Moshe M. Werber

Protective Effects of Human Recombinant Mnsod in Adjuvant Arthritis and Bleomycin-Induced Lung Fibrosis

We have previously shown that human recombinant methionyl manganese superoxide dismutase (MnSOD) is more efficient than CuZnSOD as an anti-inflammatory agent in a model of acute inflammation (Carrageenan-induced paw edema). This effect was attributed to the prolonged half-life of MnSOD in blood (t1/2 = 6 h vs. 10 min, respectively). In the present study, the two enzymes were compared in terms of their effectiveness in two systems: (1) Adjuvant-induced arthritis in rats, which is considered to be a model for the chronic situation of rheumatoid arthritis and (2) Bleomycin-induced lung fibrosis, which is a chronic situation believed to be mediated by oxygen free radicals. Rats inflicted with adjuvant arthritis were treated during the period of maximal joint swelling (Days 15-21 after adjuvant injection) with MnSOD or CuZnSOD (12 to 50 mg/kg, i.p. daily). MnSOD administration resulted in a 50-75% reduction of paw swelling, as well as inhibition of the elevation of serum globulins. A similar treatment with CuZnSOD gave merely marginal effects. In the second system, lung fibrosis was induced in rats by intratracheal administration of bleomycin. MnSOD (50 mg/kg, s.c.), administered 2 h before and then 2 and 4 days after bleomycin, markedly inhibited lung fibrosis, as evident from lung weight and collagen content measured by the 3rd week. By contrast, CuZnSOD administration did not give a significant effect. The results indicate that MnSOD is superior to CuZnSOD in the treatment of chronic inflammatory processes. In addition, they lend further support to the involvement of oxygen free radicals in bleomycin toxicity.

Induction of a dissimilatory reduction pathway of nitrate in Halobacterium of the Dead Sea. A possible role for the 2 Fe-ferredoxin isolated from this organism.

Abstract The processes involved in nitrate metabolism in Halobacterium of the Dead Sea are part of a dissimilatory pathway operating in these bacteria. The induction of both nitrate and nitrite reductases is shown to depend on the presence of nitrate and of anaerobic conditions. The gas products of the denitrification process were identified as nitrous oxide and nitrogen. Some properties of two of the enzymes involved in this process, nitrate and nitrite reductases, are reported. It is shown that the 2 Feferredoxin, which is present in large quantities in Halobacterium of the Dead Sea, can serve as an electron donor for nitrite reduction by nitrite reductase. It is suggested that the presence of a dissimilatory pathway for the reduction of nitrate in Halobacterium of the Dead Sea can be used as a tool for its classification.

Purification and characterization of a highly acidic 2Fe-ferredoxin from Halobacterium of the dead sea.

Abstract A 2Fe-ferredoxin from Halobacterium of the Dead Sea has been purified by chromatography on Sepharose and DEAE-cellulose, using decreasing concentration gradients of ammonium sulfate. Its amino acid composition reveals an extremely high excess of acidic amino acid residues: 44 glutamate and aspartate residues (of which 4 are in the amide form), compared to 6 lysines and arginines, as well as a high content of aromatic amino acids. The molecular weight of this ferredoxin was found to be 14,000 by amino acid composition, sedimentation equilibrium, and iron content. The millimolar coefficients at the maxima of the visible absorption spectrum are: 28.0 (277 nm), 12.2 (330 nm), 9.1 (420 nm), and 8.3 (465 nm). The optical properties—absorption and CD spectra in the visible region—of this ferredoxin are very similar to those of plant and algal ferredoxins, whereas its redox potential is much higher: −345 ± 5 mV (at pH 7.3, 0.5 m NaCl). Although it is reduced by illuminated chloroplasts, it cannot mediate the photoreduction of NADP in their presence. Data reported elsewhere suggest that its physiological function might be to serve as an electron donor for nitrite reduction.

Physico-chemical studies on the light chains of myosin III. Evidence for a regulatory role of a rabbit myosin light chain☆

Abstract Treatment of myosin with DTNB causes a decrease in the calcium sensitivity of actomyosin, concurrently with the release of the DTNB light chains. The removal of the calcium-binding DTNB light chains is accompanied by a loss of the calcium binding capacity of myosin. A regulatory role is ascribed to these light chains.

Tryptophan-130 is the most reactive tryptophan residue in rabbit skeletal myosin subfragment-1

Rabbit skeletal muscle myosin subfragment‐1 (S‐1) was reacted with dimethyl(2‐hydroxy‐5‐nitrobenzyl)sulfonium bromide (DHNBS) resulting in modification of 0.8 tryptophan residues per S‐1. In order to assign the most reactive tryptophan of the 5 S‐1 tryptophans, antibodies were raised in rabbits against bovine serum albumin modified with DHNBS. The antibodies reacted with the 27 kDa tryptic fragment of DHNBS‐treated S‐1, indicating that the reactive tryptophan resides on this domain. The 27 kDa fragment was isolated from DHNBS‐treated S‐1 and was further cleaved at a single cysteine residue by 2‐nitro‐5‐thiocyanobenzoic acid. This cleavage resulted in two peptides, each of them containing one tryptophan. The antibodies reacted with the smaller peptide consisting of residues 122–204. The only tryptophan residing on this peptide is Trp130, and this is therefore the most reactive tryptophan of S‐1.

Characterization of crystals of genetically engineered human manganese superoxide dismutase

The genetically engineered human manganese superoxide dismutase crystallizes in space group P2(1)2(1)2 with a = 75.51 A, b = 79.00 A, c = 67.95 A. At room temperature the crystals are not stable against radiation, so we cooled them to 90 K and collected a data set to 3 A resolution at this temperature.

Preliminary X-ray diffraction studies on 2 Fe-ferredoxin from Halobacterium of the Dead Sea.

The 2 Fe-ferredoxin from the Halobacterium of the Dead Sea has been crystallized. The space group is P6322 with one protein molecule per asymmetric unit. The cell parameters are a = b = 60.6 A, c = 127.8 A. The crystals are stable under radiation and diffract to high resolution.

The role of metal ions in the mechanism of action of hydrolytic metalloenzymes: Carbonic anhydrase

Abstract Metalloenzymes are among the most efficient enzymes. One of the mechanisms available to hydrolytic metalloenzymes consists of using the metal ion, which is embedded in the protein, as a carrier for hydroxide ions in neutral solution. Models for this mechanism are surveyed and analyzed from the point of view of the “charge effect”. The active center of carbonic anhydrase is compared to several of these models, and the similarities are pointed out. It is concluded that the “carrier for hydroxide ions” mechanism is the most plausible one for carbonic anhydrase. It is proposed that the metal ion also plays a role in the regeneration of the active center of the enzyme, i.e. the ionization of the metal-bound water molecule, after each turnover. Some general implications for the mechanism of action of other hydrolytic metalloenzymes are considered.

Nucleophilicity of isourea linkages in substituted agaroses: A direct method for the determination of alkylamino groups coupled to CNBr-activated agarose

Abstract Coupling of alkylamines to cyanogen bromide-activated agarose results in the formation of N-substituted isoureas, which are good nucleophiles. The nucleophilic attack of these isoureas on the active chromogenic ester p-nitrophenyl acetate causes the release of an equivalent amount of p-nitrophenolate. This constitutes the basis for a direct determination of alkylamino groups in substituted agaroses.

One-site reactivity of halobacterial 2Fe-ferredoxin as a plant ferredoxin substitute

A 2Fe-ferredoxin isolated from several Halobacterium species was reported to resemble in many respects chloroplast-type ferredoxins [ 1,2]. Thus, optical characteristics, the EPR spectrum, and other physical properties resembled more closely plant rather than bacterial 2Fe-ferredoxins. Ferredoxin isolated from a Halobacterium of the Dead Sea crossreacted to an extent of 15% with an algal ferredoxin (from Spirulina maxima), whereas it did not crossreact with the bacterial 2Fe-ferredoxins [3]. Moreover, a high degree of similarity was found between the amino acid sequences of halobacterial ferredoxins and that of the blue green algaNostoc muscorum [4,5]. However, as has already been pointed out, functional differences exist between halobacterial and chloroplast type ferredoxins,in particular with respect to the ability of the former to substitute for the latter in the photoreduction of NADP by illuminated chloroplasts [ 1,2,6]. In the present study we investigated these functional differences and the mechanism underlying them in detail. Halobacterial ferredoxin is shown to be rather efficient in accepting electrons from the photosynthetically produced reductant, but rather poor in its further interaction with the flavoprotein.