASOR varied among cell preparations, the effect of collagenase on (125)I-ASOR binding was examined. When collagenase-dissociated hepatocytes were re-exposed to collagenase at 37 degrees C, 10-50 percent of control binding was observed. However, by measuring the extent of (125)I-ASOR binding at 5 degrees C in the same cell population before and after collagenase dissociation, little reduction in the number of ASGP surface receptors was found. Therefore, the possibility that the time and temperature of the cell isolations allowed recovery of cell surface receptors following collagenase exposure was tested. Freshly isolated cells, dissociated cells that were re-exposed to collagenase, and perfused livers exposed to collagenase without a Ca(++)-free pre-perfusion, were found to bind 110-240 percent more(125)I-ASOR after 1 h at 37 degrees C that they did at 0 time. This recovery of surface ASGP binding activity occurred in the absence of significant protein synthesis (i.e., basal medium or 1 mM cycloheximide). Suspensions of isolated, unpolarized hepatocytes were placed in monolayer culture for 24 h and confluent cells were demonstrated to reestablish morphologically distinct plasma membrane regions analogous to bile canalicular, lateral, and sinusoidal surfaces in vivo. More than 95 percent of these cells maintained the capacity to bind, internalize, and degrade (125)I-ASOR at levels comparable to those of the freshly isolated population. ASOR-HRP (at 5 degrees C) was specifically bound to all plasma membrane surfaces of repolarized hepatocytes (cultured for 24 h) except those lining bile canalicular-like spaces. Thus, both isolated, unpolarized hepatocytes and cells cultured under conditions that promote morphological reestablishment of polarity maintain the pathway for receptor- mediated endocytosis of ASGPs.     

Application of a novel chemiluminescence-based DNA detection method to single-vector and multiplex DNA sequencing

A chemiluminescent DNA detection method is described and its application shown for both single-vector and multiplex DNA sequencing using the standard dideoxy chain-termination process. This recently developed detection method, which utilizes the light emitted by an enzyme-catalyzed dioxetane reaction, is highly sensitive and affords significant advantages in safety and speed over the traditional radioactive labeling method. When adapted to a multiplex strategy, this chemiluminescent detection method constitutes a safe, simple and rapid method for increasing the throughput of DNA sequencing procedures.     

13C NMR studies on bilayers formed from synthetic di-10-methyl-stearoyl phosphatidylcholine enriched with 13C in the N-methyl carbons

¹³C NMR relaxation measurements have been carried out on phospholipid bilayer systems formed from synthetic di-10-methyl-stearoyl phosphatidylcholine with 92% enrichment in one of the N-methyl carbons. Studies on single-walled vesicles prepared by sonication from this lipid, and on large multi-lamellar liposomes show that although T₁ values are nearly the same, T₂¹ values are markedly different. It is proposed that equivalent segmental motions in the two systems give rise to similar T₁ values. The T₂¹ values, on the other hand, are consistent with the view that the single-walled vesicles have a more disordered molecular organization than do the multi-lamellar bilayers.