Lothar Jaenicke

Cadmium/zinc-metallothionein induces DNA strand breaks in vitro

The in vitro DNA strand breaking activity of metallothionein (MT) containing Cd2+ and Zn2+ in a molar ratio of 5∶2 is described. Studies with radical scavengers and electron paramagnetic resonance spectroscopy indicate that the DNA damage might be caused by a radical species formed by the native protein (i.e., MT) charged with the heavy metal ions. No DNA strand breaks are detectable with the heat-denatured MT or with Cd2+ or Zn2+ alone. Inhibition studies using EDTA as a metal ion chelator orN-ethylmaleimide to alkylate sulfhydryl groups suggest that both the bound heavy metal ions as well as the SH groups of the various cysteine residues of MT may be involved in the MT-dependent DNA cleavage. Further characterization showed that the DNA cleavage is more likely random than sequence- or base-specific. These observations may provide a clue in the search for initial events in Cd-related carcinogenicity.

Purification and characterization of trimming glucosidase I from Saccharomyces cerevisiae

Glucosidase I was purified about 1900‐fold from yeast microsomal preparations by DEAE‐Sephacel chromatography, affinity chromatography on AH‐Sepharose 4B‐linked N‐5‐carboxypentyl‐1‐deoxynojirimycin and Con A‐Sepharose chromatography. The enzyme is a glycoprotein with a subunit molecular mass of 95 kDa. Its reaction has a pH optimum close to 6.8 and does not require metal ions. Purified glucosidase I hydrolyses the distal α1,2‐linked glucose residue from the Glc3‐Man9‐GlcNAc2 chain of its natural substrate, but is not active against Glc2‐Man9‐GlcNAc2 and aryl‐α‐glucosides. Like glucosidase I from calf liver, the yeast enzyme is strongly inhibited by 1‐deoxynojirimycin (dNM), N‐methyl‐dNM and N‐5‐carboxypentyl‐dNM with K i values of 16, 0.3 and 3μM, respectively.

ON THE STRUCTURE OF OXYBLEPHARISMIN AND ITS FORMATION FROM BLEPHARISMIN

Abstract The blepharismins ( 1 ) from Blepharisma japonicum give the corresponding oxyblepharismins ( 2 ) on irradiation in vitro and in vivo . The chemical structures of these compounds are elucidated and a mechanism is given for this unusual transformation.

Cystophorene and hormosirene, sperm attractants in Australian brown algae

found that the prominent absorbance peaks in I-IPLC chromatograms of crude extracts of cockroach corpora cardiaca correspond to the myoactive as well as to the hypertrehalosaemic fractions; myoactive and hypertrehalosaemic factors have identical amino acid compositions and the purified peptides show the same chromatographic characteristics; on a molar basis, the synthetic myoactive factors are as active in causing an increase of carbohydrates in cockroach blood as the hypertrehalosaemic hormones. These facts make it very likely that cockroach myoactive factors M I and M II and hypertrehalosaemic hormones I and II are the same compounds. Thus, after more than 20 years research, the structure of cockroach hypertrehalosaemic hormones is most likely known. Interestingly, the structure of M I (and thus of hypertrehalosaemic hormone I) is the same as the structure of neurohormone D, a factor from cockroach corpus cardiacum that has cardioexcitatory action ([10] and E. Baumann, Universit/it Jena, DDR, personal communication). Furthermore, cockroach hypertrehalosaemic hormones I and II show structural similarities to the decapeptide locust adipokinetic hormone and the octapeptide crustacean red pigment-concentrating hormone: pGlu-Leu-Asn-Phe-Ser-ProGly-Trp-NH 2 [11[. Interestingly, structurally related peptides have also been found recently in corpora cardiaca of other insect species. Although the amino acid sequence of these neuropeptides is not known yet, amino acid composition data revealed nonapeptides as the bioactive material in the stick insect Carausius morosus (G. G/ide, unpublished results) and in the moth Manduca sexta (R. Ziegler, Universitfit Berlin, FRG, personal communication). Both peptides are not identical with locust adipokinetic hormone. The structure of these nonapeptides is anxiously awaited.

Pheromone-Binding and Matrix-Mediated Events in Sexual Induction of Volvox carteri

Sexual differentiation of the reproductive cells (gonidia) of the green alga Volvox carteri f. nagariensis Iyengar is triggered by a glycoprotein inducer which is released into the medium by sexual male spheroids. If added, it induces the next generation cycle to become sexual. About 3000 pheromone molecules per spheroid will give full induction. Specific binding sites for the pheromone have been identified in the extracellular matrix in which characteristic events take place. Inducer binding and action are reversible for a certain time period until the start of cell division; thereafter, determination of the gonidia is final. Extent of sexual induction is proportional to the duration of inducer pulses. Inhibition of sexual induction reveals as essential extracellular components of the induction system high molecular acidic glycoproteins, cyclic nucleotides, Ca2+/calmodulin, and pheromone-controlled gene products. Low matrix levels of cyclic AMP and cyclic GMP seem to stimulate induction; high levels to inhibit.

Pheromone-triggered gamete release in Chorda tomentosa

1. Silverstein, R.M., et al. : Science 159, 889 (1968); Kinzer, G.W., et al. : Nature 221, 477 (1969) 2. Kohnle, U. : Diss. Univ. Freiburg i.Br. (in prep.) 3. Renwick, J.A.A., Vit6, J.P,, in: Chemie der Pflanzenschutzund Sch~idlingsbekfimpfungsmittel, Vol. 3, p. 1 (R. Wegler, ed.). Berlin: Springer 1980 4. Schurig, V., et al. : Naturwissenschaften 70, 92 (1983) 5. Stock, A.J., Borden, J.H. : Can. Entomol. 115, 539 (1983) 6. Nielssen, A.C.: Entomol. scand. 10, 219 (1979) 7. Postner, M., in: Die Forstsch/idlinge Europas, Vol. 2, p. 334 (W. Schwencke, ed.). Hamburg: Parey 1974

Site and time of formation of the sex-inducing glycoprotein in Volvox carteri

The colony-forming green alga Voluox carteri offers the opportunity to study sexual differentiation in a simple and well-controllable system. The presence (or absence) of an inducing hormone in the medium determines whether the algae grow sexually or asexually. Under the influence of this sex inducer the cleavage pattern of the reproductive cells (gonidia) is altered resulting in a sexual embryo which contains up to 64 gynogonidia (eggs) or 256 androgonidia (sperm-packets) instead of an asexual embryo containing up to 16 vegetative gonidia (details in [ 1 I). We have considerable knowledge about the biochemical nature of the sex inducer: it is a singlechain glycoprotein of 27 SOOM, [2]. The carbohydrate residues make up -40% of the molecule, and the hormone interacts strongly with certain lectins, free and Sepharose-bound (unpublished). However, as far as the origin of the sex inducer is concerned, we only know that the hormone is exclusively secreted by the male strain, appearing in the medium when spermpackets are released. Here, we try to answer the following questions: (i) Which type of cells: viz, somatic cells, androgonidia, sperm-packets; are responsible for the hormone production? (ii) When and for how long is the sex inducer excreted? (iii) Does the translation of the hormone’s protein portion and/or its glycosylation immediately precede the release? The evidence presented here indicates that the sperm-packets are the site ofhormone production and release. The sex inducer is excreted for 10 h beginning

Enantioselective synthesis of dictyopterene C 6R-(−)-butyl-2,5-cycloheptadiene the pheromone of several dictyotales (Phaeophyceae)

Abstract R-(-)-Dictyopterene C ( 1 ) is a widespread constituent of many marine brown algae (Phaeophyceae). A highly enantioselective synthesis of i and its enantiomer ent- 1 via chromatographic separation of the diastereomeric γ-hydroxyphenylethylamide intermediates 3a and 3b is described.

D-Hydroxynitrile lyase: Involvement of the prosthetic flavin adenine dinucleotide in enzyme activity

Although freely reversible, it is probably used in the plant only in the direction shown, since the formation of nitriles follows a different path [2] . The enzyme was studied by Rosenthaler [3] and eventually purified [4], when it, surprisingly, turned out to be a flavoprotein, containing FAD as the prosthetic group. The holoenzyme of 74 000 dalton is a single polypeptide with 1 mol FAD rather tightly bound cKd lo-* M-’ ; corrected value from fluorometric titration data of the apo-enzyme). The colourless inactive apoprotein, obtained by precipitation with acid ammonium sulfate according to [5] , also of 74 000 dalton, denatures at neutral pH with a half time of 20 min (25°C). It is readily reactivated by FAD, but not by FMN, or riboflavin. No change in sedimentation behaviour is observed on binding of the prosthetic group. As the cofactor is obviously required for a reaction not involving oxidation/reduction of a substrate, the question as to the function of the FAD arises: is it providing groups for the formation of reactive intermediates or is it stabilizing a catalytically active con-

Evidence for radical species as intermediates in cadmium/zinc-metallothionein-dependent DNA damage in vitro.

Toxicologic data on cadmium (Cd) indicate that intracellular metallothionein (MT) is protective for Cd exposure, whereas extracellular Cd-containing MT might be toxic. Moreover, Cd is suspected to be a carcinogen though the underlying mechanism is not known. Here we report on the genotoxic activity of cadmium/zinc-metallothionein (Cd/Zn-MT) in a cell-free test system: a concentration-dependent increase in DNA strand breaks was detected with increasing doses of Cd/Zn-MT, whereas no DNA strand breaks were observed in the presence of heat-denatured MT or Cd or Zn ions alone. Modifications of native Cd/Zn-MT by the metal ion-chelating agent EDTA or the sulfhydryl group alkylating agents N-ethylmaleimide and iodoacetamide suggest that the various cysteine residues of MT, together with the attached heavy metal ions, may be involved in the DNA cleavage reaction. Furthermore, DNA strand breaks caused by Cd/Zn-MT seem more likely to be random than sequence- or base-specific. Results from experiments with radical scavengers and electron spin resonance spectroscopy point to radical species formed by Cd/Zn-MT as mediators of the DNA damage. Thus, the actual activity of Cd/Zn-MT--whether protective or damaging--appears to depend on various parameters governed by the extra- and intracellular environment. ImagesFigure 1.