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PubMed1975

Restriction and modification in B. subtilis. Purification and general properties of a restriction endonuclease from strain R.

All Bacillus subtilis R-type strains showing the phenomena of restriction and modification contain an endonuclease that inactivates in vitro the biological activity of a variety of DNAs lacking R-specific modification, such as transfecting SPPI, SPO2 and phi105 DNA, and transforming B. subtilis 168-type DNA. The corresponding DNAs carrying R-specific modification are resistant to the enzyme. The enzyme has been purified approximately 400-fold and is essentially free from contaminating double strand-directed unspecific exo- or endonuclease activity. Only Mg2+ is required as cofactor. The substrate DNAs are cleaved at specific sites. The double-stranded fragments produced from SPP1 DNA (molecular weight 2.5 x 10(7)) have an average molecular weight of about 3 x 10(5).

Adenosine Triphosphate↗
PubMed1990

Mitochondrial modifications in a single nuclear mutant of Saccharomyces cerevisiae affected in cAMP-dependent protein phosphorylation.

This paper reports studies of bioenergetic modifications in a TTR1 single-nuclear mutant, isolated as resistant to triethyltin, an inhibitor of mitochondrial ATPase, and effective in cAMP-dependent protein phosphorylation. This mutant appears to have lost the wild-type cell ability to respond to a decrease of oxygen concentration in the growth medium by a decrease of cytochrome concentration in the cell. ATP synthesis rate in mutant cells in both the prestationary and stationary phase of growth appeared increased in comparison to wild-type cells, as too was respiration rate. A comparative study of mitochondria extracted from wild-type and from TTR1 mutant cells showed an increase in respiration rate, an increase in ATP synthesis rate, and an increase in TPP+ uptake in mutant mitochondria. The specific ATPase activity, as well as its sensitivity to TET, appears to be similar for mitochondria extracted from both strains. It was proposed that the modification of mitochondrial biogenesis in the TTR1 mutant may be due to a response of the cell to an increase in ATP hydrolysis caused by the mutation. It is also possible that the modification in cAMP-dependent protein kinase regulation which appeared to occur in this mutant affects protein(s) involved in mitochondrial biogenesis.

Adenosine Triphosphatases↗
PubMed1989

Nucleotide sequence and genetic organization of the NgoPII restriction-modification system of Neisseria gonorrhoeae.

The NgoPII restriction endonuclease, which recognizes the sequence 5'-GG decreases CC-3', differs from its isoschizomer HaeIII in being sensitive to methylation at the external cytosine residue. The entire nucleotide sequence of a cloned 3.3 kb segment of Neisseria gonorrhoeae strain P9 chromosomal DNA which harbours the NgoPII restriction-modification system has been determined. This data, coupled with sub-cloning experiments, indicates that the restriction endonuclease (R.NgoII) and modification (M.NgoII) genes are transcribed from separate promoters but are arranged in tandem, with the R.NgoPII gene being located on the 5' side of the M.NgoPII gene. Unlike all previously reported restriction systems the 3' end of the endonuclease open reading frame overlaps the 5' end of the methylase open reading frame by 8 codons. This overlap may have implications for the regulation of the NgoPII restriction-modification system.

Amino Acid Sequence↗
PubMed1984

A synaptic modification algorithm in consideration of the generation of rhythmic oscillation in a ring neural network.

In consideration of the generation of bursts of nerve impulses (that is, rhythmic oscillation in impulse density) in the ring neural network, a synaptic modification algorithm is newly proposed. Rhythmic oscillation generally occurs in the regular ring network with feedback inhibition and in fact such signals can be observed in the real nervous system. Since, however, various additional connections can cause a disturbance which easily extinguishes the rhythmic oscillation in the network, some function for maintaining the rhythmic oscillation is to be expected to exist in the synapses if such signals play an important part in the nervous system. Our preliminary investigation into the rhythmic oscillation in the regular ring network has led to the selection of the parameters, that is, the average membrane potential (AMP) and the average impulse density (AID) in the synaptic modification algorithm, where the decrease of synaptic strength is supposed to be essential. This synaptic modification algorithm using AMP and AID enables both the rhythmic oscillation and the nonoscillatory state to be dealt with in the algorithm without distinction. Simulation demonstrates cases in which the algorithm catches and holds the rhythmic oscillation in the disturbed ring network where the rhythmic oscillation was previously extinguished.

Animals↗
PubMed1992

Carbodiimide modification reduces the conductance and increases the tetrodotoxin sensitivity in batrachotoxin-modified sodium channels.

The relationship between the channel entrance and the tetrodotoxin (TTX) binding site was investigated by chemical modification at the extracellular surface of bilayer-incorporated batrachotoxin-(BTX) modified sodium channels using an impermeant carbodiimide in the presence or absence of exogenous nucleophiles. Two (classes of) groups could be modified such that the open-channel conductance was decreased while TTX binding was unaffected, and TTX did not protect against this modification. Because the final conductance level depends on the exogenous nucleophile, each covalent modification appears to involve a carboxyl group. In addition, a third (carboxyl) group could be modified such that TTX binding affinity was increased. These results suggest that the channel entrance and the TTX binding site are spatially separate, which supports previous suggestions that the mechanism by which guanidinium toxins close sodium channels involves a conformational change subsequent to toxin binding.

Animals↗
PubMed1993

Effect of chemical modification of extracellular histidyl residues on the channel properties of the nicotinic acetylcholine receptor.

We have examined the effect of chemical modification with diethyl pyrocarbonate (DEP) on the properties of acetylcholine (ACh)-activated channels in the cloned muscle-cell line BC3H-1. After protein modification, patch-clamp recordings showed alterations in the kinetics of the nicotinic acetylcholine receptor (AChR) channel. The major effect was observed in the channel mean open time, which was reduced up to about 12-fold at 466 microM DEP. The specificity of the effect was first established through comparison with both untreated cells and cells treated with inactivated DEP. Consistent with an increase in the number of unprotonated histidine residues (pKa = 6.0), this effect increased concomitantly with the pH of the reaction medium, being faster at pH 8 than at pH 6. The changes were dependent on time and DEP concentration, with an apparent EC50 = 114 microM. Modified channels also showed an increase in the number of events per burst of openings together with a decrease in burst durations. The amplitude of the channel-closed time component of about 1 ms increased with respect to the longest-duration-closed component. The number of alpha-bungarotoxin sites was slightly reduced after the modification, without affecting ligand binding affinity. The results suggest that DEP affects extracellular histidine residues involved in the ion translocation function of the AChR, but not its toxin-recognition ability. DEP could, therefore, induce a dissociation between toxin and agonist binding, as is often observed in neuronal AChR.

Bungarotoxins↗
PubMed1993

Modification of cardiac Na+ channels by anthopleurin-A: effects on gating and kinetics.

We used the whole cell patch clamp technique to investigate the characteristics of modification of cardiac Na+ channel gating by the sea anemone polypeptide toxin anthopleurin-A (AP-A). Guinea pig ventricular myocytes were isolated enzymatically using a retrograde perfusion apparatus. Holding potential was -140 mV and test potentials ranged from -100 to +40 mV (pulse duration 100 or 1000 ms). AP-A (50-100 nM) markedly slowed the rate of decay of Na+ current (INa) and increased peak INa conductance (gNa) by 38 +/- 5.5% (mean +/- SEM, P < 0.001, n = 12) with little change in slope factor (n = 12) or voltage midpoint of the gNa/V relationship after correction for spontaneous shifts. The voltage dependence of steady-state INa availability (h infinity) demonstrated an increase in slope factor from 5.9 +/- 0.8 mV in control to 8.0 +/- 0.7 mV after modification by AP-A (P < 0.01, n = 14) whereas any shift in the voltage midpoint of this relationship could be accounted for by a spontaneous time-dependent shift. AP-A-modified INa showed a use-dependent decrease in peak current amplitude (interpulse interval 500 ms) when pulse duration was 100 ms (-15 +/- 2%, P < 0.01, n = 17) but showed no decline when pulse duration was 100 ms (-3 +/- 1%). This use-dependent effect was probably the result of a decrease in the recovery from inactivation caused by AP-A which had a small effect on the fast time constant of recovery (from 4.1 +/- 0.3 ms in control to 6.0 +/- 1.1 ms after AP-A, P < 0.05) but increased the slow time constant from 66.2 +/- 6.5 ms in control to 188.9 +/- 36.4 ms (P < 0.002, n = 19) after exposure to AP-A. Increasing external divalent cation concentration (either Ca2+ or Mg2+) to 10 mM abolished the effects of AP-A on the rate of INa decay. These results demonstrate that modification of cardiac Na+ channels by AP-A markedly slowed INa inactivation and altered the voltage dependence of activation; these alterations in gating characteristics, in turn, caused an increase in gNa presumably by increasing the number of channels open at peak INa. AP-A slows the rate of recovery of INa from inactivation which is probably the basis for a use-dependent decrease in peak amplitude. Finally, AP-A binding is sensitive to external divalent cation concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Posttranslational modifications of the cytochrome P-450 monooxygenase system.

Two forms of enzymatic posttranslational modifications of the monooxygenase system are described: modification by phosphatase and modification by protein kinase. Phosphatase treatment of microsomes isolated from phenobarbital-pretreated rabbits and rats caused a marked decrease of monooxygenase activity which was paralleled by a comparable decrease of NADPH-cytochrome P-450 reductase activity while the second essential component of the system, cytochrome P-450, remained unaltered. Thus phosphatase attacks monooxygenase via reductase. Protein kinases showed the opposite preference; while cytochrome P-450 was phosphorylated, NADPH-cytochrome P-450 reductase was not. Thus the kinase affects monooxygenase via cytochrome P-450. The phosphorylation of cytochrome P-450 turned out to be a specific reaction observed only with certain cytochrome P-450 isoenzymes and certain protein kinases.

Animals↗
PubMed1996

Post-translational modifications of lantibiotics.

Several newly reported post-translational modification reactions are involved in lantibiotic biosynthesis. A short overview of the present knowledge on the post-translational modifications and on the enzymes involved in lantibiotic biosynthesis is given. The oxidative decarboxylation of the epidermin precursor peptide EpiA is described in detail. The FMN-containing oxidoreductase EpiD is involved in the formation of the C-terminal S-[(Z)-2-aminovinyl]-D-cysteine residue of epidermin: under reducing conditions the side chain of the C-terminal cysteine residue of EpiA is converted to an enethiol. EpiD has no absolute substrate specificity and can be used for modification of peptides having the C-terminal consensus motif [V/I/L/(M)/F/Y/W]-[A/S/V/T/C/(I/L)]-C.

Amino Acid Sequence↗
PubMed1985

Effects of chemical modification of amino groups by two different imidoesters on voltage-clamped nerve fibres of the frog.

Voltage clamped single nerve fibres of the frog Rana esculenta were treated with the amino groups specific reagents ethyl acetimidate and isethionyl acetimidate. Ethyl acetimidate is lipid soluble, relatively non-polar and can penetrate a membrane. Isethionyl acetimidate is lipid-insoluble, polar and membrane-impermeant. Treatment with ethyl acetimidate caused an irreversible reduction of Na currents and a shift of the voltage dependence of the steady-state sodium inactivation, h infinity (E), in the hyperpolarizing direction. The voltage dependence of sodium activation was much less affected and a small shift into the depolarizing direction was observed. By contrast, the non-permeant reagent did not reduce the sodium currents and the shifts of the h infinity (E) curve were smaller than the shifts caused by ethyl acetimidate. Furthermore, a small shift of the voltage-dependence of activation in the hyperpolarizing direction was observed. As the modification procedure with imidoesters does not cause a change of net charge, the results cannot be explained by an effect of surface charge modification; rather, they seem to be due to a direct effect of amino group modification on the voltage dependence of inactivation.

Alkanesulfonates↗
PubMed1983

Unusual features of the Ca2+-ATPase activity of myosin from fast skeletal muscle of the frog: effect of actin and SH1 thiol group modification.

The K+-ATPase and actin-activated Mg2+-ATPase activity of myosin from fast skeletal muscle of the frog, Rana esculenta or Rana temporaria, are comparable to the respective activities of rabbit fast skeletal muscle. On the other hand, the Ca2+-ATPase activity of the same preparations of frog myosin is 6-7-fold lower than that of myosin from rabbit muscle. Various control experiments indicate that the small extent of Ca2+ stimulation is an intrinsic property of frog muscle myosin. Unlike myosin from rabbit muscle, the Ca2+-ATPase activity of frog myosin is strongly activated by actin; at high actin concentrations it approaches the level of the Ca2+-ATPase activity of rabbit myosin. The levels of Ca2+-ATPase activity of frog and rabbit myosins also become comparable upon modification of myosin SH1 thiol groups; this means that the modification of the SH1 groups results in a much higher activation of the Ca2+-ATPase of frog myosin than that of rabbit myosin. The results suggest a difference in the active site conformation in frog and rabbit muscle myosins. The effects of actin and SH1 group modification are discussed in terms of allosteric changes which diminish the difference in the active site conformation of the two myosins. We have also observed a difference in the reactivity of thiol groups which are not essential for the enzymatic activity in frog and rabbit myosin, indicating structural differences in regions other than the active site.

Actins↗
PubMed1979

Bleomycin induces cataract formation in newborn rats in association with modifications of crystallin synthesis.

The antitumor antibiotic Bleomycin induces the formation of cataract when injected into newborn rats but not when injected into rats after 12 days or older. The development of these lesions is strictly associated with progressive biochemical modifications of the crystallins as demonstrated by electrophoresis on one and two-dimensional gels. When the lenses of the rats are observed histologically, the first modifications appear between 7 and 13 days after the injection of the drug concomitantly with the first biochemical modifications. Later on the fiber cells start to degenerate and there is a gradual destruction of the whole center of the lens.

Animals↗
PubMed1984

Direct modification of plasma low-density lipoproteins in interstitial inflammatory fluid of the rabbit.

Utilizing the polyvinyl sponge-implant model in the rabbit, we have previously demonstrated modification in low-density lipoproteins (LDL) in the extravascular space in association with a cellular inflammatory response. In an attempt to isolate the source of these modifications, plasma LDL was labeled with 125I, and introduced directly into the extravascular space at the time of sponge implantation. [125I] plasma LDL recovered from interstitial inflammatory fluid (IF) at 24 h after implantation demonstrated increased electrophoretic mobility as well as heterogeneity in particle size and hydrated density. These results are in agreement with our previous observations and indicate that modification in IF-LDL probably occurs after it has entered the extravascular space across the vascular wall.

Animals↗
PubMed1994

Nitric oxide and NAD-dependent protein modification.

Nitric oxide (NO) has been suggested to act as a regulator of endogenous intracellular ADP-ribosylation, based on radiolabelling of proteins in tissue homogenates incubated with [32P]NAD and NO. After the NO-stimulated modification was replicated in a defined system containing only the purified acceptor protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the hypothesis of NO-stimulation of an endogenous ADP-ribosyltransferase became moot. The NO-stimulated, NAD-dependent modification of GAPDH was recently characterized as covalent binding of the whole NAD molecule to the enzyme, not ADP-ribosylation. With this result, along with the knowledge that GAPDH is stoichiometrically S-nitrosylated, the role of NO in protein modification with NAD may be viewed as the conferring of an unexpected chemical reactivity upon GAPDH, possibly due to nitrosylation of a cysteine in the enzyme active site.

Adenosine Diphosphate Ribose↗
PubMed1982

Modifications in energy metabolism during the development of chick glial cells and neurons in culture.

Developmental changes in lactate dehydrogenase (LDH), enolase, hexokinase (HK), malate dehydrogenase (MDH), and glutamate dehydrogenase (GDH) activities were measured in cultures of pure neurons and glial cells prepared from brains of chick embryos (8 day-old for neurons, 14 day-old for glial cells) as a function of cellular development with time in culture. The modifications observed in culture were compared to those measured in brain extracts during the development of the nervous tissue in the chick embryo and during the post-hatching period. A significant increase of MDH, GDH, LDH, and enolase activities are observed in neurons between 3 and 6 days of culture, whereas simultaneously a decrease of HK values occurs. In the embryonic brain between 11 and 14 days of incubation, which would correspond for the neuronal cultures to day 3 through 6, modifications of MDH, GDH, HK, and enolase levels are similar to those observed in neurons in culture. Only the increase of LDH activity is less pronounced in vivo than in cultivated cells. The evolution of the tested enzymatic activities in the brain of the chick during the period between 7 days before and 10 days after hatching is quite similar to that observed in cultivated glial cells (prepared from 14 day-old embryos) between 6 and 18 days of culture. All tested activities increased in comparable proportions. The modifications of the enzymatic profile indicate that some maturation phenomena affecting energy metabolism of neuronal and glial elements in culture, are quite similar to those occuring in the total nervous tissue. A relationship between the development of the energy metabolism of the brain and differentiation processes affecting neuroblasts and the glial-forming cells is discussed.

Animals↗
PubMed1994

2,4,6-Trinitrobenzenesulfonic acid modification of the carboxyl-terminal region (C-domain) of calreticulin.

The role of the primary amino groups of lysine sidechains in Ca2+ binding to calreticulin was evaluated by chemical modification of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS). TNBS binding to calreticulin could be described by two steps: (i) a fast reaction, with low affinity, and (ii) a slow reaction with a relatively high affinity. Inclusion of Ca2+ and/or Mg2+ decreased both the amount of TNBS bound to calreticulin and the apparent affinity constant of the slower reaction. In contrast, the properties of the faster reaction for TNBS binding were not sensitive to Ca2+ and/or Mg2+. Analysis of TNBS binding to the carboxyl-terminal (C-domain) and aminoterminal (N-domain) of calreticulin revealed that the C-domain and N-domain are responsible for the slow and fast component of the TNBS binding, respectively. In keeping with this, in the presence of Ca2+, TNBS binding to the C-domain was significantly reduced, whereas modification of the N-domain was unaffected. TNBS modification of calreticulin significantly decreased Ca2+ binding to the low affinity/high capacity Ca2+ binding site(s) which are localized to the C-domain but had no effect on the high affinity/low capacity Ca2+ binding localized to the N domain. In the C-domain of calreticulin, which contains the low affinity/high capacity Ca2+ binding sites, acidic residues are interspersed at regular intervals with one or more positively charged lysine and arginine residues. Our results indicate that the aminogroups of the lysine sidechains in the C-domain of calreticulin have a role in the low affinity/high capacity Ca2+ binding that is characteristic of this region of the protein and which is proposed to contribute significantly to the capacity of the endoplasmic reticulum Ca2+ store.

Amino Acid Sequence↗
PubMed1994

Effect of amino group modification of ovine luteinizing hormone (oLH) by N-succinimidyl 6-[3-(2-pyridyldithio)propionate]hexanoate, a long chain N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) on immunological and biological properties: a comparative study with SPDP modified oLH.

The epsilon-NH2 groups of ovine luteinizing hormone has been modified with the long chain N-succinimidyl-3-(2-pyridyl dithiopropionate (LC-SPDP). The LC-SPDP modification primarily occurs in -NH2 groups of the alpha-subunit. Although, the sequential modification of lysine residue in alpha-subunit led to progressive reduction in the receptor binding and immunological properties but the steroidogenic activity was relatively unaffected. The immunoreactivity and receptor binding properties of LC-SPDP modified oLH molecule were more affected comparative to SPDP modified derivatives. This suggested that the increase in hydrophobic carbon chain in LC-SPDP-oLH molecules resulted into the drastic inhibition in the immunological and biological properties. However, the steroidogenic potential of LC-SPDP/or SPDP-oLH derivative was comparable. The present study clearly demonstrate that a single -NH2 group modification with LC-SPDP would generate the site for the conjugation to the toxin/carrier proteins and the resultant oLH-S-S-toxin conjugate would retain significant immunological and biological properties of the hormone molecule.

Amino Acids↗
PubMed1995

Characterization of chemotherapy-induced morphonuclear modifications in the P388 leukaemia and the MXT mammary tumour models of the mouse.

Chemotherapy-induced morphonuclear modifications were monitored in vivo by means of the digital cell image analysis of Feulgen-stained nuclei. Two experimental models were used, i.e. the P388 mouse leukaemia and the MXT mouse mammary carcinoma. The drugs used were doxorubicin, etoposide and cyclophosphamide. The results indicate that the chemotherapy induced a significant decrease in the MXT tumour growth and a significant increase in the survival of the P388 leukaemic mice. These effects were accompanied at the morphonuclear level by an increase in the nuclear area, by modifications in the DNA content in accordance with the effects of the drugs on the cell cycle and by several modifications in the chromatin texture in accordance with the model or the drugs studied. While there were neither homogeneous morphonuclear changes in all treatment groups nor clearcut correlations between the morphonuclear changes and tumour growth or the survival of the animals, the present study nevertheless shows that it is possible, at least partly, to monitor in vivo certain chemotherapy-induced effects occurring at the morphonuclear level, and subsequently to obtain information on the mode of action of the drugs.

Animals↗