Lactate dehydrogenase in two digenetic trematodes and their host

Polyacrylamide gel electrophoresis of the two digenetic trematodes, Gigantocotyle explanalum from the liver and Gastrothylax crumenifer from the rumen of the water buffalo, Bubalus bubalis revealed the presence of at least six and seven isoenzymes of lactate dehydrogenase (LDH), respectively in a partially purified enzyme preparation. The respective host tissues showed five isoenzymes of LDH, which are characteristic to the vertebrates. Both parachloromercuribenzoate and iodoacetate affected the LDH activity of the parasites and host tissues differently. Spectrophotometric analysis also showed different specific activity and susceptibility to the action of thiol inhibitors. The host LDH was quite stable at 57°C for 30 min, but that of the parasites was less stable.     

Proteins dealing with N-ethylmaleimide inhibition of pig heart mitochondrial phosphate transport system.

Our data clearly demonstrate that protective effect of phosphate and protective effect of mersalyl against NEM-inhibition of phosphate transport act at the level of two kinds of proteins. (1)Two major components are phosphate and nigericin NEM sensitive. According to our previous data [13] it has been also demonstrated that these two proteins components are valinomycin NEM sensitive (results not shown here) suggesting a relationship between these proteins and the energy linked proton translocation process. Relationships between these proteins and the phosphate translocation process are not evident and are under further investigations. (2) Two other insoluble major components localised at the level of the subparticular fraction are mersalyl NEM sensitive. We can suggest that these proteins are implicated in the translocation of phosphate in pig heart mitochondria.     

EcoRI activity: Enzyme modification or activation of accompanying endonuclease?

A study has been made of the factors and mechanism leading to appearance of the so-called EcoRI activity described by Polisky et al. (1975) in the restrictase EcoRI preparations. The preparations of purified restrictase EcoRI, precipitated at 0.9 ammonium sulphate saturation, as well as that obtained using standard techniques have been found to contain an admixture of an endonuclease which at neutral pH and high ionic strength multiply cleaves those DNAs which normally have only one recognition site for EcoRI. Under the standard conditions for EcoRI digestion this activity is found only when large amounts of freshly isolated enzyme are added to the incubation mixture and it is sharply enhanced by replacement of Mg2+ with Mn2+. The number and size of DNA fragments produced under such conditions practically do not differ from those found under the so-called EcoRI conditions, that is for alkaline pH values and low ionic strength. The optimum incubation mixture for the EcoRI activity has been found to be 10 mM Tris . HCl buffer (pH 8.8) + 2 mM Mn2+. Similar activity is induced also by addition to EcoRI solution of 40--50% glycerol or a number of organic solvents (dimethylacetamide (DMA), dimethylformamide (DMF), dimethylsulphoxide (DMSO), sulphalane (SP) in concentrations from 1 to 6%. The EcoRI activity induced by 50% glycerol or at alkaline pH values and low ionic strength is suppressed or sharply inhibited by 2--3 mM parachloromercuribenzoate (PCMB), while EcoRI is not sensitive to this agent. The DNA fragments cleaved by EcoRI have cohesive termini and can be easily ligated. It is suggested that the EcoRI activity can be due not only (or largely not) to modification of the "recognizing capacity" of the EcoRI restrictase but not activation of a latent specific endonuclease which is present in the restrictase preparation as an impurity.     

Cytochrome P450 cam: SS- dimer and -SH derivative reactivities.

N-ethyl-maleimide alkylation converts ferric P450_cam to a (succinimido-cys)₄ protein with native optical and EPR spectra but insensitive to substrate induced shift of iron to high spin with Soret absorption to higher energy and inactive in putidaredoxin ferric-ferrous reduction. On photo or chemical reduction the ferrous protein oxygenates and, with reduced putidaredoxin, converts substrate to product. Mild oxidation of P450_cam yields a disulfide dimer whose properties on alkylation of 3 sulfhydryls equal the succinimido₄ monomer; additional alkylation converts either monomer or dimer to a P420.     

Purification and study of some properties of a collagenase produced by Empedobacter collagenolyticum

A collagenase from Empedobacter collagenolyticum was extracted from the culture medium of the bacteria. The complete purification of the enzyme was achieved by successive ammonium sulfate precipitation. Sephadex G 200 gel filtration and DEAE cellulose chromatography. This collagenase is active on insoluble collagen, and on the synthetic peptide Pz-Pro-Leu-Gly-Pro-D-Arg. Its optimum activity was at 30 degrees C and at pH 7.6. A strong inhibition was observed with chelating agents such as O-phenanthroline and EDTA. Among the cations tested to restore the activity, only Ca2+ has a measurable effect. Heavy metals, Pb, Hg, Cd, Cu, Fe, Co, strongly inhibit the enzyme activity. Zn2+ is also highly inhibitory; 10 microM ZnCl2 completely inhibits the collagenase. p CMB, iodoacetate have little effect on the collagenase. This new collagenase ressembles by most of its properties the already known bacterial collagenases.