Interaction of bovine heart lactate dehydrogenase with erythrocyte lipids.

The interaction between bovine heart lactate dehydrogenase and erythrocyte lipid suspension as a function of pH, NAD, NADH, lipid and salt concentration was studied by ultracentrifugation. In the presence of erythrocyte lipid liposomes the enzyme forms two kinds of complex: lactate dehydrogenase adsorbed to liposomes and soluble lactate dehydrogenase-phospholipid complexes. The two complexes reveal different dependence of their stability on pH values. Lactate dehydrogenase decreases its specific activity when it binds to the phospholipid molecules. Efficient adsorption of lactate dehydrogenase to liposomes occurs in their pH range 6.0-8.0 and at low ionic strength. The adsorption is diminished in the presence of NAD+ but it is not influenced by NADH. Possible mechanisms of the interaction and implications for the function in vivo are discussed.

Binding of glyceraldehyde-3-phosphate dehydrogenase to phospholipid liposomes.

The binding of glyceraldehyde-3-phosphate dehydrogenase prepared from rabbit muscle to phospholipid model membranes (liposomes) as a function of pH, ionic strength, and the influence of the binding on specific activity of the enzyme was studied. The binding decreases the specific activity of the enzyme. The binding was studied by the method of association of the enzyme with liposomes during centrifugation. The existence of a dominant interaction of electrostatic character was found.

Interaction of rabbit muscle aldolase with phospholipid liposomes.

The interaction between rabbit muscle fructose diphosphate aldolase and phospholipid model membranes (liposomes) was studied by measurement of the tryptophan fluorescence of the enzyme. Interaction with liposomes decreases intrinsic fluorescence intensity of the enzyme and shifts the emission wavelength maximum to higher values. The effects appear to be strongly dependent on the nature of the phospholipid polar group and on ionic strength. Also, a reversible modification of specific activity of aldolase upon interaction with liposomes was found. It is postulated that aldolase binds to liposomes mainly by electrostatic interactions and that the binding causes a change in the conformation of the enzyme.

Interaction of bovine skeletal muscle lactate dehydrogenase with liposomes. Comparison with the data for the heart enzyme.

The effects of pH, salt concentration and the presence of oxidized and reduced forms of coenzyme on the interaction of skeletal muscle lactate dehydrogenase with the liposomes derived from the total fraction of bovine erythrocyte lipids were investigated by ultracentrifugation and were compared with those results obtained using the heart-rate isoenzyme which we have previously studied. Liposomes are good adsorptive systems for both types of isoenzyme. In the presence of erythrocyte lipid liposomes, bovine muscle and heart lactate dehydrogenases form two kinds of complex: lactate dehydrogenase adsorbed to liposomes and soluble lactate dehydrogenase-phospholipid complexes. Soluble protein-phospholipid complexes reveal different dependences of their stabilities on pH values and it seems that the nature of the binding site in either isozyme is different. In addition, absorption of the isoenzymes on the liposomes also reveals in difference in the effects of NAD and NADH. While the presence of NAD dissociates LDH-H4 from the liposomes and NADH does not influence its adsorption, NAD promotes the binding of LDH-M4, and NADH favors the dissociation.

Interaction of bovine heart pyruvate kinase with phospholipids.

The interaction between bovine heart pyruvate kinase and liposomes was investigated for various phospholipids as function of pH, and salt concentration using steady-state kinetics and ultracentrifugation. Liposomes made from erythrocyte total lipid fraction and individual phospholipids were used. Pyruvate kinase specific activity increases upon the interaction with the phospholipids. The activation is specifically sensitive to presence of phosphatidylserine in liposomes. L-serine, and phospho-L-serine which are main components of phosphatidylserine head group show also some activation effect. Efficient adsorption of pyruvate kinase to phosphatidylserine liposomes occurs in the pH range 6.0-8.0 and at low ionic strength. Interaction with phosphatidylserine liposomes results in the change of Vmax and Km values for phospho enol pyruvate without marked effect on Km value for ADP, and Hill coefficients for both substrates. The interaction does not seem to influence the cooperativity between binding sites.

Fluorescent probe studies on binding of glyceraldehyde-3-phosphate dehydrogenase to phosphatidylinositol liposomes. Further evidence for conformational changes.

Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle can be absorbed on charged lipid bilayers by electrostatic forces. Upon binding to phosphatidylinositol liposomes the enzyme modifies its conformational state as it is shown by resonance energy transfer experiments. In the presence of 2-mercaptoethanol o-phthaldialdehyde reacts with amino groups of the protein and the covalently bound fluorophore is an acceptor of excitation energy transferred from tryptophanyl residues of the protein. The observed decrease of energy transfer efficiency upon binding to phosphatidylinositol liposomes is compared with the influence of the urea on the fluorescence spectra of the labelled protein. Significance of conformational changes of the enzyme upon adsorption on liposomes in the regulating function of cell membranes is discussed.

Modification of some properties of spherical lipid membranes induced by the association with fructose-1,6-bisphosphate aldolase.

The influence of fructose-1,6-bisphosphate aldolase, as a membrane peripheral protein, on some electrical and transport properties of spherical lipid membranes was investigated. It was found that the association of the enzyme with the membrane did not effect markedly the electrical conductance or capacity of the membrane but decreased the water filtration coefficient and the cationic transferance number. The enzyme association also modifies temperature characteristics of the membrane parameters.

Fluorescence investigation on conformational state of rabbit muscle aldolase interacting with phosphatidylinositol liposomes.

Evidence of conformational changes in rabbit muscle aldolase upon binding to phosphatidylinositol liposomes and the effect of the interaction on the thermal conformational transition are reported. Interaction with phosphatidylinositol liposomes significantly decreases the aldolase tryptophanyl fluorescence and shifts the maximum wavelength to higher values. The dynamic quenching constant for the aldolase fluorescence quenching by acrylamide in the presence of liposomes is much higher than that for unmodified enzyme; this signifies an increase in accessibility of some tryptophanyl residues to small polar molecules. Indirect interaction between single phospholipid molecules, small micelles or any soluble impurities able to penetrate into the protein molecule interior does not seem to be involved in the conformational rearrangement. Native and liposome-interaction-induced conformational states reveal different temperature dependences of the tryptophan residues exposure. The implications of the modification of the conformational state of the enzyme for its function in vivo are discussed.

Effect of 1-anilinonaphthalene-8-sulphonate on phase transition temperature of dipalmitoylphosphatidylcholine liposomes.

The effect of 1-anilinonaphthalene-8-sulfonate (ANS) on the thermotropic phase transition of dipalmitoylphosphatidylcholine (DPPC) bilayers was examined by differential scanning calorimetry. The main phase transition temperature was found to be shifted to lower values in the presence of the probe. The shift strongly depends on pH and the presence of salts. This indicates that the penetration of the probe of the hydrocarbon moiety of the bilayer is influenced by coulombic interactions. Pretransition phenomena are also affected. The implications for the interpretation of experimental data of biomembrane studies are discussed.

Role of cysteine endopeptidases in cancerogenesis.

Role of cysteine endopeptidases in cancerogenesis steps: neoplastic transformation, invasion and metastasis is reviewed and discussed. Positive correlation between tumor invasiveness, as well as its metastatic potential and secretion of cysteine endopeptidase (particularly cathepsins B and L) has been documented well in literature. Based on our recent results we postulate that serum endopeptidase-like activity could be used as a marker of cancer aggressiveness in diagnostic procedures in oncology. We also propose that the cysteine endopeptidase inhibitor levels (total, active and latent) could be useful factors for recognising the activation of the organism self-defence mechanisms against cancer. In addition, our idea of use of urinary cysteine peptidase inhibitors (UCPI) as potential anticancer agents is presented and discussed.

Liposome-interaction induced conformation changes of glyceraldehyde-3-phosphate dehydrogenase.

Tryptophanyl emission spectra of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were measured after the addition of liposomes prepared of natural phospholipids: phosphatidylinositols (PI), phosphatidylserines (PS) and phosphatidylcholines (PC). The measurings were made for various molar lipid/protein ratios (100-1000). A decrease in the enzyme fluorescence intensity and a "red" shift of the emission band maximum were observed. The susceptibility of the enzyme fluorescence to liposome action strongly depended on the kind of phospholipid and changed in the sequence PI greater than PS greater than PC. The presence of liposomes affected the accessibility of tryptophan residues for the fluorescence quencher (acrylamide). The results suggested that interaction induces some specific conformation changes in the enzyme molecules which may be responsible for modification of the enzyme activity. A comparison of the modification in fluorescence characteristics with those observed during denaturation suggested that the denaturation mechanism is not operative. Other possible mechanisms of the interaction are discussed.

Temperature studies of glyceraldehyde-3-phosphate dehydrogenase binding to liposomes using fluorescence technique.

Interaction of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase with negatively charged liposomes was investigated as a function of temperature. This interaction affects the temperature-dependent conformational transition in the enzyme and exerts stabilizing effect on the protein structure. It can be seen from the fluorescence quenching experiments that the accessibility of tryptophanyl residues and isoindol probe fluorophores (covalently bound with the protein amino groups) for a dynamic quencher, acrylamide, is altered upon binding. This accessibility represented by effective quenching constant (Keff) strongly depends on temperature for unmodified enzyme and for the enzyme adsorbed on liposomes, it is nearly constant over a wide range of temperatures.

Cysteine peptidase inhibitors and activator(s) in urine of patients with colorectal cancer.

The total activity of cysteine peptidase inhibitors and activator(s) was determined in the samples of urine received from colorectal cancer patients. Patients with peptic ulcer and healthy volunteers agreed to be a control group. The studies revealed a marked difference between the values of the determined parameters for the patients with colorectal cancer and those for the control group. Determination of cysteine peptidase inhibitors in patient's urine is proposed as a new diagnostic procedure.