Time difference scanning gel chromatography: computer simulations.

The time difference profile method of gel scanning chromatography developed by Brumbaugh, Saffen and Chun (Biophysical Chemistry, 1979) has been examined by computer simulation. The method is found to produce values for centroid movements that mimic those of the system being examined but are not quantitatively correct. In all cases the time differential "centroid" is larger than that of the concentration derivative (true) centroid and move at a rate slightly faster than the true centroids. This faster rate slowly decreases towards the true rate but does not approach it within reasonable times. This distorted movement reflects the distorted emphasis given to the larger species in the time differential method. The time difference method has been shown to give an adequate measure of the axial dispersion coefficient, L, for single species systems.

Kinetic control of three-species association reactions in gel chromatography.

The gel chromatographic patterns of a monomer-dimer-tetramer system under kinetic control have been studied by computer simulation. In no case do the derivative curves of the concentration profiles exhibit more than bimodality in these systems and in some cases are found to exist as a single, almost symmetric, peak. The monomer-dimer reaction affects the dimer-tetramer reaction only slightly and the same is true for the effect of the dimer-tetramer reaction on that of the monomer-dimer. All systems can be fit, to a first approximation, by an empirical formula which suggests any reaction with a first order half life within one and one half or two orders of the transport time will be under kinetic control.

Active enzyme gel chromatography: II. Computer simulations.

The behavior of an enzyme undergoing reaction while on a gel chromatography column has been studied by computer simulation using the steady state assumtion for a system with a single enzyme-substrate complex. The profiles of the enzyme-substrate complex, product, and substrate were examined varying the parameters of kcat, flow rate, partition coefficient dispersion, and time. These investigations confirm that much information about both the active enzyme and the product may be obtained by examining the product profile alone, varying the power of applying scanning gel chromatography to active enzyme systems.

A simple method of analyzing profiles in time-difference direct optical scanning gel chromatography.

A simple method is presented for the analysis of time-difference, large-zone chromatography profiles. The method is derived from basic theory and is applicable to multicomponent as well as single-component systems. Simple computer simulations are used to demonstrate the inaccuracies of earlier, more empirical methods. This method has been tested on several proteins using an inexpensive, semi-automated, data acquisition and control system.

The determination of equilibrium constants for heterogeneous macromolecular interactions. Systems forming 2:1 complexes.

In developing a method for analyzing the heterogeneous association nA + mB in equilibrium AnBm, we have specifically investigated the case of n = 2, m = 1 for both the specific case of no appreciable intermediates and the more general case allowing intermediates. Computer-simulated three-dimensional surfaces of the 2:1 model generated from total concentrations of species A and B and the resulting weight-average molecular weights were analyzed with a Gauss-Newton nonlinear least-squares minimization routine. The surfaces generated included normalized random error of varying standard deviations imposed upon both the concentrations and weight-average molecular weights. For comparison purposes, these surfaces were analyzed not only by using the correct 2:1 model, but also by an incorrect (1:1) model and by the other (incorrect) 2:1 model. Except for those situations where the 'experimental' noise was consistently higher than the concentration of one of the species, correct K values were obtained and the correct model was easily distinguished from the incorrect model. The computer routine similarly distinguished between data correctly described as 1:1 and the same data incorrectly analyzed as either 2:1 model. For those cases in which a microscopic Ki value predicts an association such that all species involved for that particular Ki are in appreciable amounts, the Ki value is returned correctly. Correct overall equilibrium constants are also converged upon as long as adequate amounts of A2B, B and A are present.

The determination of equilibrium constants for heterogeneous macromolecular interactions.

A method has been developed to determine the association constant for a heterogeneous association of the type A + B in equilibrium AB. This method requires knowledge of the two initial concentrations and of the resulting weight-average molecular weight for each data point. Computer simulations using Gaussian-distributed error on the measured parameters show that the researcher can readily determine whether the particular concentration range chosen is appropriate for the strength of binding and therefore how reliable the calculated constant might be. It is also shown that errors in measuring molecular weight have, in general, a more profound effect than do errors in concentration.

Seedling establishment in an epiphytic orchid: an experimental study of seed limitation.

Will increased fruit and seed production in a severely pollination-limited orchid stimulate population growth? We tested whether safe sites for germination and seedling establishment are limiting for the twig epiphyte, Tolumnia variegata, by manipulating fruit set and monitoring subsequent seedling establishment for two seasons (1991-1992, 1992-1993). In the Cambalache Forest Reserve of Puerto Rico, we established 36 plots along a transect. Each plot consisted of nine trees. A center tree was designated as the site for attaching Tolumnia and manipulating fruit set. The other eight potential host trees were 1-3 and 3-5 m from the center tree in each of the cardinal directions. A 1-m length of stem 1 m from the ground was monitored for recruits on each of the nine trees of 24 fruit-enhanced plots and 12 controls (23 and 13, respectively for the 1992-1993 season). Fruit enhancement plots were divided among two treatments: one-fruit and five-fruit additions for the 1st year and one to five and more than five fruits for the 2nd year. Availability of suitable host species was not limiting. T. variegata showed little specificity for host tree species, good host trees and shrubs were common, and there was no evidence that the orchid had a preference for small branches, despite possessing the entire suite of characteristics thought to respresent "obligate" twig epiphytes. Fruit enhancement increased seed rain and seedling establishment consistently in only the high-fruit treatment plots. Most recruitment occurred near fruiting plants. Over the 2-year period, mortality was 18% for adults and 85.5% for the 1991-1992 cohort of recruits. Net recruitment was positive for both the treatment (average = 1.74) and control plots (average = 0.67). Seedling establishment at our study site was not microsite-limited. If selection for increased pollinator attraction occurs, then an increase in seed output should result in population growth.

A current assessment of the role of porcine parvovirus as a cause of fetal porcine death.

One hundred one litters containing 1 or more dead porcine fetuses were collected at an Iowa abattoir during a 2-month interval and examined for evidence of viral infection. Each of 1,137 fetuses (302 dead, 835 alive) of these litters was tested for porcine parvovirus (PPV) antigens by direct immunofluorescence microscopy (FA) of fetal lung. Antigens of PPV were detected in the lungs of most of the fetuses of 11 of the litters. The 11 FA-positive litters contained 105 dead (100 FA-positive) and 14 live (12 FA-positive) fetuses. Infectious PPV was isolated from 10 of the 11 FA-positive litters and from 3 of the 90 FA-negative litters. No cytopathogenic agents other than PPV were isolated from any of the litters. Eleven of 101 (11%) litters examined and 100 of 302 (33%) dead fetuses examined were FA positive for viral antigen, indicating that PPV remains as a major cause of porcine fetal death.

Carbon isotope ratios are correlated with irradiance levels in the Panamanian orchid Catasetum viridiflavum.

Carbon isotope composition ((13)C/(12)C) in leaves of the Panamanian epiphytic orchid Catasetum viridiflavum were measured on individuals growing in canopies over a water surface to distinguish the effects of a change in source CO(2) and humidity from those of intercellular CO(2) concentration in determining isotopic composition. Carbon isotope ratios were observed to vary by over 4 in response to changes in total daily photon flux (PFD, 400-700 nm). Changes in isotopic composition of source CO(2) or changes in humidity were not likely to have played a role in determining leaf isotopic composition. Observed changed in carbon isotope discrimination (Δ) of leaves experiencing different light levels ranged from 17 to 21. Because leaf nitrogen contents were similar among all orchids, we suggest that the carbon isotope discrimination data indicated that stomatal limitation to photosynthesis increased with increasing irradiance.

A peptide isolated from a random phage peptide library is a structural mimic to the P1, P4-diadenosine 5'-tetraphosphate binding site on its receptor.

We have previously demonstrated that a monoclonal antibody (mAb TL4), which inhibits P1, P4-diadenosine 5'-tetraphosphate (Ap4A) binding to its receptor, selected a consensus RGS tripeptide from a random phage hexapeptide library. RGS interfered with Ap4A binding to its membrane receptor [Liu, G., Bryant, R. T. & Hilderman, R. H. (1996) Biochemistry 35, 197-201]. However the mechanism by which RGS interfered with Ap4A binding to its receptor was not determined. In this communication, we demonstrate that RGS interacts with Ap4A to prevent [3H]Ap4A binding to the Ap4A membrane receptor. To further characterize the mechanism by which RGS inhibits Ap4A binding to its receptor, we used mAb TL4 to screen a 15-residue random peptide phage library and DNAs from 24 clones were sequenced. 20 clones contain a RGSSS sequence while 17 of these clones contained an identical 15-amino-acid insert (clone A). Gel-filtration studies of previously equilibrated clone A phage and [3H]Ap4A support the idea that [3H]Ap4A interacts specifically with clone A phage while RGS effectively competes for [3H]Ap4A interaction on clone A phage. These data are consistent with RGS mimicking a sequence on the receptor essential for Ap4A binding.

Identification of a unique membrane receptor for adenosine 5',5"'-P1,P4-tetraphosphate.

Adenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) has been implicated as a modulator of cell stress. We have performed binding studies which indicate that membranes from all tissues tested bind tritium-labeled Ap4A. The characteristics of Ap4A binding were determined on brain membrane homogenates after development of an optimized in vitro filter-binding assay. Ap4A binding is specific for adenylated dinucleotides and for the length of the phosphate bridge. A Kd of 0.71 microM for Ap4A was determined.

Characterization of a homogeneous arginyl- and lysyl-tRNA synthetase complex isolated from rat liver. Kinetic mechanism for lysyl-tRNA synthetase.

Bisubstrate kinetics and end product and dead end inhibition studies were performed on lysyl-tRNA synthetase isolated from rat liver. The kinetic patterns obtained are consistent with a sequential ordered mechanism of substrate addition, tRNA bound first, followed by lysine, and then by ATP. Pyrophosphate and AMP are released in a random fashion with aminoacylated tRNA the last product to dissociate from the enzyme. This is the first report of a kinetic mechanism for lysyl-tRNA synthetase.

Studies of L-arginine transport in bovine aortic endothelial cells.

We have previously demonstrated that p(1),p(4)-diadenosine 5'-tetraphosphate induces the release of NO and modulates the uptake of L-arginine by bovine aortic endothelial cells (BAEC) [Hilderman, R. H., and Christensen, E. F. (1998) FEBS Lett. 407, 320-324; Hilderman, R. H., Casey, T. E., and Pojoga, L. H. (2000) Arch. Biochem. Biophys. 375, 124-130]. In this communication we characterize the uptake of L-Arg by BAEC. L-Arg is transported into BAEC by at least two different transporter systems. One transporter system is protein synthesis dependent, and L-Arg transported by this system is incorporated into proteins. The second transporter system involved in L-Arg uptake is protein synthesis independent, and uptake occurs by facilitated diffusion. The L-Arg transported by facilitated diffusion is metabolized into L-argininosuccinate. Homologous and heterologous competition uptake studies were performed using a fixed concentration of radiolabeled L-Arg, L-lysine, and L-leucine with varying concentrations of competing nonradiolabeled amino acids. The results of these competition uptake studies are consistent with the protein-synthesis-dependent uptake of L-Arg taking place through a transporter system that is highly specific for L-Arg and with the facilitated diffusion uptake taking place through a transporter that is specific for L-Arg and L-Leu.

A re-examination of some properties of fatty acyl-CoA micelles.

Three separate techniques have been employed to estimate the critical micelle concentration: spin labeling using 6-doxylstearoyl-CoA, gel permeation chromatography, and analytical ultracentrifugation. The first method is a labeling technique. The latter two methods utilize no potentially interfering probe and provide a value for the aggregation number for palmitoyl-CoA. All three methods provide a critical micelle concentration for palmitoyl-CoA no lower than 30 to 60 microM. The latter methods provide an aggregation number near 40 and certainly no larger than 200. These values are inconsistent with the values suggested earlier (Zahler, W. L., Barden, R. E., and Cleland, W. W. (1968) Biochim. Biophys. Acta 164, 1-11). The spin-labeled analogues, 6- and 16-doxylstearoyl-CoA, were shown not to micellize, yet these analogues were good inhibitors for citrate synthase. These observations will require the re-examination of a large body of literature in which inhibition of enzymes by fatty acyl-CoA at concentrations below 30 microM was simply ascribed to the formation of micelles.

Adenylated dinucleotide binding to the adenosine 5',5'''-P1,P4-tetraphosphate mouse heart receptor.

We have demonstrated specific adenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) receptors at heart cell surfaces. Optimal Ap4A binding requires receptor activation. Other Investigators have demonstrated that Ap5A and Ap6A act as vasopressors. We now compare the binding of Ap4A, Ap5A and Ap6A on heart membranes to determine if all three ligands bind to the same receptor and their relative avidities. Anti-Ap4A receptor antibodies inhibit the binding of all three ligands. SDS-PAGE analysis of Ap4A, Ap5A and Ap6A cross-linked to membranes reveals that all three are attached to a 30 kDa peptide. The specific activity for binding to unactivated membranes is similar for all three ligands. However, after receptor activation there is a 3.4x increase in Ap4A binding and a 32.5x decrease in the KD; values remain unchanged for Ap5A and Ap6A. These data indicate that Ap4A, Ap5A and Ap6A bind to the same receptor on cardiac membranes but receptor activation enhances only Ap4A binding.

The adenosine 5',5"',P1,P4-tetraphosphate receptor is at the cell surface of heart cells.

We have previously demonstrated the existence of an adenosine 5',5"',P1,P4-tetraphosphate (Ap4A) receptor in mouse hart membrane fractions [Hilderman, R. H., Martin, M., Zimmerman, J. K., & Pivorun, E. P. (1991) J. Biol. Chem. 266, 6915-6918]. However, we did not determine the cellular localization or distribution of the receptor. In this report, the Ap4A receptor is shown to be on the cell surface of individual mouse heart cells by the following four methods: (1) intact cells show specific, saturable, and reversible binding of Ap4A; (2) monoclonal antibodies (Mabs) raised against the Ap4A receptor inhibit Ap4A binding to its receptor on intact heart cells; (3) bound Mabs are shown to be at the outer cell surface via reaction with a alkaline phosphatase conjugated goat anti-rat IgG; (4) when intact cells are labeled with the impermeable cell surface labeling reagent, (sulfosuccinimido) biotin, labeled receptor is immunoprecipitated with Mabs. Furthermore, subcellular fractionation of mouse hearts demonstrates that virtually all of the Ap4A receptor is associated with a membrane fraction with at least 77% of the active receptor on plasma membranes.