Biophysical studies of engineered mutant proteins based on calbindin D9k modified in the pseudo EF-hand

The genes for four mutant proteins from calbindin D9k, all with mutations in the N-terminal Ca2+-binding domain (pseudo EF-hand) have been synthesized and expressed in Escherichia coli. The purification scheme has been modified to minimize the formation of deamidated proteins. The set of modifications in the pseudo EF-hand is an attempt to turn this site into a structure resembling an archetypal EF-hand, with its characteristic 113Cd-NMR shift (-80 to -110 ppm) and high calcium-binding constants, whereas the C-terminal Ca2(+)-binding site (EF-hand) is kept intact in all mutant proteins. The mutant proteins studied here all have pseudo EF-hands with a lower calcium-binding constant and a higher calcium off-rate to the pseudo EF-hand than the wild-type protein. From the results obtained it is obvious that proline 20 in the pseudo EF-hand, which has been deleted or replaced by glycine in three of the mutants, has a stabilizing effect on calcium binding to that site. Furthermore, the modifications in the pseudo EF-hand seem to have only a local effect, leaving the tertiary structure of the protein and the calcium-binding properties of the unmodified site virtually unchanged.     

Similar induction of the hepatic EGF receptor in vivo by EGF and partial hepatectomy

A 2-fold increase in the level of epidermal growth factor (EGF) receptor mRNA accompanied by a similar increase in newly synthesized ligand-binding EGF-receptors was observed 2-4 h after intraportal EGF-injections and 2-4 h after partial hepatectomy. After this initial increase, the EGF-receptor levels decreased back to control levels 6-8 h after EGF-injections and below control levels 6-8 h after partial hepatectomy. EGF was also found to influence the degradation of endocytosed [125I]-EGF 3 h after injection to a similar extent as partial hepatectomy. The similar effects of EGF and partial hepatectomy suggests that EGF or EGF-like factors may be mechanistically involved in the early "promotion" stage during the pre-replicative phase of liver regeneration. The EGF-induced effects are, however, at least not alone responsible for the replicative process.     

Protein surface charges and Ca2+ binding to individual sites in calbindin D9k: stopped-flow studies

The kinetics of calcium dissociation from two groups of site-specific mutants of calbindin D9k--a protein in the calmodulin superfamily with two Ca2+ sites and a tertiary structure closely similar to that of the globular domains of troponin C and calmodulin--have been studied by stopped-flow kinetic methods, using the fluorescent calcium chelator Quin 2, and by 43Ca NMR methods. The first group of mutants comprises all possible single, double, and triple neutralizations of three particular carboxylate groups (Glu-17, Asp-19, and Glu-26) that are located on the surface of the protein. These carboxylates are close to the two EF-hand calcium binding sites, but are not directly liganded to the Ca2+ ions. Conservative modification of these negative carboxylate side chains by conversion to the corresponding amides results in a marked reduction in the Ca2+ binding constants for both sites, as recently reported [Linse et al. (1988) Nature 335, 651-652]. The stopped-flow kinetic results show that this reduction in Ca2+ affinity derives primarily from a reduction in the Ca2+ association rate constant, kon. The estimated maximum value of the association rate constant (kon(max) for Ca2+ binding to the wild-type protein is ca. 10(9) M-1 s-1. In contrast, for the mutant protein with three charges neutralized the maximum association rate constant is estimated to be only 2 X 10(7) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partition of synaptic membranes in aqueous two-phase systems at subzero temperatures by using anti-freeze solvent

The freezing point of aqueous two-phase (liquid-liquid) systems containing water, dextran and poly(ethylene glycol) has been lowered by including glycerol. Biological membranes, obtained by fragmentation of a crude synaptosomal preparation from calf brain cortex, have been included in the two-phase systems. The effects of temperature and the concentration of glycerol on the partition of the membranes within the systems have been investigated. Considerable stabilisation of the membranes was noticed when they were partitioned at -10 degrees C compared with 0 degrees C. The influences of glycerol, ethylene glycol, N,N-dimethylformamide and tetrahydrofuran on the phase-forming properties of the systems and on enzyme activities are also presented. Possible use of the above systems for studies and separation of biological membranes are discussed.     

Studies on the lecithin: cholesterol acyltransferase substrate properties of HDL as determined by its subclass distribution analysed by gradient gel electrophoresis

In order to study the impact of high-density lipoproteins (HDL) subclasses on the ability of HDL to act as substrate for lecithin: cholesterol acyltransferase (LCAT), we isolated HDL from nine normolipidemic male subjects. The HDL particle size distribution was analysed by gradient gel electrophoresis and the esterification rate of the isolated homologous HDL was compared with a pool of HDL where all the nine subjects took part. It was found that the strongest determinant for HDL cholesterol esterification rate was the inhibitory action of HDL subclass 2B.     

Functional properties of calbindin D9K mutants with exchanged Ca2+ binding sites

We have constructed three different engineered proteins based on calbindin D9K by either exchanging the two calcium binding sites within the protein or making the amino acid sequence of the two calcium binding sites identical. The individual calcium binding sites of the engineered proteins retain most of their ion binding characteristics as well as the basal structure of their Ca2+ ligand sphere in the new environment. Even the protein with its sites interchanged, a mutation involving 30 amino acids out of a total of 75, still binds calcium with an affinity as high as that of many natural EF-hand proteins.     

The role of glutamine synthetase in the regulation of nitrogenase activity (“switch off” effect) in Rhodospirillum rubrum

We have studied the changes in the activities of both nitrogenase (switch off) and glutamine synthetase in Rhodospirillum rubrum upon addition of ammonium ions or glutamine to nitrogen fixing cultures. Both activities decrease drastically and return in a parallel manner when added ammonia is metabolized. The decrease in glutamine synthetase activity does not seem to be primarily due to adenylylation of the enzyme. Addition of glutamine to cells starved for nitrogen results in inactivation of glutamine synthetase but nitrogenase is only partially switched off.Abbreviations CeMe₃NBr Cetyltrimethylammonium bromide - Hepes N-2-hydroxyethyl-piperazine-N-2 sulfonic acid - MSO methionine-D,L-sulfoximine - Tea-Dmg triethanol amine-3,3-dimethylglutaric acid     

Studies of chromophores in model membranes by polarized light-absorption spectroscopy. The orientation and binding of tetracaine and procaine.

A method for studying the orientation and binding of chromophores in macroscopically aligned membranes by polarized light absorption spectroscopy is described. Here tetracaine and procaine solubilized in a lamellar phase of octanoyl-1-glyceride (monooctanoin) and water have been investigated. Tetracaine is found to be located in the lipid region with a preferential orientation of the molecular long axis parallel to the hydrocarbon chains. The orientation of procaine, mainly residing in the water region, is very small.     

Interaction of human serum albumin with fatty acids. Role of anionic group studied by affinity partition.

The interaction of human serum albumin with fatty acids has been determined using the method of affinity partitioning in aqueous biphasic systems containing dextran, poly(ethylene glycol) and esters of dicarboxylic acids with poly(ethylene glycol). The difference in the partition of albumin in phase systems with and without the poly(ethylene glycol)-bound fatty acid group provides a measure of the interaction of fatty acids with the protein. The relative contribution of the polar and non-polar interaction to the binding of fatty acids to albumin has been estimated by comparing the present data with that obtained earlier using poly(ethylene glycol)-bound straight chain aliphatic hydrocarbons. In both cases, the aliphatic chain should contain a minimum of 8 carbon atoms to affect the partition of albumin and that the maximum effect is obtained with chains containing 16 carbon atoms. The effect of the polymer-bound fatty acid group is higher than the corresponding hydrocarbon only when the number of carbon atoms in it exceeds 12. The relative effect of polymer-bound 16-carbon chains, with and without a carboxyl group in the terminal position is independent of pH in the range 5--9.     

Purification of adenovirus messenger ribonucleic acid by an aqueous polymer two-phase system.

An aqueous polymer phase system containing 6.3% (w/w) dextran and 3.5% (w/w) poly(ethylene glycol) in 10 mM phosphate buffer (pH 8.0) was developed to select RNA-DNA hybrids from unhybridized RNA. The top phase of this phase system, which contains DNA and the RNA-DNA hybrids, can be used to purify adenovirus messenger RNA both early and late in the infectious cycle. The hybrids can be melted by heat in the top phase and the messenger RNA selected by oligo(dT)cellulose chromatography whereupon the polymers and the DNA percolate and the polyadenylated messenger RNA absorb to the column. The isolated messenger RNA appears to be almost quantitatively recovered at a purity from 70 to 90% depending on the concentration of the specific messenger RNA in the starting material. Early and late viral messenger RNA were selected on the complementary strands of adenovirus DNA according to this procedure.