Frens Peters

Numerical analysis of binding studies: A direct procedure avoiding the pitfalls of a scatchard analysis of equilibrium data for unknown binding models

It is shown on theoretical grounds that the straightforward analysis of binding data according to Scatchard may lead to erroneous results, especially when more complicated binding schemes are involved. We have demonstrated this point by presenting Scatchard plots with slight variation of experimental parameters. These inherent difficulties of Scatchard analyses can be avoided by applying a direct procedure. We have developed a program, which compares the measured quantity and the theoretical value directly and which considers the following binding models: (i) independent equivalent binding of n ligands; (ii) independent unequivalent binding of 2 ligands; (iii) positive or negative cooperative binding of 2 ligands. Other binding schemes can easily be implemented. We have used this procedure for the evaluation of equilibrium data on the complex formation of tRNA-Tyr and tyrolyl tRNA synthetase from E. coli in terms of different binding models.

A critical interpretation of experiments of binding of peptide hormone to specific receptors by computer modelling

Many investigations dealing with the interaction of peptide hormones and specific cell membrane receptors imply the existence of two classes of independent binding sites. On class is characterized by high affinity and low capacity, the other one by low affinity and high capacity. This conclusion has been derived from the fact that the Scatchard plots of binding data show a significant upward curvature. Using a more precise and critical method of evaluation these findings probably must be revised in some cases. Other conclusions taken from linear plots concerning the regulation of hormone receptors should be discussed more carefully. Some sources of errors caused by an uncritical interpretation of Scatchard plots are demonstrated.

A quantitative analysis of glucagon binding to isolated intact neonatal and adult rat hepatocytes on the basis of two different binding models

The interaction of glucagon with specific receptors has been studied in isolated intact neonatal and adult rat hepatocytes. The hormone binding measured directly with 125I-labelled glucagon was saturable and reversible. The 125I-labelled glucagon binding was inhibited by unlabelled homologous hormone at concentrations ranging from 0.5 nM to 50 microM. Two different binding models were assumed to analyse the binding data by a nonlinear least-squares procedure: (I) a single class of independent sites and (II) two classes of independent sites. The comparison of the fitted theoretical curves reveals that both binding models are in fact compatible with these data. Adult hepatocytes have a considerably higher affinity for glucagon than neonatal hepatocytes; the binding capacity of neonatal liver cells from 1-7-days-old rats proved to be markedly reduced compared with the cells from adult rats. The glucagon-induced intracellular cyclic AMP production was measured at various hormone concentrations under conditions identical to those for the determination of extracellular hormone binding. The correlation of both parameters indicates a direct connection between receptor-occupancy and adenylate cyclase stimulation of both parameters indicates a direct connection between receptor-occupancy and adenylate cyclase stimulation. These results suggest that a decreased receptor concentration in neonatal hepatocytes is responsible for the decreased cyclic AMP production.

The influence of the concentrations of elongation factors and tRNAs on the dynamics and accuracy of protein biosynthesis

Computer simulations of the elongation cycle of bacterial protein biosynthesis demonstrate that the accuracy of protein biosynthesis cannot be explained by a mechanism which involves only an initial selection and a proofreading reaction. It is suggested that only a combination of initial selection, proofreading and a retardation of non-cognate flows at the level of the EF-Tu-catalyzed GTPase reaction and the peptidyl transfer can guarantee sufficient accuracy at reasonable costs. According to this view the ribosome functions as an allosteric enzyme which, in both its affinity and enzymatic activity, responds optimally only to the cognate substrate. Detailed calculations show, furthermore, that increasing the concentration of EF-G and EF-Ts above the level prevailing in vivo only slightly increases the rate of elongation. In contrast, increasing the concentration of EF-Tu over aminoacyl-tRNA (aa-tRNA) leads to a sharp decline in the rate of elongation. While varying the concentration of EF-G has no effect on the accuracy of protein synthesis, excess of EF-Tu over aminoacyl-tRNA leads to a large increase in accuracy. These results suggest a mechanism by which the accuracy of protein biosynthesis is preserved during amino acid starvation.

DIADEM--a system for the interactive data acquisition and processing in an analytical laboratory.

A conversational program for the acquisition of experimental data in a multi-user, multi-instrument computer system is described. It assists the researcher when recording on-time data. Due to the simple structure of the dialogue, no special knowledge of computer handling is required by the experimenter. Whereas the experimental methods are versatile, a uniform concept of the dialogue and the file structure is realized.

The assessment of glucagon binding in isolated intact neonatal and adult rat liver cells

Glucagon binding has, to date, generally been measured using membrane-fragment preparations [3]. Such investigations have been complemented by studies using isolated intact cells [5], and experiments of this kind can be considered as rather more relevant with respect to the in vivo situation. Furthermore, use of the intact cellular system permits the correlation of extracellular hormone binding with resulting intracellular metabolic effects. In order to obtain large quantities of defined populations of isolated, functionally and morphologically intact cells reproducibly from rat liver, we developed a simple separation procedure: a) Parenchymal liver cells of adult (-~ 3 months) male rats were prepared by the perfusion technique with 80 U/ml collagenase I (Sigma) in HEPES buffer [4]. b) Neonatal liver cells were isolated from young ( 1 5 days) rats by digestion with 1.8 U/ml dispase I (Boehringer) or 80 U/ml collagenase I [6]. The suspensions of neonatal or adult rat liver cells were further purified by isopycnic centrifugation through a continuous Percoll gradient [1]. For this purpose, a solution of 40.5% (v/v) Percoll in Eagles MEM was centrifuged at 15,000 g for 20 rain in a fixed-angle rotor 9 After layering the cell suspensions on top of the resulting gradient, a further centrifugation was carried out for 30 min at 100 g in a swingbucket rotor. Intact parenchymal cells of neonatal and adult rat liver banded at a density (determined refractometrically or by marker beads) of 1.075 1.090 g/ml, whereas non-viable cells banded at a lower density 9 After removal from the gradient, the cells were washed twice with buffer and were then suspended in Eagles MEM supplemented with 10% calf serum. About 9 0 95 % of the cells proved to be viable as judged by Trypan-blue exclusion. The interaction between glucagon and specific receptors was studied in isolated hepatocytes of neonatal and adult rats using radioactively-labelled glucagon. In order to determine the dissociation constant Ko for the glucagon binding and the 0.030-

Chemical modification studies on the tyrosyl-tRNA synthetase/tRNA complex are in agreement with an antico-operative binding scheme

Abstract Chemical modification experiments on the tyrosyl-tRNA synthetase/tRNA t yr system of Bacillus stearothermophilus reported by Bosshard et al. (1975,1978) which have been interpreted in terms of a binding scheme with one binding site that spans two subunits have been re-evaluated by a more rigorous procedure. The results show that the experiments of Bosshard et al. are in agreement with the antico-operative binding model that has been demonstrated for the Escherichia coli system.