Invivo inactivation of γ-aminobutyric acid-α-ketoglutarate transaminase by 4-amino-5-fluoropentanoic acid

Intraperitoneal injection into mice of varying concentrations of (S)-4-amino-5-fluoropentanoic acid ((S)-AFPA) produces a dose-dependent irreversible decrease in brain γ-aminobutyric acid-α-ketoglutaric acid aminotransferase (E.C. 2.6.1.19) activity. Concomitant with this inactivation is an increase in whole brain γ-aminobutyric acid (GABA) levels. Four hours after a dose of 100 mg/kg body weight of (S)-AFPA to mice, endogenous brain GABA concentrations increase to 16 times that of the untreated animals and the enzyme activity decreases to 20% that of the controls. The binding of (S)-AFPA to GABA receptors was more than three orders of magnitude poorer than for GABA itself.     

An essential arginyl residue in yeast hexokinase.

The inactivation of yeast hexokinase A (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) by phenylglyoxal obeys pseudo first-order kinetics. Formation of a reversible enzyme-reagent complex prior to modification is suggested by the observed saturation kinetics. Loss of activity correlates with the incorporation of 1 mol of [14C]phenylglyoxal per mol 50 000 dalton subunit. No significant conformational change occurs concomitantly. Inactivation is attributable to modification of an arginyl residue. The pattern of protection by substrates and analogs favors an interaction of this essential residue with the terminal phosphoryl group of ATP or glucose 6-phosphate.     

An essential arginyl residue at the nucleotide binding site of creatine kinase.

Treatment of rabbit muscle creatine kinase (EC 2.4.3.2) with either butanedione in borate buffer or phenylglyoxal in Veronal buffer decreases enzymatic activity correlating with the modification of a single arginyl residue per subunit of the dimeric enzyme. Very little activity is lost when modification is performed in the presence of MgATP or MgADP. Nucleotide binding to the modified enzyme is virtually abolished as determined by ultraviolet difference spectroscopy. The data suggest that an arginyl residue plays an essential role in the enzymatic mechanism of creatine kinase, probably as a recognition site for the negatively charged oligophosphate moiety of the nucleotide.     

An essential arginyl residue in the tonoplast pyrophosphatase from etiolated mung bean seedlings

Tonoplast membrane of etiolated mung bean (Vinga radiata. L.) seedlings contained H⁺-translocating pyrophosphatase (PPase). Modification of tonoplast vesicles and partially purified PPase from etiolated mung bean seedlings with arginine-specific reagents, phenylglyoxal (PGO) and 2,3-butanedione (BD), resulted in a marked decline in H⁺-translocating PPase activity. The half-maximal inhibition was brought about by 20 millimolar PGO and 50 millimolar BD for membrane bound and 1.5 millimolar PGO and 5.0 millimolar BD for soluble PPase, respectively. The substrate, Mg²⁺-pyrophosphate, provided partial protection against inactivation by these reagents. Loss of activity of partially purified PPase followed pseudo-first order kinetics. The double logarithm plots of pseudo-first order rate constant versus reagent concentrations gave slopes of 0.88 (PGO) and 0.90 (BD), respectively, suggesting that the inactivation may possibly result from reaction of at least one arginyl residue at the active site of H⁺-translocating PPase.     

Identification of an essential arginine residue in the beta subunit of the chloroplast ATPase

The ATPase activity of soluble chloroplast coupling factor (CF1) was irreversibly inactivated by phenylglyoxal, an arginine reagent. Under the conditions of inactivation, 2.48 mol of [14C]phenylglyoxal were incorporated per 400,000 g of enzyme when the ATPase was inactivated 50% by the reagent. Isolation of the component polypeptide subunits of the [14C]phenylglyoxal-modified enzyme revealed that the distribution of moles of labeled reagent/mol of subunit was the following: alpha, 0.37; beta, 0.40; gamma, 0.08; delta, none; epsilon, 0.03. CNBr treatment of the isolated alpha and beta subunits and fractionation of the peptides by gel electrophoresis revealed that the radioactivity bound to the alpha subunit was nonspecifically associated with several peptides, while a single peptide derived from the beta subunit contained the majority of the radioactivity associated with this subunit. After treating the isolated beta subunit with trypsin and Staphylococcus aureus protease, a major radioactive peptide was isolated with a sequence Arg-Ile-Thr-Ser-Ile-Lys. This sequence, when compared with the primary structure of the CF1 beta subunit as translated from the gene (Zurawski, G., Bottomley, W., and Whitfeld, P. R. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6260-6264) indicated that the arginine marked with the asterisk, the predominant residue modified by phenylglyoxal when the ATPase activity of CF1 is inactivated by the reagent, is Arg 312.     

Polyamine metabolism and function in brain

Putrescine, spermidine and spermine are simple alipathic polycations of ubiquitous occurrence. The pathways of biosynthesis and catabolism, and changes of the concentrations of these compounds in brain under various conditions are discussed.The pharmacological properties of the polyamines have not yet revealed functions which are characteristic only for the CNS, but preliminary evidence suggests structural roles in membranes and a modulatory function in certain neuronal systems.     

Moderate concentrations of 4-O-methylhonokiol potentiate GABAA receptor currents stronger than honokiol

Background

Magnolia bark preparations from Magnolia officinalis of Asian medicinal systems are known for their muscle relaxant effect and anticonvulsant activity. These CNS related effects are ascribed to the presence of the biphenyl-type neolignans honokiol and magnolol that exert a potentiating effect on GABA_A receptors. 4-O-methylhonokiol isolated from seeds of the North-American M. grandiflora was compared to honokiol for its activity to potentiate GABA_A receptors and its GABA_A receptor subtype-specificity was established.

Methods

Different recombinant GABA_A receptors were functionally expressed in Xenopus oocytes and electrophysiological techniques were used determine to their modulation by 4-O-methylhonokiol.

Results

3 μM 4-O-methylhonokiol is shown here to potentiate responses of the α₁β₂γ₂ GABA_A receptor about 20-fold stronger than the same concentration of honokiol. In the present study potentiation by 4-O-methylhonokiol is also detailed for 12 GABA_A receptor subtypes to assess GABA_A receptor subunits that are responsible for the potentiating effect.

Conclusion

The much higher potentiation of GABA_A receptors at identical concentrations of 4-O-methylhonokiol as compared to honokiol parallels previous observations made in other systems of potentiated pharmacological activity of 4-O-methylhonokiol over honokiol.

General significance

The results point to the use of 4-O-methylhonokiol as a lead for GABA_A receptor potentiation and corroborate the use of M. grandiflora seeds against convulsions in Mexican folk medicine.     

Involvement of an essential arginyl residue in the coupling activity of Rhodospirillum rubrum chromatophores.

The arginine reagents phenylglyoxal and 2,3-butanedione in borate buffer completely inhibited photophosphorylation and Mg-ATPase of Rhodospirillum rubrum chromatophores. The inactivation rates followed apparent first order kinetics. Oxidative phospho-rylation and the light-dependent ATP-P_i exchange reactions ofR. rubrum chromatophores and the Ca-ATPase activity of the soluble coupling factor were similarly inhibited by 2,3-butanedione in borate buffer. The apparent order of reaction with respect to inhibitor concentrations for all these reactions gave values of near 1 suggesting that inactivation was the consequence of modifying one arginine per active site. ATP synthesis and hydrolysis by R. rubrum chromatophores were strongly protected against inactivation by ADP and ATP, respectively, and by other nucleotides that are substrates of the reactions but not by the products. Similarly, the Ca-ATPase of the soluble coupling factor was protected by ATP but not by ADP. Inactivation of chromatophores reactions by butanedione in borate buffer was more rapid in the light than in the dark. The results suggest that the catalytic sites for ATP synthesis and hydrolysis on the chromatophore coupling factor are different and both contain an essential arginine.     

Regulation of nuclear uptake of "activated" receptor-glucocorticoid complex by pyrophosphate

The desialylated human chorionic gonadotropin α- and β-subunits were combined with their native complementary subunits and the thyrotropic activities of the recombinants were compared to those of native and desialylated human chorionic gonadotropin using human thyroid membranes. All the combined forms interacted with the thyrotropin receptor-adenylate cyclase system, but only those with sialic acid residues present on the α-subunit were able to activate the enzyme. These data support the concept that the α-subunit contains the domain through which this hormone activates adenylate cyclase.     

Arginyl residue modification of the sarcoplasmic reticulum ATPase protein.

Glucocerebrosidase, a membrane bound enzyme, can be solubilized by acetone precipitation and the resultant soluble enzyme activity demonstrated in the presence of acidic phospholipid, e.g. phosphatidylserine. This is the first report of the detergent-free solubilization of glucocerebrosidase.     

Elevated serum and hepatic tyrosine aminotransferase in voles chronically infected with Trypanosoma brucei gambiense.

Chronic infections of voles (Microtus montanus) with Trypanosoma brucei gambiense result in marked elevations in both serum and hepatic tyrosine aminotransferase. Serum enzyme levels correlate poorly with hepatic levels but well with parasitemia. Because the trypanosome itself contains high levels of this same enzyme, it is hypothesized that lysis of extravascular parasites, possibly due to agglutination by variant-specific antibody, releases parasite enzymes into host tissues, resulting in the observed increase in serum enzyme levels. The possible role of increased tyrosine transamination in the behavioral syndrome of trypanosome-infected voles is considered.     

Transaminases in rat retina during development

Aspartate, alanine and tyrosine aminotransferase activities have been determined in rat retina during postnatal development. Aspartate transaminase activity is rather low at birth; however, it increases as a function of age, reaching its maximum value at the 23rd day. Alanine transaminase activity increases more rapidly than aspartate transaminase, reaching its maximal value 15 days after birth, but, unlike the latter, this activity considerably decreases during further development. Neonatal tyrosine aminotransferase activity increases by 3 fold between the 5th and 15th day of life.The behaviour of the three enzymes investigated has been related to functional and morphological differentiation of retinal cellular layers.     

Hydrogen exchange into soluble spinach chloroplast coupling factor during heat activation of its ATPase

Exchange of 500–600 atoms of ³H per mol of solubilized spinach chloroplast coupling factor (CF₁) occurs when the enzyme is incubated for 4 min in ³H₂O at 63°C. These ³H atoms are bound in parts of the protein where exchange is hindered by the three-dimensional structure at 25°C. Back-exchange at 25°C shows complex kinetics, with at least two kinetic components having half-times of 1.4 and 40 h, respectively. Back-exchange from the denatured enzyme is extremely rapid with an apparent half-time of the order of 20–30 s. The time courses for exchange and ATPase activation are very similar at 63°C, and reasonably close at 25°C. Both reactions have an optimum temperature of 60°C when measured after 4 min. Activation of ATPase requires a strong reducing agent to be present, but this is not needed for hydrogen exchange. It is suggested that an open conformation of CF₁ induced by heat may be a required intermediate for the rapid activation of ATPase, being a sporadic and rare occurrence at 25°C but also a required step in ATPase activation. This open conformation could be related to that induced in bound CF₁ by thylakoid membrane energization.     

Fasciola hepatica: the presence of particle-associated and soluble nonspecific acid phosphatases.

The kinetic and physical properties of acid phosphatases in the lysosomal and microsomal fractions of F. hepatica were found to be similar, indicating that they are one and the same enzyme. In contrast, the biochemical properties of the soluble acid phosphatase (EC 3.1.3.2) were quite different from those of the lysosomal and microsomal fractions. This indicated the presence of two distinct forms of the enzyme one particle associated and the other soluble. Electrophoretic heterogeneity of these two types of acid phosphomonoesterase was seen. Two bands of activity were observed in both lysosomal and microsomal fractions and three bands in the soluble fraction.     

Sulphydryl groups in photosynthetic energy conservation. IV. Inhibition of the ATPase of chloroplast coupling factor 1 by sulphydryl reagents

1. o-Iodosobenzoate and 2,2′-dithio bis-(5-nitropyridine) inhibited by about fifty per cent the ATPase activity of heat-activated chloroplast coupling factor 1 only when present during the heating but were without effect when added before or after the activation. Reversion of this inhibition was only obtained by a second heat treatment with 10 mM dithioerythritol.2. The inhibition of the Ca²⁺-ATPase of coupling factor 1 by o-iodosobenzoate or 2,2′-dithio bis-(5-nitropyridine) was not additive with similar inhibitions obtained with the alkylating reagents iodoacetamide and N-ethylmaleimide.3. The heat-activated ATPase of o-iodosobenzoate-treated coupling factor 1 had a higher K_m for ATP, without modification of V. The modified enzyme was desensitized against the allosteric inhibitor ADP.     

In vitro protein synthesizing activity of rat liver as influenced by a physiological dose of cortisone and dibutyryl cyclic AMP.

A single administration of a low dose of cortisone acetate (0.2 mg/100 g) into adrenalectomized rats reduces the cell-free protein synthesizing activity of isolated total liver polysomes by 60 % after 90 min. DBcAMP also inhibits markedly the protein synthesizing activity but its effect is due to an inactivation of soluble fraction. Combined administration of both drugs yields polysomes active more than twice than those of untreated control. Inhibition by the glucocorticoid was restricted only to the membrane-bound polysomes.