Dipeptidyl peptidases in the soleus muscle of the rat before and after treatment with 5-hydroxytryptamine

A moderate peptidase activity against L-lysyl-L-proline-4-methoxy-beta-napththylamide was detected histochemically in unfixed sections of soleus muscle fibres of inbred male Wistar rats using two variants of the semipermeable membrane technique. One variant involved simultaneous coupling with tetrazotised 3,3'-dimethoxybenzidine, the other post-coupling. The activity at pH 6 increased approximately three-fold in many fibres showing signs of insult in rats that had been given a single low dose of 5-hydroxytryptamine (10 mg/kg body weight) 48-72 h earlier. The hydroxytryptamine treatment was found to induce a selective myopathy. Some of the increased peptidase activity within insulted muscle fibres appeared to arise from invading mononuclear cells, but the majority seemed endogenous to muscle fibres. The peptidase activity persisted in some fibres 21-28 days after 5-hydroxytryptamine administration, by which time the whole muscle appeared histologically normal. The variation of the activity of the peptidase with pH in the presence of various inhibitors was investigated in both control and insulted muscle fibres. From its sensitivity and behaviour towards Zn2+, Hg2+, Cu2+, puromycin, benzethonium chloride and phenylmethylsulphonyl fluoride and its indifference towards Co2+, Cd2+, Mn2+ and o-phenanthroline, it is concluded that the activity can be attributed to a mixture of at least two peptidases, dipeptidyl peptidase II and an unidentified neutral dipeptidyl peptidase. The possible role of the peptidase(s) in muscle regeneration in discussed.     

Mitochondrial alpha-glycerol phosphate dehydrogenase activity in IIA fibres of the rat lateral gastrocnemius muscle; the effect of Ca2+ and ATP

Summary Mitochondrial -glycerol phosphate dehydrogenase is an important enzyme, but it is difficult to extract and purify. We have measured the activity of this enzyme in single type IIA skeletal muscle fibres under initial rate conditions by microdensitometry of the formazan reaction product.The K_m (1.6mm) for the substrate (l--glycerol phosphate) was lower than reported for the extracted enzyme. Further, at low substrate concentrations (3mm), the enzyme was allosterically activated by free Ca²⁺ concentrations of 1 m or greater, and half-maximal stimulation occurred at 0.3 m free Ca²⁺. In the absence of Ca²⁺, there was negative cooperativity of substrate binding with a Hill constant of 0.57, but no cooperativity occurred in the presence of calcium. ATP (10mm) inhibited enzyme activity in the presence of Ca²⁺ but not in its absence.     

Biochemical characterization of glutamine synthetase from the diazotrophic cyanobacterium,Anabaena doliolum

In cyanobacteria, the glutamine synthetase-L-glutamine-2-oxoglutarate aminotransferase (GS-GOGAT) pathway is the major ammonia-assimilating route. The GS ofAnabaena doliolum was synthesized more under N₂-fixing conditions, followed by ammonium, nitrate, and nitrite as nitrogen sources. The activities of both the glutamine synthetase, Mg²⁺-dependent biosynthetic and Mn²⁺-dependent -glutamyl transferase were optimum at pH 7. The active site of the enzyme bears sulfhydryl (-SH) groups; this was confirmed with the-SH group inhibitors, para-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM). The biosynthetic and -glutamyl transferase activities showed specificity for the divalent cations, Mg²⁺ and Mn²⁺, respectively. The other divalent cations Co²⁺, Cu²⁺, and Ni²⁺ were poor substitutes. This enzyme also required these divalent cations to stabilize its structure and function under extreme conditions such as high and low temperatures and urea denaturation. The glutamate analogl-methionine-d,l-sulfoximine, inactivated the enzyme, whereas the GOGAT inhibitor, azaserine, had no effect on the enzyme activity. The GS enzyme required de novo protein synthesis.     

Release of serotonin from nerve endings byl-5-hydroxytryptophan,α-methyl-m-tyramine,and elevated potassium ions

The release of [³H]5-hydroxytryptamine ([³H]5-HT) byl-5-hydroxytryptophan (L-5-HTP),-methyl-m-tyramine (-MMTA), and elevated levels of K⁺ was studied using crude synaptosomal preparations (P₂) isolated from the telencephalon of the rat and pigeon. Studies were conducted in vitro in the presence of either 2×10^–5 M tranylcypromine, which inhibited the MAO activity of both the extrasynaptosomal mitochondria and the mitochondria contained within the nerve endings (intrasynaptosomal mitochondria), or 2×10^–5 M nialamide, which inhibited the MAO activity of the extrasynaptosomal mitochondria under the experimental conditions used. In the P₂ fraction isolated from the rat, either 55 mM K⁺, 0.10 mMl-5-HTP, or 0.03 mM-MMTA significantly increased the release of [³H]5-HT above control levels, regardless of which MAO inhibitor was present in the medium. In the presence of tranylcypromine, this increased release by 55 mM K⁺ or 0.10 mMl-5-HTP was partially suppressed if Ca²⁺ was omitted from the medium. In the presence of nialamide, the release by 55 mM K⁺ was completely prevented if Ca²⁺ was omitted; the release byl-5-HTP was only partially affected. The release of [³H]5-HT by-MMTA did not appear to be markedly affected by removal of Ca²⁺, regardless of which MAO inhibitor was present. Very similar data were obtained in the presence of nialamide using the P₂ fraction isolated from the telencephalon of the pigeon, with the exception that 0.10 mMl-5-HTP caused an increase in the release of [³H]5-HIAA (which was not calcium-dependent) instead of [³H]5-HT. The data are discussed in     

Activation of two types of fibres in ferret,Mustela putorius furo,cremaster muscle

Summary Some contractile, histochemical, morphological and electrophysiological properties of ferret, Mustela putorius furo, cremaster muscle have been estimated. Histochemical fibre typing revealed the presence of two types of fibres (type I 66.2%, type II 33.8%). Morphometry performed on ATPase-stained transverse sections showed that type I was composed of a large amount (40%) of small(<400 m²) cells. In mammalian Ringer two groups of fibres could be recognized on the basis of the values of resting potential (-69.7 mV and-59.1 mV) and intracellular sodium activity (8.3 mmol·l^-1 and 14.1 mmol·l^-1, respectively). In experiments on fibre bundles, the elevation of extracellular potassium concentration to 15–200 mmol·l^-1 produced contractures that consisted of a well-defined transient or phasic tension followed by a sustained or tonic tension. Properties of activation and inactivation of the tension analysed in small bundles of cut fibres (lengths 0.5–1.0 cm) were of fast- and slow-twitch type for phasic and tonic phase, respectively. In contrast to the phasic component of K contractures, the tonic phase was abolished by Ca²⁺ withdrawal and inhibited by Ni²⁺, Cd²⁺, Co²⁺, Gd³⁺ and gallopamil (D600). In Ca²⁺-free medium the sustained tension was restored by adding Sr²⁺. It is concluded that in ferret cremaster muscle the presence of slow-twitch fibres would give rise to the tonic component of the K contracture in which an extracellular source of activator Ca²⁺ is involved. The ability of these fibres to contract with a maintained tension for prolonged periods of time might participate in the temperature regulation of the testes.Abbreviations a _i ^Na intracellular sodium activity - ATPase myosin adenosine triphosphatase - D600 gallopamil - E _m membrane potential - E _r resting potential - EDL muscle, extensor digitorum longus muscle - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - e.c. excitation-contraction - SDHase succinate dehydrogenase - NADHase nicotinamide adenine, dinucleotide hydrogen-diaphorase - SOL muscle, soleus muscle - T time constant of relaxation - TEACI tetraethylammonium chloride - [Ca]_o, [K]_o, [Na]_o extracellular calcium, potassium, sodium concentration     

Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Summary Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed and increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and -N-acetyl-d-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and -glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the German Research Foundation (Sfb 174)     

Pharmacological characterization of glutamate binding sites in cultured cerebellar granule cells and cortical astrocytes

Membranes prepared from cerebellar granule cells and cortical astrocytes exhibited specific, saturable binding ofl-[³H]glutamate. The apparent binding constant K _d was 135 nM and 393 nM and the maximal binding capacity B_max 42 and 34 mol/kg in granule cells and astrocytes, respectively. In granule cells the binding was strongly inhibited by the glutamate receptor agonists kainate, quisqualate, N-methyl-d-aspartate (NMDA),l-homocysteate and ibotenate, and the antagonistdl-5-aminophosphonovalerate. In astrocytes, only quisqualate among these was effective.l-Aspartate,l-cysteate,l-cysteinesulphinate and -d-glutamylglycine were inhibitors in both cell types. The binding was totally displaced in both cell types byl-cysteinesulphinate with IC₅₀ in the micromolar range. In astrocytes the binding was also totally displaced by quisqualate, but in granule cells only partially by NMDA, kainate and quisqualate in turn. It is concluded from the relative potencies of agonists and antagonists in [³H]glutamate binding that cerebellar granule cells express the NMDA, kainate and quisqualate types of the glutamate receptor, while only the quisqualate-sensitive binding seems to be present in cortical astrocytes.     

Production and properties of asparaginase from a new Erwinia sp

Asparaginase production by a mesophilic strain ofErwinia sp. was examined; the maximum of activity was found at 40°C and pH 8.5. Among the various carbon sources, mannitol was shown to be the best for production of activity. Inorganic nitrogen sources were better than the organic ones. The enzyme activity was not inhibited by 10 mmol/L metal ions (Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Co²⁺, Ni²⁺, Zn²⁺); the activity was strongly inhibited by addition of EDTA.l-Arginine,dl-alanine,l-asparagine andl-glutamine stimulated thel-asparaginase production by 3.9, 1.7, 4.3 and 4.0 fold, respectively. The combination ofl-arginine,l-asparagine andl-glutamine synergistically stimulated the asparaginase up to 5.8 fold.     

l-threonine production by l-aspartate- and l-homoserine-resistant mutant of Escherichia coli

Summary Growth and l-threonine productivity of l-threonine producer Escherichia coli H-4290 were inhibited by precursor amino acids, l-homoserine and l-aspartate. l-Threonine hyper-producers were isolated among the mutants resistant to l-homoserine and l-aspartate. Mutants H-4351 (Hom^r) and H-4578 (Hom^r, Asp^r) accumulated 22.2 g/l and 24.3 g/l of l-threonine in test tube cultures, while the parental strain H-4290 accumulated 18.2 g/l. The enzyme level of aspartokinase I (first enzyme of the threonine operon) was enhanced 2.3 times (H-4351) and 3 times (H-4578) that of H-4290. Mutant H-4578 accumulated 76 g/l of l-threonine in a 2-l jar fermentor after 75 h cultivation.Abbreviations DAP diaminopimeric acid - Met ^l poor growth in methionine-free medium - AHV -amino--hydroxyvaleric acid - Thr-N^- lack of ability to utilize l-threonine as a nitrogen source - Rif rifampicin - Lys+Met^r resistant to l-lysine and dl-methionine     

Photoproduction of ammonium by Chlamydomonas reinhardtii cells immobilized in barium alginate: a reactor feasibility study

Summary Chlamydomonas reinhardtii cells immobilized in Ba-alginate beads provide a stable and effective system for photoproducing ammonium from nitrite in a culture medium containing l-methionine-d,l-sulphoximine. The process was studied in either air-lift, fluidized or packed-bed reactors, the last one providing the most suitable system with a volumetric activity of 2700 mol NH _inf+ ^sup4 ·1^–1 per hour.     

Gene cloning and biochemical characterization of eryngase,a serine aminopeptidase of Pleurotus eryngii belonging to the family S9 peptidases

Pleurotus eryngii serine aminopeptidase that has peptide bond formation activity, redesignated as eryngase, was cloned and expressed. Eryngase has a family S9 peptidase unit in the C-terminal region having a catalytic triad of Ser, Asp, and His. In the phylogenetic relations among the subfamilies of family S9 peptidase (S9A, prolyl oligopeptidase; S9B, dipeptidyl peptidase; S9C, acylaminoacyl peptidase; S9D, glutamyl endopeptidase), eryngase existed alone in the neighbor of S9C subfamily. Mutation of the active site Ser524 of the eryngase with Ala eliminated its catalytic activity. In contrast, S524C mutant maintained low catalytic activity. Investigation of aminolysis activity using l-Phe-NH₂ as a substrate showed that S524C mutant exhibited no hydrolysis reaction but synthesized a small amount of l-Phe-l-Phe-NH₂ by the catalysis of aminolysis. In contrast, wild-type eryngase hydrolyzed the product of aminolysis l-Phe-l-Phe-NH₂. Results show that the S524C mutant preferentially catalyzed aminolysis when on an l-Phe-NH₂ substrate.     

Proton transfer in and polarizability of hydrogen bonds in proteins. Tyrosine-lysine and glutamic acid-lysine hydrogen bonds — IR investigations

The OH N O^– H⁺N hydrogen bonds formed between tyrosine and lysine, and between glutamic acid and lysine residues are studied by infrared spectroscopy considering the following systems: (l-lys)_n + phenol, copoly (l-lys, l-tyr)_n, (l-lys)_n + (l-tyr)_n and (l-lys)_n + (l-glu)_n. The phenol-lysine hydrogen bonds are largely symmetrical in the average if the pK_a of the protonated lysine is 2.2 units larger than that of the phenols. In the case of the hydrogen bonds between tyrosine and lysine residues in copoly (l-lys, l-tyr)_n and (l-lys)_n + (l-tyr)_n, the weight of the proton limiting structure OH N is 80–90%, and that of the polar O^– H⁺N structure 10–20%. Double minimum proton potentials occur but the proton is preferentially present at the tyrosine residues. In the (l-lys)_n + (l-glu)_n system, the protons are present at the lysine residues. Thus, these hydrogen bonds have very large dipole moments (about 10 D). With the lysine-phenole hydrogen bonds, hydration shifts the proton transfer equilibrium a little in favour of the polar proton limiting structure O^– H⁺N. These hydrogen bonds are broken to a large extent, however, when only about 3 water molecules are present per lysine residue. When less water is present, as in the copoly (l-lys, l-tyr)_n and (l-lys)_n + (l-tyr)_n systems, these hydrogen bonds are, however, formed quantitatively. Thus — as discussed in this paper — the tyrosine-lysine hydrogen bonds can participate in proton conducting hydrogen bonded systems — as, for instance, present in bacteriorhodopsin — performing the proton transport through hydrophobic regions of biological membranes.     

Short communication: Purification and properties of a glucose-forming amylase of Lactobacillus brevis

An extracellular glucose-forming amylase was produced by Lactobacillus brevis isolated from Kagasok tea. The enzyme was purified 70-fold and had optimal activity at 55°C and pH 6.5. Its K _m value for starch was 0.27 mg ml^-1 and its M _r was approx. 75,900 Da. The activity of the enzyme was enhanced by Ca²⁺, Mg²⁺, Na⁺ or K⁺ and inhibited by EDTA, KCN, citric acid and l-cysteine.     

Endogenous γ-l-glutamylglutamate is a partial agonist at the N-methyl-d-aspartate receptors in cultured cerebellar granule cells

-l-Glutamylglutamate (LGG), an endogenous constituent of the brain, reduced the glutamateevoked increase in intracellular Ca²⁺ in cultured cerebellar granule cells. The extent and properties of this inhibition were different at different Mg²⁺ concentrations. The intracellular Ca²⁺ response to NMDA was slightly enhanced by 0.1 mM LGG in normal (1.3 mM) Mg²⁺ medium, but in Mg²⁺-free medium LGG was stimulatory at low (0.1–1 M) NMDA and inhibitory at high (0.1–1 mM) NMDA concentrations. In the absence of Mg²⁺, LGG alone increased cytosolic free Ca²⁺ and depolarized the cells. These effects were potentiated by glycine and blocked by extracellular Mg²⁺, 2-amino-5-phosphonopentanoate (APV), 7-chlorokynurenate, 3-amino-1-hydroxypyrrolidin-2-one (HA-966) and 5,7-dinitroquinoxaline-2,3-dione (MNQX). The results indicate that LGG is a partial NMDA agonist. On the other hand, the non-NMDA antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) also inhibited the effects of LGG. This indicates an involvement of non-NMDA receptors in the actions of LGG. The consequent depolarization may also contribute to the activation of NMDA receptor-governed ionophores.     

Folding versatility of the C-terminal tetrapeptide amide sequence of the lipopeptaibol antibiotics trichodecenin and trichogin

Summary An X-ray diffraction analysis ofZ-l-Leu-Aib-Gly-l-Ile-l-Leu-OMe, containing the N-acylated tetrapeptide amide sequence-l-Leu-Aib-Gly-l-Ile-, showed that in the crystal state the carbonyl group preceding thel-Leu¹ residue acts as the acceptor of two C=OH–N intramolecular H-bonds, which give rise to an-l-Leu¹-Aib²-type-III' -turn and an-l-Leu¹-Aib²-Gly³-l-Ile⁴--turn, respectively. A second (type-I') -turn encompasses the-Aib²-Gly³-sequence. This is the third type of folding motif known for that tetrapeptide sequence, considering also those already published for the C-terminal segment of the lipopeptaibol antibiotics trichodecenin I and trichogin A IV.     

Apical plasma membrane-bound enzymes of rabbit uterine epithelium

Summary In order to monitor changes in the apical cell membrane of rabbit uterine epithelium which are postulated to be a precondition for trophoblast attachment, the marker enzymes: alkaline phosphatase, aminopeptidase M, -glutamyl transferase and dipeptidyl peptidase IV were investigated during the periimplantation phase. Endometrium of early pregnancy (implantation chamber, interblastocyst endometrium; 5–8 days post coitum, d p.c.) was compared with specimens obtained at hCG-induced pseudopregnancy (p. hCG) to distinguish between membrane changes regulated by maternal plasma steroid hormones and such which might be induced locally by blastocyst-derived signals.All enzymes tested showed their main activity at 5 d p.c./p. hCG. The weakest reaction in this series of stages was generally found at 8 d p.c. (interblastocyst segments) or at 8 d p. hCG. In contrast to the rest of the epithelium, the implantation chamber retained high activity of dipeptidyl peptidase IV, and the activity of alkaline phosphatase even raised here again at 7 and 8 d p.c. indicating a direct local influence of the blastocyst on the luminal epithelium. The results suggest that 1) considerable changes occur in the composition of the apical plasma membrane of the uterine epithelium when the endometrium enters the receptive state, 2) the overall trend is towards a loss of apical-type characteristics of this membrane domain and 3) the changes are modulated both systemically (by plasma steroid hormone levels) and locally by signals from the implanting blastocyst.Abbreviations d p.c. days post coitum - d. p. hCG days post hCG injection - hCG human chorionic gonadotropin - aP alkaline phosphatase - ATPase adenosine triphosphatase - Ca²⁺-ATPase Ca²⁺-activated adenosine triphosphatase - APM aminopeptidase M - GGT -glutamyl transferase - DPP IV dipeptidyl peptidase IV - PCMB p-chloromercuric benzoate - DFP di-isopropylfluorophosphate - DMF dimethylformamide     

Silver ions induce a rapid Ca2+ release from isolated intact bovine rod outer segments by a cooperative mechanism

Summary Micromolar concentrations of silver ion activate large Ca²⁺ fluxes across the plasma membrane of intact rod outer segments isolated from bovine retinas (intact ROS). The rate of Ag⁺-induced Ca²⁺ efflux from intact ROS depended on the Ag⁺ concentration in a sigmoidal manner suggesting a cooperative mechanism with a Hill coefficient between 2 and 3. At a concentration of 50 m Ag⁺ the rate of Ca²⁺ efflux was 7×10⁶ Ca²⁺/outer segment/sec; this represents a change in total intracellular Ca²⁺ by 0.7mm/outer segment/sec. Addition of the nonselective ionophore gramicidin in the absence of external alkali cations greatly reduced the Ag⁺-induced Ca²⁺ efflux from intact ROS, apparently by enabling internal alkali cations to leak out. Adding back alkali cations to the external medium restored Ag⁺-induced Ca²⁺ efflux when gramicidin was present. In the presence of gramicidin, Ag⁺-induced Ca²⁺ efflux from intact ROS was blocked by 50 m tetracaine orl-cis diltiazem, whereas without gramicidin both blockers were ineffective. Bothl-cis diltiazem and tetracaine are blockers of one kinetic component of cGMP-induced Ca²⁺ flux across ROS disk membranes. The ion selectivity of the Ag⁺-induced pathway proved to be broad with little discrimination between the alkali cations Li⁺, Na⁺, K⁺, and Cs⁺ or between Ca²⁺ and Mg²⁺. The properties of the Ag⁺-induced pathway(s) suggest that it may reflect the cGMP-dependent conductance opened in the absence of cGMP by silver ions.     

The peptidase activity of human serum butyrylcholinesterase: Studies using monoclonal antibodies and characterization of the peptidase

Purified human serum butyrylcholinesterase, which exhibits cholinesterase, aryl acylamidase, and peptidase activities, was cross-reacted with two different monoclonal antibodies raised against human serum butyrylcholinesterase. All three activities were immunoprecipitable at different dilutions of the two monoclonal antibodies. At the highest concentration of the antibodies used, nearly 100% of all three activities were precipitated, and could be recovered to 90–95% in the immunoprecipitate. The peptidase activity exhibited by the purified butyryl-cholinesterase was further characterized using both Phe-Leu and Leu-enkephalin as substrates. ThepH optimum of the peptidase was in the range of 7.5–9.5 and the divalent cations Co²⁺, Mn²⁺, and Zn²⁺ stimulated its activity. EDTA and other metal complexing agents inhibited its activity. Thiol agents and -SH group modifiers had no effect. The serine protease inhibitors, diisopropylfluorophosphate and phenyl methyl sulfonyl fluoride, did not inhibit. When histidine residues in the enzyme were modified by diethylpyrocarbonate, the peptidase activity was not affected, but the stimulatory effect of Co²⁺, Mn²⁺, and Zn²⁺ disappeared, suggesting the involvement of histidine residues in metal ion binding. These general characteristics of the peptidase activity were also exhibited by a 50 kD fragment obtained by limited -chymotrypsin digestion of purified butyrylcholinesterase. Under all assay conditions, the peptidase released the two amino acids, leucine and phenylalanine, from the carboxy terminus of Leu-enkephalin as verified by paper chromatography and HPLC analysis. The results suggested that the peptidase behaved like a serine, cysteine, thiol-independent metallopeptidase.     

L-lysine is a barbiturate-like anticonvulsant and modulator of the benzodiazepine receptor

Our earlier observations showed thatl-lysine enhanced the activity of diazepam against seizures induced by pentylenetetrazol (PTZ), and increased the affinity of benzodiazepine receptor binding in a manner additive to that caused by -aminobutyric acid (GABA). The present paper provides additional evidence to show thatl-lysine has central nervous system depressant-like characteristics.l-lysine enhanced [³H]flunitrazepam (FTZ) binding in brain membranes was dose-dependent and stimulated by chloride, bromide and iodide, but not fluoride. Enhancement of [³H]FTZ binding byl-lysine at a fixed concentration was increased by GABA but inhibited by pentobarbital between 10^–7 to 10^–3M. While GABA enhancement of [³H]FTZ binding was inhibited by the GABA mimetics imidazole acetic acid and tetrahydroisoxazol pyridinol, the enhancement by pentobarbital andl-lysine of [³H]FTZ binding was dose-dependently increased by these two GABA mimetics. The above results suggest thatl-lysine and pentobarbital acted at the same site of the GABA/benzodiazepine receptor complex which was different from the GABA binding site. The benzodiazepine receptor antagonist imidazodiazepine Ro15-1788 blocked the antiseizure activity of diazepam against PTZ. Similar to pentobarbital, the anti-PTZ effect ofl-lysine was not blocked by Ro15-1788. Picrotoxinin and the GABA, receptor antagonist bicuculline partially inhibitedl-lysine's enhancement of [³H]FTZ binding with the IC₅₀s of 2 M and 0.1 M, respectively. The convulsant benzodiazepine Ro5-3663 dose-dependently inhibited the enhancement of [³H]FTZ binding byl-lysine. This article shows the basic amino acidl-lysine to have a central nervous system depressant characteristics with an anti-PTZ seizure activity and an enhancement of [³H]FTZ binding similar to that of barbiturates but different from GABA.     

Aminopeptidase A is angiotensinase A

Summary Quantitative histochemical measurements of aminopeptidase A (APA; E.C.3.4.11.7) were done kinetically in the kidney glomeruli of rat and mouse with an instrumental setup consisting of a microdensitometer and a computer-supported morphometric system. The histochemical demonstration of APA was carried out using the simultaneous azo coupling technique (purest-grade Fast Blue B as coupling agent and -l-glutamic acid-4-methoxy-2-naphthylamide as substrate). The methodological studies show that APA activity is calcium-ion-dependent and increases linearly with the thickness of the tissue section (3–12 m) and that the time-course of APA activity as determined by linear regression is linear only for the first 1 to 2 min of the reaction. — Kinetic measurements indicate a 40% decrease in APA activities when -l-glutamic acid-4-methoxy-2-naphthylamide (-l-Glu-MNA) is replaced by -l-aspartic acid-4-methoxy-2-naphthylamide. When -l-Glu-MNA is replaced with l-alanine-4-methoxy-2-naphthylamide, which is a substrate of aminopeptidase M (APM) only very low reaction rates are measurable (about 1.4% of those with -l-Glu-MNA). 100 and 130 mM NaCl in the incubation medium increase APA activities by approximately 16%–17%. — To clarify the functional importance of APA in the kidney, their activities were measured under the influence of angiotensins. The glomerulus was selected as the measuring site, for besides APA it contains no APM or other peptidases that could degrade angiotensins (the glomerular dipeptidyl peptidase IV is not inhibited by angiotensin II). Using the Lineweaver-Burk plot, we determined a K _m of 0.16 mM for the APA in rat glomeruli and 0.14 mM in mouse glomeruli. The V _max in mouse glomeruli is 1.6 times higher than in rat glomeruli. Ang iotensin I, II and III competitively inhibit APA in the rat and mouse glomeruli. — With quantitative histochemical techniques it was possible to show that APA is equivalent to angiotensinase A (splitting off the N-terminal aspartic acid from angiotensin I and II).Supported by the Deutsche Forschungsgemeinschaft (SFB 105)