Amphetamine-metabolites of deprenyl involved in protection against neurotoxicity induced by MPTP and 2'-methyl-MPTP.

The ability of 1-deprenyl to protect against the parkinsonian effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been attributed to the inhibition of conversion of MPTP to MPP+ (1-methyl-4-phenylpyridinium) catalyzed by MAO-B. We report here that deprenyl-treatment in mice has an additional neuroprotective element associated with the rapid metabolization of 1-deprenyl to 1-methamphetamine and 1-amphetamine. 1-Methamphetamine and 1-amphetamine inhibit MPP(+)-uptake into striatal synaptosomes prepared from rats. Post-treatment by 1-deprenyl, 1-methamphetamine, 1-amphetamine (at times when MPTP is no longer present in the striatum of mice) protects against neurotoxicity in C57BL mice by blocking the uptake of MPP+ into dopaminergic neurons, and even against the neurotoxicity induced by 2'CH3-MPTP, which is partly bioactivated by MAO-A. These findings may have clinical implications since deprenyl has recently been found to delay the progression of Parkinson's disease.

Dissimilar mechanisms of action of anticalmodulin drugs: quantitative analysis.

A novel molecule from the arylalkylamine family of drugs, KHL-8430, has been identified as a potent and specific inhibitor of calmodulin activity. The effect of this drug on calmodulin-mediated enzymatic actions has been analyzed to exemplify how to model the mechanism of action of a functional calmodulin antagonist. The approach used includes both binding and enzyme kinetic studies. In both types of experiments, the effects of drugs on calmodulin-phosphofructokinase [ATP:D[fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11] and calmodulin-phosphodiesterase (3':5' cyclic nucleotide phosphodiesterase, EC 3.6.1.3) interactions have been investigated. We have found that KHL-8430, in contrast to trifluoperazine, a classical anticalmodulin drug, competes with neither phosphofructokinase nor phosphodiesterase for calmodulin binding, yet it liberates phosphofructokinase from calmodulin inhibition and phosphodiesterase from calmodulin stimulation. The anticalmodulin activity occurs at lower KHL-8430 than trifluoperazine concentrations. These findings might establish the functional importance of these differences in the specificity of these drugs. The synthesis of the data suggests that (i) whereas trifluoperazine antagonizes both phosphofructokinase and phosphodiesterase binding to calmodulin, KHL-8430 interacts with calmodulin complexed with enzymes; (ii) KHL-8430 binds to the calmodulin-phosphofructokinase complex with an affinity constant of 0.8 microM, whereas the binding constant of trifluoperazine is 2.5 microM (iii) within the ternary complex the dimeric form of the kinase preserves activity that is otherwise inactive; and (iv) the binding of trifluoperazine and KHL-8430 to calmodulin exhibits negative cooperativity. The approach used in this study makes it possible to screen for the calmodulin antagonist effect of other drugs as well.

Substance P and neurokinin A, two coexisting tachykinins stimulating the release of [3H]5-HT from rat cerebral cortical slices.

Substance P (SP) and neurokinin A (NKA), two coexisting neuropeptides of the tachykinin family, stimulated the basal (5 mM K+) and evoked (40 mM K+) release of [3H]5-HT (5-hydroxytryptamine) from tissue slices of the rat cerebral cortex. Spantide ([DArg1,-DTrp7,9,Leu11]SP; 10(-5) M) inhibited the effects of SP but potentiated the effects of NKA. The effects of SP and NKA appear to be exerted at distinct receptors but involve a common post-receptor mechanism as no full additivity of the SP- and NKA-mediated stimulation of [3H]5-HT could be observed. The effects of the 3 tachykinins, SP, NKA and NKB, are compared with respect to stimulation of the release of [3H]5-HT.

Tachykinin regulation of serotonin release: enhancement of [3H]serotonin release from rat cerebral cortex by neuromedin K and substance P acting at distinct receptor sites.

Neuromedin K and substance P (SP) enhanced the basal (5 mM K+) and potassium-evoked (40 mM K+) release of [3H]5-hydroxytryptamine ([3H]5-HT) from slices of the rat cerebral cortex. The effects of the two tachykinins were additive. The SP antagonist span-tide ([D-Arg1, D-Trp7.9, Leu11]-SP) (10(-6) M) inhibited the stimulatory effect of SP but not of neuromedin K on the release of [3H]5-HT. These findings suggest that the two tachykinins exhibit their effects on serotonin release at distinct receptor sites.

Microcompartmentation of cAMP in wild-type and memory-mutant dunce strains of Drosophila melanogaster.

The "enzyme-probe" method [Solti M, Friedrich P: Eur J Biochem 95:551, 1979] has been applied to characterize the cyclic AMP pool in wild-type Canton-S and memory-mutant dunceM11 strains of Drosophila melanogaster. The kinetics of cyclic AMP breakdown in whole fly homogenates by endogenous cyclic nucleotide phosphodiesterase(s) indicate that the cyclic AMP pool is divided into free and bound fractions. The bound fraction in Canton-S and dunceM11 is 0.5 and 1.5 pmole/mg fly, respectively. Considering the total cyclic AMP content of the two strains, 1.6 and 10 pmole/mg fly, respectively, we conclude that the bulk of excess cyclic AMP in the mutant is free nucleotide.

Differences in protein phosphorylation in vivo and in vitro between wild type and dunce mutant strains of Drosophila melanogaster.

The protein phosphorylation patterns of wild type and dunce mutant strains of Drosophila melanogaster, as detected by sodium dodecylsulfate-gel electrophoresis and autoradiography, have been compared. After labelling in vivo with 32Pi or in vitro in homogenates with [gamma-32P]ATP, radioactive bands at and above apparent polypeptide mol. wt approximately 110,000 were more pronounced in dunce fly heads than in wild type heads. When labelling in vitro, in dunceM11 there appeared a radioactive band at apparent mol. wt approximately equal to 53,000 that was faintly visible in the wild strain. The same band could be intensified in both strains by adding cyclic AMP to the homogenate or by performing homogenization in the presence of theophylline. The data suggest that the mol. wt approximately equal to 53,000 protein is a substrate for cyclic AMP-dependent protein kinase.

Cyclic nucleotide phosphodiesterases in larval brain of wild type and dunce mutant strains of Drosophila melanogaster: isoenzyme pattern and activation by Ca2+/calmodulin.

Like adult heads and whole flies, larval brains of wild type Drosophila melanogaster contain two major soluble cyclic nucleotide phosphodiesterases, forms I and II. Larval brains of the learning-defective mutant strain, dunceM11, contain only the form I enzyme. In both wild type and dunce strains the form I enzyme is activated by Ca2+/calmodulin. A time-dependent loss of this Ca2+ activation was observed.

Localization of glyceraldehyde-3-phosphate dehydrogenase in intact human erythrocytes. Evaluation of membrane adherence is autoradiographs at low grain density.

Human erythrocytes were treated with highly tritiated [3H]iodoacetate under conditions when half of the label became attached to glyceraldehyde-3-phosphate dehydrogenase. After fixation, the cells were subjected to electron microscopic autoradiography. For the evaluation of distribution of grains, which were relatively few, a computerized method was developed. Statistical analysis of data showed the significant adherence of grains to the cell membrane. The results support the view that glyceraldehyde-3-phosphate dehydrogenase is localized near the membrane in the intact erythrocyte.

The 'enzyme-probe' method for characterizing metabolite pools. The use of NAD-glycohydrolase in human erythrocyte sonicate as a model system.

1. An approach for testing the homogeneity of metabolite pools is described. An alien enzyme that can attack the metabolite in question is introducted into the system studied. By analyzing the time-course of decomposition of the metabolite it can be decided whether the pool is homogeneous in respect of reactivity towards the probe-enzyme or can be divided into fractions of different reactivities. 2. The information obtainable from such experiments is illustrated by the case of human erythrocyte sonicate as model system with NAD-glycohydrolase as probe-enzyme. The nicotinamide adenine dinucleotide pool in the concentrated sonicate could be resolved into three fractions (I, II and III) with half-lives of about 1, 7 and 240 min, respectively. Fraction I is free NAD, fraction II is NAD bound to glyceraldehyde-3-phosphate dehydrogenase, and fraction III is coenzyme strongly bound to some, so far unidentified, protein. Sonicate glycolysis seems to require only fraction II and is unable to use fraction III under the experimental conditions applied. 3. The scope of application of the enzyme-probe method is discussed.

Partial reversible inactivation of enzymes due to binding to the human erythrocyte membrane.

Hypotonic human erythrocyte ghosts, devoid of the original glyceraldehyde-3-phosphate dehydrogenase content of the red cell, bind added glyceraldehyde-3-phosphate dehydrogenases, isolated from human erythrocytes, rabbit and pig muscle, as well as rabbit muscle aldolase. There are only slight differences in the affinities towards the various glyceraldehyde-3-phosphate dehydrogenases. On the other hand, glyceraldehyde-3-phosphate dehydrogenases are bound much stronger than aldolase; in an equimolar mixture the former can prevent the binding of the latter, or replace previously bound aldolase at the membrane surface. Binding is always accompanied by the partial inactivation of enzymes, which can be reverted by desorption. Unwashed ghosts rich in hemoglobin seem to have a more pronounced inactivating effect on bound glyceraldehyde-3-phosphate dehydrogenase. In isotonic media ghosts, whether white or unwashed, reseal and do not interact with the enzymes.

Cross-linking and coupling of rabbit muscle aldolase and glyceraldehyde-3-phosphate dehydrogenase by glutaraldehyde.

The mode of cross-linking of rabbit-muscle aldolase and glyceraldehyde-3-phosphate dehydrogenase by glutaraldehyde was studied. The about 5 A long reagent can partly cross-link subunits within the tetramers, whereas it is readily able to make intermolecular cross-links producing polymeric enzyme species. Of the two enzymes, glyceraldehyde-3-phosphate dehydrogenase has a greater tendency to polymerize in the presence of glutaraldehyde. In the case of aldolase, the inter- and intramolecular cross-links between subunits can be distinguished by SDS gel-electrophoresis. The copolymerization pattern of the two enzymes indicates that, though the formation of mixed polyenzymes can be detected by affinity chromatography on human erythrocyte ghosts, under the conditions tested these proteins do not form heterologous enzyme complexes that could be trapped by glutaraldehyde.

Some structural features of rabbit muscle aldolase as derived from its limited proteolysis.

The peptides released during the limited tryptic proteolysis of rabbit muscle aldolase (Biszku et al., 1973) were located in the primary structure. The pattern of peptide liberation, peptide bond splitting and activity decrease in compatible with two structural models for the truncated tetrameric product, named aldolase-T. According to the more probable model aldolase-T has the structure A+A+B++B++. Subunits B++ are deprived of the segments comprising residues 1-27, 42-71 and 306-364 of the intact enzyme and are inactive. The fragment comprising residues 28-41 is non-covalently attached to these subunits. Subunits A+ are depleted only of peptides 1-27 and 324-332 and retain 70% activity. In these subunits the fragment comprising residue 333-364 remains non-covalently bound. The molecular weights of the truncated subunits, determined with polyacrylamide-gel electrophoresis in the presence of sodium dodecylsulfate support the above conclusions. Aldolase-T can be reversibly denatured at pH 2 or in 4 M urea. The recovery of enzymatic activity after decreasing urea or acid concentration indicates the non-covalent rebinding of fragment 333-364. This fragment is named the "T-peptide" of trypsin-treated aldolase. It is suggested that segments 1-27 and 324-364 are not necessary for the renaturation process. Since aldolase-T is a tetramer it seems that large parts of the N- and C-terminal regions of the enzyme are not involved in the intersubunit interactions. The C-terminal region of aldolase, starting around residue 324, appears to be necessary to the structure of the active site. In contrast to this, the N-terminal region up to residue 27 and probably to residue 60, is not part of the active center.