A molecular thermodynamic approach to predict the secondary structure of homopolypeptides in aqueous systems.

Under physiological conditions, many polypeptide chains spontaneously fold into discrete and tightly packed three-dimensional structures. The folded polypeptide chain conformation is believed to represent a minimum Gibbs energy of the system, governed by the weak interactions that operate between the amino acid residues and between the residues and the solvent. A semiempirical molecular thermodynamic model is proposed to represent the Gibbs energy of folding of aqueous homopolypeptide systems. The model takes into consideration both the entropy contribution and the enthalpy contribution of folding homopolypeptide chains in aqueous solutions. The entropy contribution is derived from the Flory-Huggins expression for the entropy of mixing. It accounts for the entropy loss in folding a random-coiled polypeptide chain into a specific polypeptide conformation. The enthalpy contribution is derived from a molecular segment-based Non-Random Two Liquid (NRTL) local composition model [H. Renon and J. M. Prausnitz (1968) AIChE J., Vol. 14, pp. 135-142; C.-C. Chen and L. B. Evans (1986) AIChE J., Vol. 32, pp. 444-454], which takes into consideration of the residue-residue, residue-solvent, and solvent-solvent binary physical interactions along with the local compositions of amino acid residues in aqueous homopolypeptides. The UNIFAC group contribution method [A. Fredenslund, R. L. Jones, and J. M. Prausnitz (1975) AIChE J., 21, 1086-1099; A. Fredenslund, J. Gmehling, and P. Rasmussen (1977) Vapor-Liquid Equilibrium Using UNIFAC, Elsevier Scientific Publishing Company, Amsterdam], developed originally to estimate the excess Gibbs energy of solutions of small molecules, was used to estimate the NRTL binary interaction parameters. The model yields a hydrophobicity scale for the 20 amino acid side chains, which compares favorably with established scales [Y. Nozaki and C. Tanford (1971) Journal of Biological Chemistry, Vol. 46, pp. 2211-2217; E. B. Leodidis and T. A. Hatton (1990) Journal of Physical Chemistry, Vol. 94, pp. 6411-6420]. In addition, the model generates qualitatively correct thermodynamic constants and it accurately predicts thermodynamically favorable folding of a number of aqueous homopolypeptides from random-coiled states into alpha-helices. The model further facilitates estimation of the Zimm-Bragg helix growth parameter s and the nucleation parameter sigma for amino acid residues [B. H. Zimm and J. K. Bragg (1959) Journal of Chemical Physics, Vol. 31, pp. 526-535]. The calculated values of the two parameters fall into the ranges suggested by Zimm and Bragg.     

Up-regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in murine peritoneal exudate macrophages by both GM-CSF and IL-3.

Murine peritoneal exudate macrophages (PEM) display multiple CSF receptors. In this study, the expression of granulocyte-macrophage (GM)-CSF receptors in PEM was studied. PEM displayed over 5000 single type, high affinity GM-CSF receptors/cell with a Kd = 38 to 42 pM and an apparent molecular mass of 86,000 Da. Treatment of PEM with low, but not high, concentrations of recombinant murine (rMu) GM-CSF continuously for 24 h resulted in a marked up-regulation of GM-CSF receptors in PEM. A similar up-regulation of GM-CSF receptors also was detected in PEM cultures treated with rMuIL-3 (1-100 ng/ml) for 24 h or longer, regardless the doses of rMuIL-3 added in this case. Scatchard analysis of equilibrium binding showed that the enhanced binding activities in both cases were due to an increase in total number of GM-CSF receptors rather than changes in receptor affinity. Contrariwise, treatment with recombinant human macrophage-CSF (greater than 100-1000 ng/ml) partially inhibited the expression of GM-CSF receptors in PEM. Removal of rMuGM-CSF from culture medium 24 h after treatment led to a further up-regulation of GM-CSF receptors over a 4 to 24-h period, depending on the doses of initial treatment. On the other hand, removal of rMuIL-3 from culture medium after prolonged treatment did not result in further increase in GM-CSF receptors. The protein synthesis inhibitor cycloheximide abrogated GM-CSF receptor up-regulation induced by both rMuIL-3 and rMuGM-CSF, whereas actinomycin D inhibited only the second (8-24 h) phase of GM-CSF receptor up-regulation induced by exposure to high concentrations rMuGM-CSF (10 ng/ml). These findings suggest that rMuGM-CSF and rMuIL-3 up-regulate GM-CSF receptors in PEM in part through similar or identical metabolic pathways and provide further evidence of a close linkage between IL-3 and GM-CSF receptors.     

Abscisic Acid increases terrestrial plant cell resistance to hydrostatic pressure

Cells of the terrestrial plant species bromegrass (Bromus inermis L.) are not naturally adapted to withstand the hydrostatic pressures encountered in aquatic environments. However, after treatment with the natural plant growth hormone abscisic acid (75 micromolar), bromegrass cells survived a hydrostatic pressure of 101.3 megapascals, approximating the limits of ocean depth (10,860 m). The increased resistance to hydrostatic pressure from 1 to 7 days of abscisic acid treatment paralleled the induced elevation of cell tolerance to freezing stress.     

Quantitative immunoassay of total cellular GAP junction protein connexin32 during liver regeneration using antibodies specific to the COOH-terminus.

A radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were used to determine relative concentrations of liver connexin32 (CX32) in rats. The RIA and ELISA utilize synthetic peptides corresponding to regions of the carboxyl-terminus and antibodies raised in rabbits against these peptides. Assuming that affinities of antisera are similar for peptide and native CX32, total cellular CX32 was found to exceed the amount of gap junction protein at the cell surface calculated from morphometric analyses by 1.5-2.0 fold. This finding raises the possibility that some of the protein is present in cytoplasmic compartments or as occult precursors in the plasma membrane. Studies of CX32 content in regenerating rat liver support this conclusion and show a time course of loss and recovery of CX32 that agrees with those reported in studies using other techniques.     

Enzymatic sulfation of gastrin in rat gastric mucosa

An enzyme which catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to gastrin (G17) was identified in rat gastric mucosal cells. The enzyme activity was detected in the 105,000xg supernatant fraction. Formation of gastrin sulfate was shown by using 125I-gastrin and non-radioactive PAPS. The product was sensitive to acid hydrolysis, arylsulfatase treatment and removed by gastrin antibody, but not changed by treatments with chondro-4-sulfatase and chondro-6-sulfatase. The product had a molecular weight of 2050 daltons, close to the molecular weight of G17 sulfate, and, therefore, indicating the sulfated product is not APS derived from the degradation of PAPS. The enzyme activity showed a Km value of 5 microM for PAPS and a pH optimum of 6.0. The activity was not detected in the liver preparation.     

The form II fructose 1,6-bisphosphatase and phosphoribulokinase genes form part of a large operon in Rhodobacter sphaeroides: primary structure and insertional mutagenesis analysis

Fructose 1,6-bisphosphatase (FBPase) and phosphoribulokinase (PRK) are two key enzymes of the reductive pentose phosphate pathway or Calvin cycle of photosynthetic carbon dioxide assimilation. Early studies had indicated that the properties of enzymes isolated from photosynthetic bacteria were clearly distinct from those of enzymes obtained from the chloroplasts of higher plants [for a review, see Tabita (1988)]. The eucaryotic enzymes, which are light activated by the thioredoxin/ferredoxin system (Buchanan, 1980), were each shown to contain a putative regulatory amino acid sequence (Marcus et al., 1988; Porter et al., 1988). The enzymes from photosynthetic bacteria are not controlled by the thioredoxin/ferredoxin system but exhibit complex kinetic properties and, in the case of PRK, there is an absolute requirement of NADH for activity. In the photosynthetic bacterium Rhodobacter sphaeroides, the structural genes of the Calvin cycle, including the genes that encode FBPase (fbp) and PRK (prk), are found in two distinct clusters, and the fbp and prk genes are closely associated in each cluster. In the present investigation, we have determined the nucleotide sequence of the fbpB and prkB genes of the form II cluster and have compared the deduced amino acid sequences to previously determined sequences of light-activated enzymes from higher plants and from other eucaryotic and procaryotic sources. In the case of FBPase, there are several regions that are conserved in the R. sphaeroides enzymes, including a protease-sensitive area located in a region equivalent to residues 51-71 of mammalian FBPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Using group testing to estimate a proportion, and to test the binomial model

Group testing has been extensively studied as an efficient way to classify units as defective or satisfactory when the proportion (p) of defectives is small. It can also be used to estimate p, often substantially reducing the mean squared error (MSE) of p and cost per unit information. Group testing is useful for larger p in the estimation problem than in the classification problem, but for larger p more care must be taken in choosing the group size (k); k being too large not only increases MSE (p), but adversely affects the robustness of p to both errors in testing (misclassification) and errors in the assumed binomial model. Procedures that retest units from defective groups, if even feasible, are shown to reduce cost per unit information very little in the estimation problem, but can provide useful information for testing the model. Methods are given for using data from tests of unequal-sized groups to estimate p and for testing the validity of the binomial model.     

Free ADP levels in transgenic mouse liver expressing creatine kinase. Effects of enzyme activity, phosphagen type, and substrate concentration

ADP is an important regulator of hepatic metabolism. Despite its importance the level of free ADP in the liver remains controversial. Recently, we engineered transgenic mice which express high levels of creatine kinase in liver. The reaction catalyzed by creatine kinase was assumed to be at equilibrium and used to calculate a free ADP level of 0.059 mumol/g wet weight. In this report we test the equilibrium assumption by studying the free ADP level as a function of enzyme activity or substrate content. Over a 5-fold range of creatine kinase activity, from 150-800 mumol/min/g wet weight, there was no change in the free ADP level. The average value of ADP for these mice was 0.061 +/- 0.016 mumol/g wet weight. Similarly, altering hepatic creatine content from 1.6 to 30 mumol/g wet weight had no effect on the calculated total free ADP level. The average value of ADP for the creatine levels was 0.048 +/- 0.015 mumol/g wet weight. Finally, the free ADP level was calculated using the equilibrium with cyclocreatine rather than creatine as substrate. The equilibrium of the reaction with cyclocreatine lies 30 times more toward phosphorylation than does the equilibrium with creatine. A free ADP level of 0.063 +/- 0.031 mumol/g wet weight was calculated using cyclocreatine. This value is not different from that found with creatine. These results show that the equilibrium assumption used to calculate free ADP levels in transgenic mouse liver is valid, and the presence of creatine kinase does not affect ADP levels.