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.     

Hydrophobic affinity partition of spinach chloroplasts in aqueous two-phase systems

The surface properties of spinach chloroplasts, both of intact chloroplasts with surrounding envelope and broken chloroplasts consisting of the inner lamellar system, have been studied by partitioning them between two aqueous phases, especially using counter-current distribution technique. The two-phase system consists of poly(ethyleneglycol), dextran and water. The two polymers are enriched in opposite phases and by binding deoxycholate or palmitate to one of the polymers the affinity of chloroplasts for the corresponding phase is strongly enhanced. The partition of the two classes of chloroplasts, however, is not affected to the same degree and the affinity of the chloroplast envelope for deoxycholate and palmitate is stronger than that of the lamellar system. This has been correlated to the chemical composition of the two types of membranes. By studying the effect of salts on the partition it has been found that the lamellar system bears a larger number of negative charges as compared to the envelope of the intact chloroplast.     

Roles of creatine in the regulation of cardiac protein synthesis

The observation that increased muscular activity leads to muscle hypertrophy is well known, but identification of the biochemical and physiological mechanisms by which this occurs remains an important problem. Experiments have been described (5, 6) which suggest that creatine, an end product of contraction, is involved in the control of contractile protein synthesis in differentiating skeletal muscle cells and may be the chemical signal coupling increased muscular activity and the increased muscular mass. During contraction, the creatine concentration in muscle transiently increases as creatine phosphate is hydrolyzed to regenerate ATP. In isometric contraction in skeletal muscle for example, Edwards and colleagues (3) have found that nearly all of the creatine phosphate is hydrolyzed. In this case, the creatine concentration is increased about twofold, and it is this transient change in creatine concentration which is postulated to lead to increased contractile protein synthesis. If creatine is found in several intracellular compartments, as suggested by Lee and Vissher (7), local changes in concentration may be greater then twofold. A specific effect on contractile protein synthesis seems reasonable in light of the work of Rabinowitz (13) and of Page et al. (11), among others, showing disproportionate accumulation of myofibrillar and mitochondrial proteins in response to work-induced hypertrophy and thyroxin-stimulated growth. Previous experiments (5, 6) have shown that skeletal muscles cells which have differentiated in vitro or in vivo synthesize myosin heavy-chain and actin, the major myofibrillar polypeptides, faster when supplied creatine in vitro. The stimulation is specific for contractile protein synthesis since neither the rate of myosin turnover nor the rates of synthesis of noncontractile protein and DNA are affected by creatine. The experiments reported in this communication were undertaken to test whether creatine selectively stimulates contractile protein synthesis in heart as it does in skeletal muscle.     

Purification of membrane proteins from Acholeplasma laidlawii by agarose suspension electrophoresis in Tween 20 and polyacrylamide and dextran gel electrophoresis in detergent-free media.

Four of the membrane proteins from Acholeplasma laidlawii that are soluble in the nonionic detergent Tween 20 have been purified by preparative electrophoretic techniques utilizing different supporting media. The last purification step for two of the major proteins was a preparative polyacrylamide gel electrophoresis performed in the absence of any detergent. The proteins were recovered by continuous elution. The purity of the fractions was examined by analytical polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. Two of the minor proteins were purified by dextran gel electrophoresis as the final step, which was also performed in a detergent-free buffer. The separation was followed by scanning the dextran gel in ultraviolet light. The proteins were recovered by slicing the gel and degrading the gel slices with dextranase. The homogeneity of the fractions was checked by electroimmunoassay.     

A quantitative model for partition in aqueous multiphase systems.

A model for the partition of charged molecules in aqueous multiphase systems has been developed. The partition coefficient of one component, or the overall partition coefficient of a number of components, between two arbitrary phases is expressed in terms of the difference in electrical potential between the phases (due to electrolytes present in the system), the net charges of the partitioned components and their partition coefficients in a (sometimes hypothetical) uncharged state. The fraction of material in one phase has also been described as a function of the net charges of the partitioned components. The model fits well to experimental data for partition of chromate, pyridine, ribonuclease A, two types of CO-hemoglobin and an enzyme mixture (yeast lysate) in three-phase systems consisting of poly(ethylene glycol), dextran, Ficoll and water. Minor deviations from the model are construed to be a pH-dependent uptake of ions. The data have also been used to detect differences in solvation of similar proteins, as well as the presence of several forms of some glycolytic enzymes present in yeast lysate.