Evidence for involvement of metalloendoproteases in a step in sea urchin gamete fusion.

Membrane fusion events are required in three steps in sea urchin fertilization: the acrosome reaction in sperm, fusion of the plasma membrane of acrosome-reacted sperm with the plasma membrane of the egg, and exocytosis of the contents of the egg cortical granules. We recently reported the involvement of a Zn2+-dependent metalloendoprotease in the acrosome reaction (Farach, H. C., D. I. Mundy, W. J. Strittmatter, and W. J. Lennarz. 1987. J. Biol. Chem. 262:5483-5487). In the current study, we investigated the possible involvement of metalloendoproteases in the two other fusion events of fertilization. The use of inhibitors of metalloendoproteases provided evidence that at least one of the fusion events subsequent to the acrosome reaction requires such enzymes. These inhibitors did not block the binding of sperm to egg or the process of cortical granule exocytosis. However, sperm-egg fusion, assayed by the ability of the bound sperm to establish cytoplasmic continuity with the egg, was inhibited by metalloendoprotease substrate. Thus, in addition to the acrosome reaction, an event in the gamete fusion process requires a metalloendoprotease.

Oxidative damage, plasma antioxidant capacity, and glucemic control in elderly NIDDM patients.

A study of oxidative damage was made in elderly noninsulin-dependent diabetes mellitus (NIDDM) patients. A statistically significant increase in glucose and fructosamine was found in fasting NIDDM patients, as well as an increase in the oxidation induced by tert-butyl hydroperoxide. The Total Reactive Antioxidant Potential (TRAP) of the plasma was much reduced (p < .02) and the uricemia was unchanged. The erythrocytes of diabetic patients show greater basal oxidation products (p < .05), and the susceptibility of the diabetic erythrocytes to oxidation injury was also shown to increase in the oxidation induced by t-BOOH (p < .05). Linear regression studies showed that TRAP was associated directly with uric acid (p < .05) and inversely with fructosamine and with glucose (p < .03 and p < .05 respectively) in patients with NIDDM, but not in the controls. The levels of fructosamine were found to be related to the basal damage of the red blood cells (direct correlation, p < .001). This study suggest an useful approach to diabetic oxidative stress for clinical settings.

ESR spectra of quasirandomly oriented centers: application to radiation damage centers in bone.

The angular dependence of the ESR line shape from polycrystalline samples due to incomplete randomness in the orientation of the crystallites is calculated for centers with axial symmetry. The effect of a preferential axis of orientation is considered and the results are applied to radiation produce centers in bone.

A spectrophotometric method for the determination of hydroperoxides in liposomes.

A modification of the Asakawa-Matsushita iodometric assay method for the determination of the content of lipid hydroperoxides was developed which permits the simultaneous processing of many samples of high lipid content. The method has the advantages of simplicity as well as good reproducibility, so it is not necessary to process standards with each determination. Our technique exceeds the sensitivity attained with other spectrophotometric determinations reported in the literature. The method requires the total elimination of water from the samples, and this was accomplished using an azeotropic mixture of ethanol:water of 96:4. The results obtained with liposomes indicate that the method is applicable to biological material limited to small volume samples, ranging 5-50 microliters. We want to emphasize that this method permits the study of the peroxidation process as function of time.

New limit on the neutrinoless betabeta decay of 130Te.

We report the present results of CUORICINO, a search for neutrinoless double-beta (0nu betabeta) decay of 130Te. The detector is an array of 62 TeO2 bolometers with a total active mass of 40.7 kg. The array is cooled by a dilution refrigerator shielded from environmental radioactivity and energetic neutrons, operated at approximately 8 mK in the Gran Sasso Underground Laboratory. No evidence for (0nu betabeta) decay was found and a new lower limit, T(1/2)(0nu) > or = 1.8 x 10(24) yr (90% C.L.) is set, corresponding to [m(nu)] < or = 0.2 to 1.1 eV, depending on the theoretical nuclear matrix elements used in the analysis.

The soluble metalloendoprotease required in myoblast fusion remains intracellular.

The fusion of myoblasts to myotubes requires an endogenous soluble metalloendoprotease. To determine whether this protease is released by fusing myoblasts, or stays within the cell, we examined the effects of membrane-impermeant and a membrane-permeant metalloendoprotease inhibitors. Membrane-permeant 1,10-phenanthroline, and membrane-impermeant bathophenanthroline disulfonic acid both inhibited soluble metalloendoprotease activity in homogenized myoblasts with equal potency. However, while 1,10-phenanthroline inhibited fusion, bathophenanthroline disulfonic acid had no effect. In addition, metalloendoprotease activity could not be detected in the media of fusing myoblasts, but was in the cells. These observations support the conclusion that the soluble metalloendoprotease required in fusion remains within the myoblast.

Coenzyme active site occupancy as an indicator of independence of the subunits of mitochondrial aspartate aminotransferase.

The enzyme, aspartate aminotransferase, is a dimer consisting of two identical subunits which contain overlapping subunit regions ( Eichele , G., Ford, G.C., Glor , M., Jansonius , J.N., Mavrides , C., and Christen , P. (1979) J. Mol. Biol. 133, 161-180), suggesting the possibility of subunit interactions. The structurally similar cytosolic isozyme exhibits noncooperative binding of pyridoxal 5'-phosphate ( Boettcher , M., and Martinez -Carrion, M. (1975) Biochemistry 14, 4528-4531; Relimpio , A., Iriarte , A., Chlebowski , J.F., and Martinez -Carrion, M. (1981) J. Biol. Chem. 256, 4478-4488) in which the apoenzyme/holoenzyme hybrid dimer shows a distinctive thermal stability. Using a nonequilibrium isoelectric focusing technique, it can be shown that mitochondrial aspartate aminotransferase also binds cofactor in a noncooperative random fashion. However, differential scanning calorimetry (DSC) thermograms show different characteristics from the cytosolic form. These differences are interpreted in terms of unique subunit interactions in this isozyme. Heating to the various DSC transition temperatures shows that the anomalous DSC thermograms in partially coenzyme-saturated apoenzyme preparations are due to a selective dissociation of apoenzyme subunits into monomers which are irreversibly denatured. The remaining holoenzyme monomers reassociate and form stable holoenzyme dimers. The net result is retention of the initial concentration of holoenzyme subunits present in any given mixture. Random occupancy of active sites and similar electrophoretic and DSC patterns upon heating of partially saturated apoenzyme preparations is observed whether the coenzyme, pyridoxal phosphate or pyridoxamine phosphate alone, or borohydride-reduced Schiff's bases of coenzyme-substrate analogue derivatives are used as active site directed ligands. The latter resemble covalent enzyme-substrate intermediates.

Properties of the active site lysyl residue of mitochondrial aspartate aminotransferase in solution.

Two vitamin B6 derivatives, N-bromoacetylpyridoxamine (BAPM) and its phosphate ester have been found to be affinity-labeling reagents for mitochondrial aspartate aminotransferase (EC 2.6.1.1). These derivatives were first shown to react with a critical sulfhydryl group in tryptophan synthase (Higgins, W., and Miles, E. W. (1978) J. Biol. Chem. 253, 4648-4652). In the apoaminotransferase, BAPM has now been found to inactivate by covalently modifying a critical lysyl residue, preventing reconstitution of the apoenzyme by pyridoxal 5'-phosphate. The dependence of the rate of inactivation upon the concentration of the reagent is consistent with a rapid equilibrium binary complex formation prior to the inactivation reaction. Both the dissociation constant for this complex and the rate of the reaction leading to inactivation are dependent on pH. BAPM binds best from pH 7.5 to 8.5. The rate of inactivation increases from pH 6 to 9. Succinate and phosphate competitively bind to the apoenzyme, protecting against BAPM inactivation. The C-5'-phosphorylated derivative is rapidly and tightly bound by the apotransaminase to form an inactive, noncovalent adduct. This bound reagent subsequently alkylates Lys-258. The rate of this covalent incorporation increases from pH 6 to 9 and is greater than the rate of BAPM modification at all pH values. The effect of pH on the reaction rates of both pyridoxal derivatives is interpreted to indicate protonation of Lys-258 at neutral pH values. These derivatives may also be analogs to a reaction intermediate different from those observed in other affinity-labeling studies. The ionization states of the Lys-258 epsilon-amino group apparently vary with the nature of the affinity label. These variations can be explained in terms of changing ionization states of Lys-258 in the steps of catalysis as well as in terms of the occupancy of charged sites on the protein by active site-directed substrates or inhibitory compounds.

Specific labeling of the active site of cytosolic aspartate aminotransferase through the use of a cofactor analogue, N-(Bromoacetyl)pyridoxamine.

The cofactor analogue N-(bromoacetyl)pyridoxamine (BAPM) has been employed to inactivate the cytosolic isozyme of apo-aspartate aminotransferase. Inactivation is the result of covalent bond formation in the (bromoacetyl)-pyridoxamine-transferase complex, via the epsilon-amino group of a lysyl residue at the active site. The stoichiometry of this inactivation is one molecule of (bromoacetyl)pyridoxamine per subunit of the transaminase dimer. Trace amounts of inorganic phosphate protect the enzyme from BAPM inactivation. In the absence of phosphate, inactivation demonstrates time, concentration, and pH dependence with an apparent pK for the target group of about 8.5 or higher. A tryptic peptide from the alpha subform has been obtained containing the carboxymethyl derivative of lysine-258, identifying this particular residue as the reactive group in the region of cofactor binding. Evidence is presented indicating that the pK of Lys-258 appears to be highly dependent upon the electrostatic state of neighboring groups in the active site region. Hence, experimentally obtained values vary according to the chemical nature and charge of the modifying agent or probe.

Reductive methylation as a probe of the heat-labile alpha-bungarotoxin-acetylcholine receptor membrane complex: evidence for surface interactions.

alpha-Bungarotoxin (alpha-Bgt) is a potent postsynaptic neurotoxin which blocks neurotransmission by binding very tightly to the acetylcholine-receptor (AcChR) protein. We have previously shown (P. Calvo-Fernandez, and M. Martinez-Carrion (1981) Arch. Biochem. Biophys. 208, 154-159) that alpha-Bgt free in its native solution conformation incorporates 12 methyl groups when reductively methylated using formaldehyde and sodium cyanoborohydride. We now show that when the alpha-Bgt molecule is bound to the AcChR contained in native membranes prepared from Torpedo californica electroplax, the number of accessible methylation sites is significantly reduced. This favors a model of alpha-Bgt-AcChR interaction involving significant numbers of lysyl moieties distributed over a reasonably large surface of the toxin molecule. In addition, this paper presents a novel procedure for the rapid and nondestructive dissociation of the toxin-AcChR membrane complex which takes advantage of the thermal instability of the complex.

Evidence for the involvement of metalloendoproteases in the acrosome reaction in sea urchin sperm.

An essential initial step in fertilization in the sea urchin Strongylocentrotus purpuratus is an intracellular membrane fusion event in the sperm known as the acrosome reaction. This Ca2+-dependent, exocytotic process involves fusion of the membrane of the acrosomal vesicle and the plasma membrane. Recently, metalloendoproteases requiring divalent metals have been implicated in several Ca2+-dependent membrane fusion events in other biological systems. In view of the suggested involvement of Zn2+ in the sea urchin sperm acrosome reaction (Clapper, D.L., Davis, J.A., Lamothe, P.J., Patton, C., and Epel, D. (1985) J. Cell Biol. 100, 1817-1824) and the fact that Zn2+ is a metal cofactor for metalloendoproteases, we investigated the potential role of this protease in the acrosome reaction. A soluble metalloendoprotease was demonstrated and characterized in sperm homogenates using the fluorogenic protease substrate succinyl-alanine-alanine-phenylalanine-4-aminomethylcoumarin. The protease was inhibited by the metal chelators EDTA and 1,10-phenanthroline, and activity of the inactive apoenzyme could be reconstituted with Zn2+. The metalloendoprotease substrate and inhibitors blocked the acrosome reaction induced either by egg jelly coat or by ionophore, but had no effect on the influx of Ca2+. These observations suggest that inhibition occurs at a step independent of Ca2+ entry. Overall, the results of this study provide strong indirect evidence that the acrosome reaction requires the action of metalloendoprotease.

Oxidative stress and membrane fluidity in erythrocytes from patients with hemolytic uremic syndrome.

In order to investigate the implications of oxidative disturbances in the hemolysis associated with the Hemolytic Uremic Syndrome (HUS), basal levels of lipid peroxidation products, the response to t-butyl hydroperoxide induced damage and membrane fluidity were assayed by the technique of electron spin resonance in erythrocytes spin labeled with 5-Doxyl stearic acid obtained from eight children with HUS, during the 1st, 2nd, 4th and 12th weeks after diagnosis. During the acute phase of the disease, red blood cells (RBC) showed increased initial lipid peroxidation products, a higher susceptibility to oxidative insult and a lower membrane fluidity. All parameters reached control values the 12th week after diagnosis. The results suggest that in the acute phase of HUS, RBCs are exposed to an oxidative imbalance that could contribute to hemolysis directly through oxidative damage and/or by decreasing membrane fluidity.