Contrasts in the actions of protein antibiotics on deoxyribonucleic acid structure and function.

The protein antibiotics neocarzinostain (NCS), macromomycin (MCR), and auromomycin (AUR), which is closely related to MCR, have been compared for their in vitro and in vivo actions on deoxyribonucleic acid (DNA). NCS, markedly stimulated by 2-mercaptoethanol, is much more active in inducing strand scissions in superhelical pMB9 and linear duplex lambda DNA than AUR, which is slightly inhibited by 2-mercaptoethanol. Purified MCR, even at very high levels, does not give any significant amount of cutting with either DNA substrate. 2-Propanol stimulates the activity of NCS but inhibits that of AUR. On the other hand, the antioxidant alpha-tocopherol strongly inhibits DNA breakage by both drugs. The intercalating drugs ethidium bromide, daunorubicin, proflavin, and actinomycin D at low concentrations inhibit DNA scission by AUR. The levels of intercalators required to inhibit NCS activity to comparable levels are about 10 times higher than those for AUR. Although MCR has virtually no in vitro DNA cutting activity, it is, like AUR and NCS, cytotoxic, as measured by the inhibition of DNA synthesis and induction of DNA strand breakage in HeLa cells.     

Effect of crosslinking on mitochondrial structure and function

Rat liver mitochondria were treated with ethylacetimidate

1 Abbreviations: SDS, sodium dodecylsulfate; DMS, dimethylsuberimidate; EA, ethylacetimidate; MBI, methylbutyrimidate; TMPD, N, N′, N′-tetramethyl-p-phenylenediamine.

and methylbutyrimidate, monofunctional imidates, and with dimethylsuberimidate, a bifunctional imidate, and the effects on structure and function studied. Mitochondria treated with 5 mM dimethylsuberimidate or greater did not respond osmotically when placed in deionized water. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed that at concentrations > 5 mM dimethylsuberimidate nearly all mitochondrial polypeptides failed to enter 6% gels, indicating crosslinking of both membrane and soluble proteins. Extensive amidination by ethylacetimidate and methylbutyrimidate had little effect on ascorbate-tetramethylphenylenediamine oxidase while extensive inhibition resulted from dimethylsuberimidate treatment. The possible involvement of molecular motion in electron transport is discussed.     

Effect of the molecular structure of natural anthracycline antibiotics on their relative hydrophobicity

Relative hydrophobicities of anthracycline antibiotics, adriamycin, rubomycin and carminomycin, have been measured by the two-phase distribution method. Two different biphasic systems were used for this purpose. Possible reasons of discrepancies between results obtained and other authors, data are discussed. It was established that the relative hydrophobicities of the compounds investigated contradict the theory of increment additivity. The results are compared with quantum-chemical calculations.     

Effect of gamma-radiation on the structure and function of yeast membrane

A decrease in the influx of several amino acids was observed following gamma-irradiation. At low dose (2.5 Gy), which does not affect cell survival, a stimulation in the uptake was visible; moreover, sulphydryl loss and lipid peroxidation were also evident. With further increase in the dose of radiation, a parallel increment in the loss of sulphydryl groups and production of malonaldehyde was observed. Radioprotectors like L-cysteine and dithiothreitol were shown to shield the radiation-induced loss of sulphydryl and damage to transport and survival. Reduced glutathione, on the other hand, exhibited protection at the level of sulphydryl damage only. N-ethylmaleimide, a well known hypoxic cell radiosensitizer, enhanced the radiosensitivity with respect to survival; it, however, had no effect on amino acid transport. Oxygen enhancement of radiation damage to transport and cell survival and the radioprotection by sodium formate under these circumstances, and more so by anoxia, were demonstrated. The results indicate that the manifestation of damage to membrane structure and function precedes any observable loss of survival.     

Influence of ovaries and photoperiod on reproductive function in the mare.

A 16 h daily photoperiod hastened the onset of the ovulatory season (first ovulation); gonadotrophin and follicular changes prior to the onset were similar in intact light-treated and control mares. A preovulatory decline in FSH concentrations before the onset of the ovulatory season preceded the decrease in number of follicles (15--25 mm) and the rise in LH concentrations which was temporally associated with the growth of an ovulatory follicle. Seasonal changes of FSH and LH concentrations were found in ovariectomized mares and were influenced by photoperiod. During the anovulatory season, there was no ovarian influence on gonadotrophin concentrations. However, during the ovulatory season the ovaries exerted a positive influence on seasonally elevated LH concentrations during oestrus and a negative influence during dioestrus. The ovaries exerted a negative influence on seasonally elevated FSH concentrations throughout the oestrous cycle. The onset of the ovulatory season occurred at the time of the first sustained increase in LH concentrations resulting from positive seasonal (increasing photoperiod) and ovarian influences.     

Effect of methyl methacrylate on mitochondrial function and structure

• 1.1. Treatment of isolated rat liver mitochondria with methyl methacrylate (MM) produced membrane disruption as evidenced by the release of citrate synthase, and changes in the ultrastructure of mitochondria.
• 2.2. At concentration 0.1%, MM uncoupled oxidative phosphorylation as evidenced by stimulation of state 4 respiration supported either by pyruvate plus malate or succinate (+rotenone) and ATP-ase activity in intact mitochondria.
• 3.3. At concentration 1% MM stimulated ATP-ase activity in intact mitochondria and succinate (+rotenone) oxidation at state 4 and was without effect on this substrate oxidation at state 3.
• 4.4. MM inhibited pyruvate plus malate oxidation either at state 3 or in the presence of uncoupling agents.
• 5.5. MM inhibited the NADH oxidase of electron transport particles at a concentration which failed to inhibit either succinic oxidase or the NADH-ferricyanide reductase activity.
• 6.6. The data presented suggest that in the isolated mitochondria MM inhibits NADH oxidation in the vicinity of the rotenone sensitive site of complex I.
• 7.7. The general conclusion is that MM may block an electron transport and to uncouple oxidative phosphorylation in rat liver mitochondria. The overall in vitro effect would be to prevent ATP synthesis which could result in cell death under in vivo conditions.

     

Effect of glycation on alpha-crystallin structure and chaperone-like function

The chaperone-like activity of alpha-crystallin is considered to play an important role in the maintenance of the transparency of the eye lens. However, in the case of aging and in diabetes, the chaperone function of alpha-crystallin is compromized, resulting in cataract formation. Several post-translational modifications, including non-enzymatic glycation, have been shown to affect the chaperone function of alpha-crystallin in aging and in diabetes. A variety of agents have been identified as the predominant sources for the formation of AGEs (advanced glycation end-products) in various tissues, including the lens. Nevertheless, glycation of alpha-crystallin with various sugars has resulted in divergent results. In the present in vitro study, we have investigated the effect of glucose, fructose, G6P (glucose 6-phosphate) and MGO (methylglyoxal), which represent the major classes of glycating agents, on the structure and chaperone function of alpha-crystallin. Modification of alpha-crystallin with all four agents resulted in the formation of glycated protein, increased AGE fluorescence, protein cross-linking and HMM (high-molecular-mass) aggregation. Interestingly, these glycation-related profiles were found to vary with different glycating agents. For instance, CML [N(epsilon)-(carboxymethyl)lysine] was the predominant AGE formed upon glycation of alpha-crystallin with these agents. Although fructose and MGO caused significant conformational changes, there were no significant structural perturbations with glucose and G6P. With the exception of MGO modification, glycation with other sugars resulted in decreased chaperone activity in aggregation assays. However, modification with all four sugars led to the loss of chaperone activity as assessed using an enzyme inactivation assay. Glycation-induced loss of alpha-crystallin chaperone activity was associated with decreased hydrophobicity. Furthermore, alpha-crystallin isolated from glycated TSP (total lens soluble protein) had also increased AGE fluorescence, CML formation and diminished chaperone activity. These results indicate the susceptibility of alpha-crystallin to non-enzymatic glycation by various sugars and their derivatives, whose levels are elevated in diabetes. We also describe the effects of glycation on the structure and chaperone-like activity of alpha-crystallin.     

Effect of polyethylene glycols on the function and structure of thiol proteases

Thiol proteases are industrially significant proteins with catalytic efficiency. The effect of low, medium and high molecular-weight poly (ethylene glycol) (PEG- 400, 6000 and 20000) on the stability of thiol proteases (papain, bromelain and chymopapain) has been studied by activity measurements using synthetic substrate. Structural studies performed on papain by far UV circular dichroism spectroscopic measurements indicate that there is loss in secondary structure of the protein in presence of increasing concentration of PEGs. Intrinsic fluorescence measurements lead us to conclude that tryptophan residues of protein encounter non-polar microenvironment in presence of PEG solvent while acrylamide quenching shows greater accessibility of tryptophan residues of papain in presence of PEGs. Extrinsic fluorescence measurements lead us to conclude that PEGs bind to the hydrophobic sites on the protein and thus destabilize it. Thermal denaturation studies show that melting temperature of papain is decreased in presence of PEGs. Possible mechanism of destabilization is discussed next. The results imply that caution must be exercised in the use of PEGs with thiol proteases or hydrophobic proteins in general, for different industrial applications, even at room temperature.     

Effect of alcohols on the structure and function of D-amino-acid oxidase.

The absorption spectrum of D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3) was significantly perturbed by various alcohols; typical fine structures were observed in the visible absorption bands, accompanied by blue shifts of the peaks. Both fluorescence intensity and fluorescence polarization were increased upon the addition of alcohols, indicating that the coenzyme is not liberated from the apoenzyme but the hydrophobicity of the environment of the enzyme-bound flavin is increased. Upon the addition of alcohols, the circular dichroism of the enzyme was markedly modified in the visible and near-ultraviolet regions, while that of the apoenzyme in the near- and far-ultraviolet regions was scarcely modified, indicating a change in the interaction between the flavin coenzyme and protein. Both the apparent maximal velocity and the apparent Michaelis constant of the enzyme were increased by the addition of alcohols. The presence of alcohols tends to dissociate the dimer of this enzyme into the monomer, but the dissociation does not fully explain the increase in the maximal velocity of the enzyme by alcohols, because the increase in the maximal velocity caused by alcohols is larger than that expected from the dissociation. Since the rate of formation of the purple intermediate was decreased by alcohols in both the dimer and the monomer, the increase in the maximal velocity could be ascribed to an increase in the rate of dissociation of the enzyme-product complex. This increase could be ascribed to the protein conformational change, which is probably provoked by combination of alcohols with the enzyme at a locus other than that for substrate binding.     

Effect of freezing and thawing on the structure and function of cytochrome oxidase

The effect of freezing-thawing on cytochrome oxidase from cattle heart was studied. The enzyme was characterized for its activity, absorption spectra, temperature perturbation differential spectra and conformational transitions. A decrease in the activity and conformational changes depend on the composition of the buffer system and the presence of potassium and sodium chlorides in the solution.     

Effect of Bcl-2 overexpression on mitochondrial structure and function

Overexpression of the antiapoptotic Bcl-2 protein enhances the uptake of fluorimetric dyes sensitive to mitochondrial membrane potential, suggesting that Bcl-2 changes the mitochondrial proton gradient. In this study, we performed calibrated measurements of mitochondrial respiration, membrane potential, deltapH, and intramitochondrial [K+] in digitonin-permeabilized PC12 and GT1-7 neural cells that either do not express human Bcl-2 (control transfectants) or that were transfected with and overexpressed the human bcl-2 gene to evaluate whether Bcl-2 alters mitochondrial inner membrane ion transport. We found that although Bcl-2-overexpressing cells exhibit higher fluorescence responses to membrane potential, pH, and K+-sensitive dyes, this increased response is due to an enhanced accumulation of these dyes and not an increased mitochondrial membrane potential, deltapH, or [K+]. This result is supported by the presence of equal respiratory rates in Bcl-2+ and Bcl-2- cells. Possible structural alterations in Bcl-2+ mitochondria that could account for increases in fluorescent dye uptake were evaluated using flow cytometry particle sizing and light scattering determinations. These experiments established that Bcl-2-overexpressing mitochondria present both increased volume and structural complexity. We suggest that increased mitochondrial volume and structural complexity in Bcl-2+ cells may be related to many of the effects of this protein involved in the prevention of cell death.     

Effect of acute nutritional deprivation on diaphragm structure and function

The influence of 90 h of acute nutritional deprivation (ND) on the cross-sectional areas of muscle fibers and the contractile and fatigue properties of the adult rat diaphragm were determined. Isometric contractile properties and fatigue resistance of the diaphragm were measured by means of an in vitro nerve-muscle strip preparation. Contractions were evoked by using phrenic nerve stimulation (left hemidiaphragm) or direct muscle stimulation (right hemidiaphragm) in the presence of curare. Acute ND resulted in a 20% reduction in body weight. No significant decrements in diaphragm or soleus weights were noted in the ND animals compared with controls (CTL), whereas the weight of the medial gastrocnemius was reduced by 20% in the ND animals. Peak twitch and tetanic tensions (normalized for the weight of the diaphragm strip) were not reduced in ND compared with CTL animals after either nerve or muscle stimulation. The fatigue index of the diaphragm was significantly reduced in ND animals only after nerve stimulation. After the fatigue test, there was rapid recovery of the additional fatigue noted with nerve stimulation. The proportions of type I and II muscle fibers of the diaphragm were similar in the CTL and ND animals. No differences in diaphragm cross-sectional areas were noted for either type I or II muscle fibers in the CTL and ND animals. It is concluded that acute ND has no effect on diaphragm contractility or morphometry and only an inconsequential influence on diaphragm fatigue.