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Our knowledge on the preparation and physicochemica1 properties of metallothionein has improved in recent years [l-l 51. On the other hand, a highly polymeric sulphur and copper rich protein called neonatal hepatic mitochondrocuprein was described by Porter [ 16-221. Although this protein bears the name cuprein, it proved completely different from those intracellular cupreins containing 2 g-atoms of each of copper and zinc and which display superoxide dismutase and singlet oxygen decontaminating activities (for a review see [23] ). The neonatal type cuprein normally being present under physiological conditions in newborn species was also found in mitochondria of the livers of adults suffering from Wilson’s disease. Apart from these polymeric sulphur rich copper proteins some low molecular weight species of similar compositions were found [22,24,25] . It was assumed that the copper is in the cuprous state [24,25] and bound with the cysteine sulphur. Due to the similar amino acid composition of both the metallothionein and neonatal hepatic mitochondrocuprein Porter suggested some relationship to metallothionein [22]. In this context we were highly interested in the possibility of a direct conversion of metallothionein into Cu-thionein. The partially loaded Cu-thionein was expected to polymerize via disulphide bridges in the presence of oxygen to form the ‘neonatal type

Metallothionein descriptive for both its unique metal binding capacity and its high sulphur content belongs to a group of most actively studied metalloproteins [l-l 51. Some metal ions including Zn2+, Cd*’ and to a minor extent Hg’+ and Cu’ appeared to be preferentially bound by the protein portion [4]. In general, however, the above metal ions are present as arbitrary mixtures in metallo-thioneins isolated from the different biological tissues. Evidence of naturally occurring monodispers cysteine rich proteins exclusively loaded with either zinc or copper ions was recently shown [ 16-191. The functional side of all these metalloproteins remains obscure. It was sug gested that apart from the metal-free thionein they may act as regulatory components in the biochemistry of the above metal ions. For example, the mercury binding capacity of the thioneins is extraordinarily high [4,5] and it was thought that this kind of mercury chelation would avoid the deleterious reactivity of free Hg”. In this context we were especially interested in preparing a monodispers Hg-thionein from rat liver metallo-thionein (Cd-, Zn-thionein). Cd’+ and Zn*+ were successfully displaced by excessive HgC12 employing the gel filtration technique [20] . The purified protein contained 8 g atoms of Hg’+. The X-ray photoelectron spectra revealed a marked chemical shift of the S2p1,2,3,2 core electrons by 1 eV compared to the binding energy of the corresponding sulphur 2p level of native metallo-thionein. This phenomenon supports the conclusion of a strong polarisation of the mercury

The formation of a ternary complex using cupric ions, basic amines of biological significance, and nucleotides was examined employing high-resolution nuclear magnetic resonance (NMR) with 13C and 31P and circular dichroism. The reaction of Cu2+ with each ligand was studied separately and as the ternary complex. From 13C-NMR data the assignment of the respective carbon atoms adjacent to a copper-binding atom was clearly shown. It is intriguing to note that even in such a ternary complex the N-7 of the purine ring, as well as the phosphate residue (measured by 13P-NMR spectroscopy) provide the preferred binding sites for the cupric ion. Marked interactions of the chelated cupric ion with the ribose moiety in the ternary complex were deduced from circular dichroic measurements. No such reactivity was seen when dAMP was employed. The existence of a ternary Cu · AMP · amine complex opens a number of new aspects regarding the reactivity of metal ions in the biochemistry of nucleic acids.

Intraperitoneal injections of insulin into rats appeared to increase the rate of nuclear protein biosynthesis in rat liver. A one hour pulse of insulin stimulated the [14C]-DL-leucine incorporation up to 50% compared to control values. A maximum (100% increase) was passed after 3 - 5 hours and the original rate of nuclear protein biosynthesis was obtained after 10 hours. The reactivity of insulin has been shown to be progressively diminished employing decreasing insulin concentrations. If [14C] -DL- leucine was replaced by [3H] -L-lysine no rise of nuclear protein biosynthesis, attributable to insulin, could be observed.

Die Umsetzung von Selenoxidchlorid mit Methylamin liefert in geringer Ausbeute das dimere Selenylmethylimid (OSeNCH3)2 neben einem hochpolymeren, unloslichen Produkt. Das monomere konnte nicht nachgewiesen werden. Im Dimeren liegt ein Vierring Se2N2 vor. Auch in den hochpolymeren Produkten, welche beim langsamen Anwarmen des Dimeren entstehen, erfolgt die Polymerisation wahrscheinlich uber den Stickstoff. Beim raschen Erhitzen fuhrt eine innermolekulare Verbrennung zur Verpuffung. The reaction of SeOCl2 with methyl amine leads to small yields of dimeric (OSeNCH3)2 and larger amounts of an insoluble polymer. The monomeric form has not been found. In the dimeric one, a four-membered Se2N2 ring exists which undergoes further polymerisation on raising the temperature. On rapid heating a sudden deflagration by intramolecular combustion occurs.

Female rats have been employed for a study of the reactivity of zinc ions on RNA and protein biosynthesis of liver nuclei. Intraperitoneal injections of 20 μmoles of this metal ion have been found to stimulate RNA biosynthesis up to 80% and a 40% increase of protein synthesis was observed under the same conditions. The precursor for RNA synthesis was [6-14C] -orotic acid and [l-14C]-ᴅʟ-leucine served as a label for nuclear protein. Low Zn2®-concentrations showed no reactivity, even when zinc-deficient rats were used. DNA-dependent RNA-polymerase activity was progressively diminished when increasing amounts of Zn2⊕-ions were added in vitro to the incubation mixtures. However, RNA-polymerase activity was strongly stimulated following a 10 h pulse of Zn2® prior to the isolation of liver nuclei.

Der Einflus des 4-Hydroxy-pentenals auf die Biosynthese von monomeren und polymeren Nucleotiden wird anhand zellfreirr Systeme untersucht. Es werden vor allem Angriffspunkte an der Nucleotidphosphorylierung und an der DNA-Polymerase gefunden. Bei Untersuchungen der Wirksamkeit des HPE in einem zellfreien Ribonucleotid-Reduktase-System kann kein deutlicher Effekt an der ADP-Reduktase gesehen werden. Wie die Biosynthese der DNS eines naturlich proliferierenden Gewebes in Gegenwart von HPE verlauft, wird an regenerierender Rattenleber gepruft.

SummaryYoung rats on a diet containing not more than 0.001 ppm boron were used to study the effect of administered boric acid on the synthesis of nuclear liver RNA. A 30-min pulse of [6-14C]-orotic acid resulted in a higher incorporation of 14C in liver RNA when borate was given intraperitoneally. This increased incorporation was diminished when the diet contained 1 ppm boron as boric acid. An attempt to determine the effect of borate on the synthesis of liver DNA, using [6-3H]-thymidine, gave unsatisfactory results. It is apparent that borate increases the rate of synthesis of nuclear liver RNA.

The name metal chelatase has been deliberately chosen for the enzyme ferrochelatase (protohaem ferro-lyase, EC 4.99.1 .l.). This enzyme preferentially catalyses the insertion of Fe’+ ions into porphyrins to form haems. However, Co*+ and Zn*+ are almost as active as Fe*+. Many divalent cations including Mg”, Ca*+, Ni*‘, Cd*‘, Pb*+ and Hg*+ inhibits this enzyme process [l-4] . The rate of non-enzymic incorporation of metal ions under the same experimental conditions is practically zero. Metal chelatase has a wide distribution in a great number of aerobic cells. Its purification, however, is difficult and this was attributable to its chemical nature. Metal chelatase appears to be a structurebound lipoprotein although a second water soluble enzyme has been detected in Rhodopseudomonas spheroides [ 51. A 40 fold enrichment was obtained from rat liver mitochondria employing a rather time consuming procedure [6] and a similar degree of purification is reported here using the inner membrane fraction of freshly isolated rat liver mitochondria. Kinetic studies were performed and are of interest in relation to different K,,, values reported for the insertion of Fe*+ into either protoporphyrin (8 PM) or mesoporphyrin (36 PM). These data suggest an influence of the porphyrin binding site on the metal binding site. The measurements, however, had to be carried out under anaerobic conditions which were not always reproducible. Therefore, Co*+ was employed

Partially hepatectomized rats have been employed for a study of the reactivity of zinc ions on DNA, RNA and protein biosynthesis of liver nuclei. Intraperitoneal injections of this metal ion increased the incorporation rate of [6- 14 C]orotic acid into rapidly synthesized nuclear RNA while [ 3 H]thymidine uptake of nuclear DNA was progressively diminished with increasing Zn 2+ concentrations. No change of nuclear protein biosynthesis was observed. RNA polymerase activity of isolated enzymatically active whole liver nuclei from Zn 2+ injected rats remained constant compared to control groups. However, when zinc ions were added in vitro to liver nuclei from uninjected rats RNA polymerase activity was strongly inhibited. Intraperitoneal injections of low Zn 2+ concentrations have been found to stimulate the incorporation rate of [ 3 H]thymidine into nuclear liver DNA of zinc deficient rats. This result appears to be significant since the same Zn 2+ concentrations were ineffective on the DNA biosynthesis of rats restricted to a normal Zn 2+ providing stock diet.

An enzymatisch aktiven Leberzellkernen aus Ratten wurde die Reaktivitat von Hg2+, Mn2+, Fe3+ und Cr3+ auf die nukleare Proteinbiosynthese gepruft. Hg2+-Ionen hemmen den14C-Aminosaure-Einbau vollstandig, wahrend Mn2+ und Fe3+ eine schwache Stimulierung verursachen. Die14C-Einbaurate ist nach Cr3+-Zusatzen stark erhoht. Es konnte gezeigt werden, dass die Cr3+-Stimulierung nicht durch Adsorption zu erklaren ist.

Apo-erythrocuprein was prepared by passing freshly isolated bovine erythrocuprein through an EDTA-equilibrated Sephadex G-25 column at pH 3.8 for 8–10 h. The apoprotein proved to be homogeneous and metal free as examined by several physicochemical methods. The fine structure of the ultraviolet absorption spectrum was very distinct. The molar coefficient of absorption at 259 nm was only about one third (ϵ259 = 3670) of the corresponding value of the native protein (ϵ259 = 9840). The apoprotein was electrophoretically homogeneous and displayed one symmetrical boundary as determined by sedimentation velocity measurements. The s20,w value was 3.0 S. Absolutely no Zn2+ and Cu2+ were detected either with atomic absorption spectroscopy or, in the case of Cu2+, using electron spin resonance (EPR). The apoprotein was fairly stable in the pH range of 2.5–9.5, provided the exposure to the more extreme pH values did not exceed 5 min. From magnetic circular dichroism (MCD) data it was concluded that the protein contained no tryptophan. Furthermore, MCD measurements were found to be an elegant method for the detection of 2 moles of tyrosine per mole of apo-erythrocuprein. The reconstitution of erythrocuprein by incubating the apoprotein together with Cu2+ and/or Zn2+ was successful under both aerobic and anaerobic conditions. However, the quality of the aerobic reconstituted erythrocuprein was not fully satisfactory as shown by absorption spectra in the visible region, EPR and enzyme assay. From optical titrations of the apoprotein using Cu2+ and Zn2+ it was concluded that Cu2+ is able to produce both a profound increase of ultraviolet absorption and a change of the absorption profile, while Zn2+ was less active. However, Zn2+ was able to cause a shift of the visible absorption maximum by 50 nm. From enzymic studies using the reduction of cytochrome c by O2− it was concluded that 4 metal ions, two of which must be Cu2+, are required for optimal superoxide dismutase activity. No measurable differences between the native and the reconstituted protein were apparent at all if the reconstitution was performed anaerobically using a second Sephadex G-25 column which contained Cu2+ and Zn2+ in the upper section.

The preparation of pure metallothionein from Cd-pretreated rats and chicken was performed using short-time treatment with chloroform/ethanol followed by ion-exchange and gel chromatography. The protein contained 7 g-atoms metal ions per 12000 g protein. The Zn to Cd ratio was 1:2.4 ± 0.1. The Cd, Zn-thionein remained homogeneous during Sephadex G-25, G-50 and G-75 gel filtration, during polyacrylamide disc-gel electrophoresis and in the analytical ultracentrifuge. The high content of cysteine residues (approx. one-third of the total residues) was consistent with the cysteine content of metallothionein isolated from human or equine tissues. The Cd, Zn-thionein was of considerable temperature stability. The physicochemical properties were examined by ultraviolet spectroscopy, circular dichroism (CD) measurements and X-ray photoelectron spectroscopy. The millimolar absorption coefficient of the native protein was at 250 nm ɛ250= 80.6 mM−1· cm−1. Virtually no aromatic amino acids were present. The ɛ250 value of the apoprotein prepared by metal displacement using HCl was ɛ250= 13.2 mM−1· cm−1. When the ultraviolet spectrum of the apoprotein was recorded at pH 6.6 a distinct peak was detectable at 255 nm (ɛ250= 18.7 m M−1· cm−1). The existence of inter- or intramolecular disulphide formation was confirmed by CD measurements employing the polyethyleneglycol esters of di-tert-butyloxy-carbonyl-l-cystine and tert-butyloxycarbonyl-l-cysteine as model compounds for high-molecular-weight and water-soluble cysteine compounds, respectively. From CD data it was concluded that Cd, Zn-thionein exists mainly as a random-coil peptide. Some indication of the binding of Cd and Zn in the native protein with ⊖S–R moieties was obtained from ultraviolet data. Final proof of the exclusive coordination of these metal ions with cysteine sulphur was successful using both circular dichroism measurements and especially X-ray photo-electron spectroscopy. The last method proved most convenient for determining the binding energies of the core electrons of Cd (Cd 3d3/2= 411.0 eV and Cd 3d5/2= 404.4 eV), Zn (2 p3/2= 1021.0 eV) and S (2p = 161.7 eV). The corresponding binding energies for S in the cystine complexes with Zn2+ and Cd2+ were 163.0 eV and 162.8 eV, respectively.

Erythrocuprein is both a most powerful and specific metalloprotein able to decontaminate highly reactive ′ Δ g singlet oxygen being exclusively generated by K 3 CrO 8 . Even in concentrations up to 2·10 −10 M erythrocuprein displayed a clearly detectable singlet oxygen decontaminating activity. In contrast to the considerable superoxide dismutase activity of low molecular weight copper chelates (4%) the singlet oxygen decontaminating activity of these model compounds was remarkably low by 4 orders of magnitude compared to the respective activity of native erythrocuprein. From these data it must be concluded that in aerobic biological systems erythrocuprein is acting as a singlet oxygen decontaminating enzyme rather than as a superoxide dismutase. This conclusion was made in the light of the well established specificity of the enzyme and the deleterious action of ubiquitous singlet type oxygen species even in the absence of superoxide.

The intracellular distribution of 65Zn in rat liver has been studied in vivo and in vitro 1 The highest 65Zn uptake can be observed in the nuclear fraction. A maximum occurs after approx. 5 hours which is in contrast to the rapid rise of detectable 65Zn in the whole homogenate appearing immediately after the intraperitoneal injections of this metal ion. Further, 65Zn is retained by the nuclei for a much longer period. When Zn-deficient rats are employed 65Zn uptake is considerable enhanced at the beginning. However, the total 65Zn radioactivity of whole liver homogenate is only slightly increased. 2 The uptake of 65Zn by isolated liver nuclei suggests a strong temperature dependence in vitro. No 65Zn uptake at all can be measured when nuclei are employed which have been stored at –25° for two months. 3 An attempt has been made to isolate a double stranded DNA-65Zn complex from liver nuclei derived from 65Zn injected rats or from nuclei incubated with 65Zn. However, high ionic strength buffers or phenol displace 65Zn from the polynucleotide during the isolation procedure. 4 The intracellular 65Zn distribution in the liver portions remaining after partial hepatectomy in rats is in accordance with the results obtained above.

A system has been established to study the 65Zn2+ uptake into enzymatically active whole rat-liver nuclei in vitro. The incubation conditions have been elucidated and it appears that 37°, 20 min and a Zn 2+ nuclei ratio of 1 nmole Zn2+ per 2.1 μg nuclear DNA are optimal. The rate of 65Zn2+ taken up by the nuclei has been found to deviate in the presence of different bioligands such as amino acids or nucleic acid derivatives. A marked stimulation of 65Zn2+ uptake can be observed with phosphate ions and nucleoside monophosphates. Ultrasonic treatment of nuclei suggests that most of the 65Zn2+ being absorbed in vitro might be located in the nuclear membrane. If a 10-h pulse of 65Zn2+ was applied to living rats a considerable amount of 65Zn2+ could be detected in the soluble fraction containing the nucleoplasma.

Lipid peroxidation in mitochondrial membranes results in loss of respiratory control, lack of contractibility and final lysis of mitochondria [l-3]. It may be induced by incubation of isolated mitochondria in the presence of oxygen with either chaotropic agents [4] or autoxidizable substances like ascorbate [2] or thiol compounds [3]. Besides, formation of lipid peroxides in livers of tocopherol deficient rats has been observed under in vim conditions [5 ] and may, therefore, have a pathophysiological significance. Two enzymes, GSH peroxidase (C-factor I) [l] and catalase (C-factor II) [ 11, were found to prevent peroxidation of unsaturated lipids in suspensions of mitochondria or “high amplitude swelling” which is related to lipid peroxidation [2, 3,6, 71. These observations suggest an involvement of H202 which may for instance be built according to the equation

THERE have been numerous reports dealing with the anti-tumour activity of Escherichia coli L-asparaginase in the therapy of human leukaemia. It has been postulated that the agent is without toxic action on normal tissues and that it is specific for asparagine dependent tumour cells, but neither of these assumptions has been proved1,2. When L-asparaginase was used in the treatment of human cancer, a satisfactory effect could be seen only with acute lymphoblastic leukaemia3–5, but many investigators emphasized the toxicity of L-asparaginase. An alteration of liver function2,5–7 has been demonstrated by the finding of hepatic lipoidosis; a marked increase in bromsulphthalein retention, and in serum levels of cholesterol, triglycerides and phospholipid; a decrease in human serum proteins and the demonstration of various clotting disorders in the course of therapy. Other authors8 observed the total inhibition of the early wave of mitotic activity in regenerating rat liver approximately 30 h after partial hepatectomy. We decided to study the effect of L-asparaginase preparations of high specific activity on DNA synthesis in rat liver nuclei, and to determine nuclear DNA and RNA polymerase activity in rat liver under the influence of L-asparaginase.

Erythrocuprein was isolated from bovine blood by precipitation with organic solvents and chromatography (Sephadex G-75 and DEAE-Sephadex A-50). The preparation was electrophoretically pure, had an absorption ratio A 259 nm A 680 nm = 31.5 and contained 2 atoms of copper and 2 atoms of zinc per mole. Preincubation at 45° in the presence of 1% sodium dodecyl sulphate resulted in a striking change of the electrophoretic properties of the protein: The molecular weights of the two bands observed were calculated to be 16 000 (monomeric species) and 64 000 (tetrameric species). Surprisingly, no dimeric form was obtained. However, the monomeric unit was obtained exclusively by preincubation with sodium dodecyl sulphate at 100° for 1 min. Upon cooling of erythrocuprein solutions to 77°K a marked increase of the absorption in the visible region was observed and the shoulder at 430 nm was more distinct. The EPR signal of Cu 2+ in the protein showed axial symmetry. Superhyperfine splittings were observed in the g ⊥ region, indicating possible coordination of the copper to one or more nitrogen atoms. Reductive titration with dithionite gave evidence of a transfer of two electrons per molecule or of one electron per Cu 2+ . The optical rotatory dispersion and magnetic optical rotatory dispersion spectra, recorded from 220 to 600 nm, show only one trough (225 nm) or peak (around 225 nm), respectively. In the spectral range from 200 to 250 nm the circular dichroism (CD) spectrum shows only one negative Cotton effect at λ = 208 nm which is modified only slightly in sodium dodecyl sulphate or urea. From these spectral characteristics it is suggested that the helical content of erythrocuprein is low. It is demonstrated that magnetic circular dichroism is a tool for resolving overlapping CD and absorption bands in the region of the copper d → d transition.

X-ray photoelectron spectra were recorded to obtain information regarding the oxidation state of some metallic and non-metallic elements in erythrocuprein. Investigations of various Cu and Zn complexes and salts show marked differences between the binding energies of ionic and complexed bound metals and their ligands. From the intensities of the X-ray photoelectron spectra of the Cu 2p 3 2 and Zn 2p 3 2 levels of erythrocuprein, it can be concluded that Cu is located at the surface of this metalloprotein, whereas Zn must be bound more inside the molecule. X-ray photoelectron spectroscopy promises to be a most valuable new analytical technique for studies on metalloproteins. It is particularly suitable for those metal ions which cannot be detected by electron spin resonance or Mossbauer spectroscopy. In addition to the investigation of metal ions in proteins, other elements, e.g. S present in relatively low concentrations, can be detected.

2. P r e p a r a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.1. I so la t ion from E r y t h r o c y t e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.2. I so la t ion from Liver, H e a r t or Bra in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.3. Convers ion in to the Apopro te in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.4. P r e p a r a t i o n of the Subun i t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Ascites tumour cells have been employed to study the reactivity of Zn++ on nucleic acid biosynthesis. 10−4M Zn++ caused a selective inhibition of DNA synthesis of intact cells. The rate of RNA- and protein-biosynthesis, however, remained unchanged. The activity of DNA polymerase as well as DNA dependent RNA polymerase was strongly affected by Zn++ in vitro.