A. Barth

Multiple molekulare Formen einer Aminopeptidase aus Euglena gracilis

Summary The aminopeptidases from Euglena gracilis Klebs, strain Z, were investigated by polyacrylamide gel electrophoresis and affinity chromatography. Nine multiple moleculare forms (AP) from (Leucine)-aminopeptidase (AP6) were characterized by disc electrophoresis. They differ in molecular weight and in specificity against aminoacid naphthylamides. A disaggregation of (Leucine)-aminopeptidase (AP6) into active subunits API and APII by EDTA can be demonstrated.

Aminopeptidasen in Brassica napus: I. Isolierung einer relativ alaninspezifischen Aminopeptidase

Summary Germinated rape seeds contain five aminopeptidases of different specificity. One of those aminopeptidases, a relative alanine specific aminopeptidase, was isolated. This enzyme, purified by gel filtration on Sepharose 4B and Sephadex G-200, by ion exchange chromatography on DEAE-Sephadex A-50 and by preparative disc-electrophoresis, was shown to be electrophoretic homogeneous. The purified aminopeptidase was most active with L-alanine-4-(phenylazo)-phenylamide as the substrate. Glycine-, L-leucine- and L-lysine-4-(phenylazo)-phenylamides as well as few dipeptide- 4-(phenylazo)-phenylamides were also hydrolyzed but with lower rates. The molecular weight, determined by gel chromatography, was 79000, the isoelectric point was found at the p H value 5.25.

Zum Aminopeptidasemuster von Euglena gracilis1)

Summary The aminopeptidases in crude extracts prepared from Euglena gracilis were investigated by Polyacrylamide gel electrophoresis. Twelve different amino β-naphthylamide substrates were used for the determination of the aminopeptidase activity. Euglena crude extracts exhibit a number of aminopeptidases: Five bands of activity can be demonstrated. Four aminopeptidases were isolated and partially characterized. The isoenzymes (Alanine)-AP1 (IP 5.1) and (Alanine)-AP2 (IP 4.8) with the same molecular weight of 68,000 were separated by filtration on DEAE-Sephadex A-50.

Beschreibung einer Proliniminopeptidase aus Euglena gracilis: Description of a Proline Iminopeptidase from Euglena gracilis

Summary A proline iminopeptidase from Euglena gracilis was purified by a multi-step procedure and an approximately 70 to 80-fold purification was achieved. The proline iminopeptidase is capable of cleaving off N-terminal L-prolyl- and L-hydroxyprolyl residues from arylamide. The enzyme is completely homogen in polyacrylamide gel elektrophoresis. The enzymatic activity was not metal dependent (exept Hg ++ ) and was completely unaffected by p-chloromercuribenzoate and chloroacetylderivates. By kinetic analysis of L-proline-4-(phenylazo)-phenylamide hydrolysis with proline iminopeptidase a Km value of 1 × 10 −5 Mol/l, a p H-optimum of 7.5 and a temperature optimum of 55°C were detected.

Isolierung und Charakterisierung einer L-Leucin-aminopeptidase (LAP) und einer L-Alaninaminopeptidase (AAP) aus Euglena gracilis

Summary Two aminopeptidases (LAP and AAP) have been isolated from cytoplasma of Euglena gracilis Klebs (1224-5/25 strain Z). The aminopeptidases were purified from the cytoplasmatic fraction by precipitation with ammonium sulphate (40–80% saturation), by dialysis against 0.01 m Tris/HCl buffer pH 7.0 and by filtration on Sephadex G-200 and OM-Sephadex C-25. The pH optimum for AAP is 8.0 and for LAP is 7.2. By means of AAP and LAP the investigation of the hydrolysis of a series of amino acid-4-(phenylazo)-phenylamides with varying amino acid residue showed that L-alanine- and L-Ieucine-4-(phenylazo)-phenylamide are the best substrates. No enzymatic hydrolysis of the amino acid-4-(phenylazo)-phenylamides like D-alanine-, D-leucine-, D-valine-, L-cystine-, L-isoleucine-, D-phenylalanine-, γ-amino butyric acid- and sarcosine-4-(phenylazo)-phenylamide could be detected. The equation of the initial rate for the liberation of the first hydrolysis product (4-phenylazo-phenylamine) can be represented in both cases by the Michaelis-Menten law, modified by Briggs and Haldane.

Beiträge zur molekularen Morphologie der multiplen Aminopeptidaseformen aus Euglena gracilis Quartärstruktur von AP 6 und AP 4

Summary Electron micrographs of highly purified relative methionine-specific AP6 give an insight of the multiple aminopeptidase form from Euglena gracilis. By this way, conclusions can be drawn on the possible molecular morphology of the other enzyme forms of the aminopeptidase system, e.g. the proline-specific AP4* and possibly also of the relative alanine-specific charge isomers AP1 and AP2 The quaternary structure of AP4 explains the dynamics of the transition AP6 ⇄ AP4* as well as the different stabilities of the multiple amino-peptidase form AP6 and the charge isomers AP1 and AP2.

Chemical Structure and Growth Regulating Activity Relationship of Some Piperidinoacetanilides: I. Growth Retarding Activity and the Influence on Choline Esterase

Summary The present study deals with the growth retarding activity of piperidinoacetanilide hydrochlorides and N-methyl-piperidinoacetanilide iodides on pea and their influence on eholine esterase activity. The compounds possess a growth retarding activity ,which exceeds the activity of chlorocholine chloride. On the other hand the piperidinoacetanilide hydrochlorides and the N-methyl-piperidino-acetanilide iodides inhibit choline esterase activity. The orders of the action of substituents in meta position in the aromatic ring system for inhibition of the choline esterase activity and for retardation of plant growth are approximately equal.

Chemical Structure and Growth Regulating Activity Relationship of Some Piperidinoacetanilides: II. Inhibition of Chlorophyll Degradation in Detached Leaves and the Influence on Chlorophyllase Activity

Summary Piperidinoacetanilide hydrochlorides and N-methyl-piperidinoacetanilide iodides retard the degradation of chlorophyll in the process of senescence of detached radish leaves. N-Methyl-piperidinoacetanilide iodides possess a strong activity for retardation of chlorophyll degradation in detached barley leaves. The selectivity of action of this compounds on retardation of chlorophyll degradation is determined by the nature of the substituents and their position in the anilide ring. Piperidinoacetanilide hydrochlorides inhibit the chlorophyllase activity in radish leaves. The inhibitory activity is expressed more strongly in in vitro experiments than in experiments in vivo.

Multiple Molecular Forms of Aminopeptidases in Poppy Seedlings (Papaver somniferum L., cv. “Dubsky”)

Summary During the germination of poppy seeds (studied up to 92 h) the aminopeptidase activity in hypocotyl and cotyledons increased dependent on the germination time, whereas the activity decreased in the endosperm. By polyacrylamide gel electrophoresis of the enzyme extracts and subsequent incubation with Ala-β-Na 4 aminopeptidase forms (AP 4 , R F = 0.32; AP 3 , R F = 0.53; AP 2 , R F = 0.64; AP 1 , RF = 0.74) were found in the seeds and in the embryo after germination for 48 h, but only one aminopeptidase form (AP 1 , R F = 0.53) was found in the endosperm and in the roots. By use of Met- or Tyr-β-NA as substrates a “low-molecular” aminopeptidase form (AP 1 , R F = 0.74) was detected in seeds as well as in the embryo, endosperm and radicula. In the filtrate of ultrafiltration (Amicon filter: UM 10) of the crude homogenate an inhibitor fraction was detected acting on the activity of 3 aminoeptidase forms.

Aminopeptidasen in Brassica napus 1) II. Untersuchungen zum Einfluß von Metallionen, über Aminosäure-Zusammensetzung und katalytische Eigenschaften der relativ alaninspezifischen Aminopeptidase

Summary Evidence is presented that the relative alanine specific aminopeptidase does not exist as a metalenzyme-complex. Bivalent cations decrease the enzyme activity in the following order: Mg 2+ 2+ 2+ 2+ 2+ 2+ .– The kinetic parameters of the hydrolysis of p -substituted L-alanine-anilides are correlated with the electronic properties of the substituents. The results can be interpreted as evidence for nucleophilic properties of the active site. Sulfhydryl groups are important for the maintenance of the catalytic activity, although it is not clear whether they are part of the active center. The decline of the enzyme activity curve is hisghest up to two sulfhydryl groups being blocked by Hg 2+ or Ag 2+ . Reducing sulfhydryl compounds do not act as stabilizers or activators of the aminopeptidase. The amino acid composition of the aminopeptidase is characterized by a relative high content of hydrophobic amino acid residues and of residues with ionisable side chains.