Acta biochimica et biophysica Hungarica最新文献

High potential iron-sulphur protein (HiPIP) has been purified to electrophoretic homogeneity from the photosynthetic bacterium Thiocapsa roseopersicina. The protein has a single polypeptide chain (molecular mass 10 kDa) containing one 4Fe-4S cluster. The midpoint redox potential (E = 0.35 V), isoelectric points and pH profile, as well as the absorption, circular dichroism and Mössbauer spectroscopic properties in the reduced and oxidized states have been determined. The protein is in the reduced state as isolated; upon oxidation by ferricyanide there are characteristic changes in its visible absorption and circular dichroism spectra. HiPIP contains no alpha helix, about half of the polypeptide chain assumes beta sheet conformation. Pronounced structural differences between the oxidized and reduced states have been observed in the aromatic amino acid and Fe-S cluster spectral regions. Mössbauer spectra of the HiPIP in the two redox states reveal further differences. The possible contribution of aromatic amino acid residues, to the redox transition is discussed.

1H-NMR and X-ray conformation studies of new muscarinergic dibenzodioxazocines have been carried out. It is suggested that EGYT-2347 (2-chloro-12-/2-piperidino-ethyl/-dibenzo [d,g] [1, 3, 6] dioxazocine hydrochloride) may exist in solution in at least two distinct conformations, unlike other tricyclic or non-tricyclic compounds having antimuscarinergic activity. One of these conformations possessing an asymmetric, twisted central hetero-ring confined between two phenyl rings is probably the energetically more stable form, while the other having a butterfly-like structure, with mirror symmetry-related phenyl rings as in phenothiazines seems to be more suitable for receptor binding. The importance of the hydrophobic pocket at the receptor site was revealed by the good correlation of the calculated and measured hydrophobic parameters to the muscarinic activity of these newly synthesized and other known muscarinergic compounds.

Human neutrophil granulocytes, when placed into hypoosmotic media, first swell, than show a regulatory volume decrease (RVD) reaction. This RVD is the most effective at pH 7.5 when inosine is used as a metabolic substrate. The RVD reaction is blocked by the inhibitors of the calcium-induced K+ transport, such as quinine, quinidine, trifluoperazine (TFP), as well as by those of the conductive Cl-transport (dipyridamole, SITS, oligomycin C). Removal of external calcium by EGTA, or addition of the calcium channel inhibitor, verapamil, reduce the RVD. Ouabain affects RVD only after long preincubation and furosemide or phloretin have hardly any effect on this process. In a hypoosmotic KCl medium neutrophils show a TFP-, and SITS-sensitive secondary volume increase. All these data indicate the opening of Ca2+-induced K+ channels and of conductive Cl- channels in hypoosmotically shocked neutrophils. Under these conditions, however, direct measurement of cytoplasmic calcium by Indo-1 does not show any major change in the overall cytoplasmic free Ca2+ levels. In neutrophils, the reduced calcium signal evoked by formyl-methionyl-leucyl-phenylalanine (fMLP) after a hypoosmotic shock, suggests that cellular calcium metabolism is altered under these conditions.

Myosin subfragment-1 was modified with pyridoxal-5'-phosphate, a lysine modifying agent. Approximately two lysines could be blocked with a concomitant decrease in the actin-activated Mg2(+)-ATP-ase activities of S-1 remained unchanged. This selective inhibition of actin-activated Mg2(+)-ATP-ase activity of S-1 by pyridoxal-5'-phosphate was further characterized by kinetic studies. The double reciprocal plot revealed no change in the Vmax, while KM increased from 15 microM to 36 microM indicating that the modification reduced the actin affinity of S-1. The effects of pyridoxylation of S-1 were compared to those of 2, 4, 6 trinitrobenzene-sulfonate modification of S-1. The two types of lysine modification are strikingly different. The reactive lysine residue (Lys 83) remained unmodified after pyridoxylation of S-1 thus the effects of trinitrophenylation could be revealed independently in the double-modified sample. Furthermore after trinitrophenylation the S-1 fragments were found to be protected against partial tryptic digestion in the presence of nucleotides.

It was found that milk samples from cows with mastitis have markedly higher selenium concentrations and higher standard deviations than those of healthy cows. A good correlation was found between the severity of the disease and increase of selenium concentration.

The applicability of density gradient ultracentrifugation using fixed angle rotors for the determination of sedimentation coefficient and molecular weight of proteins was studied. Ovalbumin, bovine serum albumin, IgG-, IgA- and IgM-globulin proteins, as standards were, among others, centrifuged in a fixed angle rotor (Beckman type 50) in 5-20% (w/v) sucrose density gradient for 300 min, at a speed of 40,000 rpm, at 5 degrees C. After centrifugation, the sedimentation diagram of the above-mentioned proteins was taken by a spectrophotometer equipped by a flow-through cuvette and a compensator. The sedimentation path was measured on the diagrams. A calibration curve was drawn by plotting the sedimentation coefficients of the standard proteins against the sedimentation paths. This curve, for which the equation y = 1.74x-1.6 is valid, was used for the determination of the sedimentation coefficients of several globular proteins under the conditions described above. The values obtained this way agree within +3-10% with those determined with analytical ultracentrifuge. If the logarithms of proteins molecular weight used as standards were drawn against the logarithms of their sedimentation paths a calibration curve was obtained. This curve for which the equation ln y = 2.022 ln x + 8.71 is valid can be used for the determination of molecular weights of globular or nearly globular proteins.

The possible mechanism of the inhibitory effect of endotoxin on adenylate cyclase was investigated. Plasma membranes were isolated from porcine thyroid gland and rat liver. The effects of endotoxin on adenylate cyclase activity was determined. The adenylate cyclase activity followed in the presence of various concentrations of guanosine-imidodiphosphate (5 x 10(-8)-10(-6) M) and NaF (0.1-10 mM) was markedly inhibited by endotoxin. Moreover, the basal activity of adenylate cyclase was inhibited similarly. The inhibition was concentration dependent. At 400 micrograms/ml of endotoxin a 60% inhibition of adenylate cyclase both without and in the presence of activators was detected. The inhibitory effects of endotoxin and its radiodetoxified derivative were similar. Neither of the two investigated endotoxin preparations tested had any effect on the 3H-guanosine-imidodiphosphate binding activity of adenylate cyclase (neither on the binding capacity, nor on the Kd value). Forskolin activated adenylate cyclase was also inhibited markedly by both endotoxin and radiodetoxified endotoxin. These results suggest that beyond the other membrane effects there is a probability of direct inhibitory effect of endotoxin on the catalytic subunit of adenylate cyclase.

Based on experimentally determined glycosidase molecular forms, their specificity and apparent Hill coefficients against trehalose (1.4) and sucrose (0.6), respectively, in honeybee haemolymph, a theoretical model is proposed involving differential types of non-random aggregation of a single enzyme protomer. This basic unit contains one trehalose-specific site and two asymmetrical subsites: one holds a catalytic zone and both share a proper affinity to any substrate non-specific binding zone. Then, the predicted aggregation possibilities of the promoter to dimers, trimers and tetramers very closely account for all the experimentally determined properties of the enzymes. Moreover, the hypothesis that the enzyme aggregation may be directed by the particular substrate present in major concentrations in the medium is supported by the observed differences in enzyme polymorphism following pre-incubation at high concentrations of either trehalose or sucrose in the medium.

More than 40 orthovoltage X-ray therapy units are monitored by calibrated thermoluminescent dosimeters (TLD). Three pairs of LiF:Mg,Ti and CaSO4:Dy pellets are irradiated in air in a polypropane capsule. The range between 0.2 to 3.0 mm Cu the half-value layer (HVL) is determined from the CaSO4-LiF ratio. In this range, the response of the LiF dosimeters is corrected for energy dependence. We are using a fixed sensitivity for LiF in the MVL range of 2 mm Al to 0.2 mm Cu. In the first two years 173 monitorings were performed by using 485 badges. Usually not more then two different radiation qualities (in the HVL ranges 2 mm Al to 0.2 mm Cu and 0.9-2.5 mm Cu) or two beams used on a therapy unit are measured by using 2-3 badges per monitoring. In 134 cases (77.5%) the dose rates were within acceptable limits (+/- 10%). In 23 cases (13.3%) a failure of the X-ray unit was detected. We think that our system is simple enough and may be used for the dosimetric monitoring of orthovoltage X-ray therapy units. In some cases even the type of malfunction can be determined.

The protein synthesis in dissected whole larval brains of Drosophila melanogaster has been monitored by [35S] methionine incorporation, as revealed by two-dimensional gel-electrophoresis and fluorography. In wild type brains, drugs known to increase cAMP level increased the labelling of at least two proteins in the Mr range 30 to 120 kD and pI range 4.8 to 6.2. One of these proteins, Mr = 78 kD and pI = 5.9, was also enhanced in the dunceM11 memory-mutant, which has an elevated cAMP level, whereas it was hardly affected in the rutabaga memory-mutant, which has a subnormal cAMP level. It is suggested that cAMP-induced alterations in protein composition and/or turnover of nerve cells may contribute to the development of memory deficit in the dunce strains.