A one-site, two-state model for the binding of anions in photosystem II

Photosystem II membranes, dialyzed against a Cl(-)-free buffer to remove bound Cl-, lost about 65% of the control activity. A light-intensity study of the Cl(-)-free membranes showed that all PS II centers were able to evolve oxygen at about 35% of the control rate when measured in Cl(-)-free medium. The Cl(-)-depleted membranes were immediately (< 15 s) reactivated to 85-90% of the original activity by the addition of fairly high concentrations of Cl- (Kd = 0.5 mM), but both Cl- and the activity were promptly lost when the membranes immediately after reactivation were diluted in a Cl(-)-free medium. However, stabilization of Cl(-)-binding could be accomplished by prolonged incubation in the presence of Cl-. The transition to stable binding, followed using 36Cl-, occurred over several minutes. The stable binding was further characterized by a Kd of 20 microM and a t1/2 for dissociation of about 1h [Lindberg et al. (1993) Photosynth. Res. 38, 401-408]. The effects on S2 signals of removal of Cl- were studied using EPR. The depletion of Cl- was accompanied by a shift in intensity toward the g = 4.1 signal at the expense of the multiline signal. When Cl- or Br- but not F- was added to the depleted PS II membranes, the original distribution of the signals was immediately (< 30 s) restored. We propose that Cl(-)-binding responsible for high oxygen-evolution activity and normal EPR properties of the S2 state may occur either as high affinity (Kd = 20 microM) and slowly exchanging (t1/2 = 1 h), or as low affinity (Kd = 0.5 mM) and rapidly exchanging (t1/2 < 15 s). Our results suggest that Br- but not F- has a mode of binding similar to that of Cl-. The high-affinity state is the normal state of binding, but once Cl- has been removed, it will first rebind as low-affinity, rapidly exchanging followed by conversion into a high-affinity, slowly exchanging mode of binding.     

Toxicokinetics and biochemistry of cadmium with special emphasis on the role of metallothionein

Cadmium is a toxic metal with extremely long biological half-time of 15-20 years in humans. It has for decades been known that cadmium exposure can cause a variety of adverse health effects, among which kidney dysfunction, lung diseases, disturbed calcium metabolism and bone effects are most prominent. Following long term exposure the kidney is the critical organ. Cadmium and its compounds give rise to lung cancer after inhalation and have been classified as human carcinogens. Metallothionein (MT) is a low-molecular -weight protein, 6500Da with high cysteine content and high metal affinity, which plays a major role in the kinetics and metabolism of cadmium. The balance between CdMT and non-bound Cd in renal tissue has been shown to be of crucial importance for expression of toxicity. The most well studied metallothioneins are metallothioneins I and II with their isoforms which are expressed in almost all mammalian tissues. Metallothionein III is expressed in brain and is rich in zinc. Since the blood-brain barrier keeps Cd outside the CNS, reported neurotoxic effects of Cd during development are likely to be secondary to an interference of Cd with Zn-metabolism and not a direct effect of Cd on brain cells. It is therefore of importance to investigate whether neurotoxicity induced by cadmium is related to mechanisms involving MT III in brain.     

Studies of Ca2+ binding in spinach photosystem II using 45Ca2+

The Ca(2+)-binding properties of photosystem II were investigated with radioactive 45Ca2+. PS II membranes, isolated from spinach grown on a medium containing 45Ca2+, contained 1.5 Ca2+ per PS II unit. Approximately half of the incorporated radioactivity was lost after incubation for 30 h in nonradioactive buffer. About 1 Ca2+/PS II bound slowly to Ca(2+)-depleted membranes in the presence of the extrinsic 16- and 23-kDa polypeptides in parallel with restoration of oxygen-evolving activity. The binding was heterogeneous with dissociation constants of 60 microM (0.7 Ca2+/PS II) and 1.7 mM (0.3 Ca2+/PS II), respectively, which could reflect different affinities of the dark-stable S-states for Ca2+. The reactivation of oxygen-evolving activity closely followed the binding of Ca2+, showing that a single exchangeable Ca2+ per PS II is sufficient for the water-splitting reaction to function. In PS II, depleted of the 16- and 23-kDa polypeptides, about 0.7 exchangeable Ca2+/PS II binds with a dissociation constant of 26 microM, while 0.3 Ca2+ binds with a much weaker affinity (Kd > 0.5 mM). The rate of binding of Ca2+ in the absence of the two extrinsic polypeptides was significantly higher than with the polypeptides bound. The rate of dissociation of bound Ca2+ in the dark, which had a half-time of about 80 h in intact PS II, increased in the absence of the 16- and 23-kDa polypeptides and showed a further increase after the additional removal of the 33-kDa protein and manganese. The rate of dissociation was also significantly faster in weak light than in the dark regardless of the presence or absence of the 16- and 23-kDa polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Social insurance cost of standard discectomy and percutaneous nucleotomy. A retrospective study of 87 social insurance claim files of male blue collar workers

To evaluate the effect of cadmium intoxication on renal transport systems for organic anions and cations, transport of p-aminohippurate (PAH) and tetraethylammonium (TEA) were studied in renal cortical plasma membrane vesicles isolated from cadmium-intoxicated rats. Cadmium intoxication was induced by daily injections of CdCl2 (2 mg Cd/kg.day sc) for 2-3 weeks. Renal plasma membrane vesicles were prepared by Percoll gradient centrifugation and magnesium precipitation method. Vesicular uptake of substrate was determined by rapid filtration technique using Millipore filter. The cadmium treatment resulted in a marked attenuation of Na(+)-dependent, alpha-ketoglutarate (alpha KG)-driven PAH uptake in the basolateral membrane vesicle (BLMV), and this was due to a reduction in Vmax and not K(m). The Na(+)-alpha KG symport activity of the BLMV was not affected by 2-week cadmium treatment, but it was significantly inhibited by 3-week cadmium treatment. On the other hand, the alpha KG-PAH antiport activity of the BLMV appeared to be markedly suppressed in 2-week as well as 3-week cadmium-treated animals. The cadmium treatment inhibited the proton gradient-dependent TEA transport in the brush-border membrane vesicle (BBMV), and this was associated with a reduction in Vmax with no change in K(m). These results indicate that cadmium exposures may impair the capacities for organic anion transport in the proximal tubular basolateral membrane and organic cation transport in the luminal membrane. The cadmium effect on organic anion transport is attributed mainly to an inhibition of dicarboxylate-organic anion antiport system.     

胞外高聚物吸附富集-火焰原子吸收光谱法测定痕量镉

以细菌Pseudomonas sp.产生的胞外高聚物PS-2作为吸附剂,利用火焰原子吸收光谱法研究了该吸附剂对镉的吸附性能,提出了用胞外高聚物PS-2作为吸附剂,富集、分离测定环境水样中痕量Cd（Ⅱ）的新方法。结果表明,在pH 6.0,PS-2用量为0.30 g时,吸附率可达到99.5 %,静态吸附容量为35.97 mg/g。以10 mL 1 mol/L HCl 作为解脱剂,可将吸附在PS-2上的镉定量洗脱。此方法的检出限为2.4 ng/L,相对标准偏差为2.5 % (c=0.30 μg/mL, n=9)。将其应用于自来水、玉带河水中痕量Cd（Ⅱ）的测定,方法回收率在95%～101%之间,测定结果与标准方法测定值吻合。     

Effect of metal ion substitutions in concanavalin A on the binding of carbohydrates and on thermal stability

Isothermal titration calorimetry (ITC) measurements were performed on the binding of alpha methyl-D-mannopyranoside, D-mannopyranose, alpha methyl-D-glucopyranoside, and D-glucopyranose (Glu) to cobalt, nickel, and cadmium substituted concanavalin A (Con A) derivatives at pH = 6.9 and at 25 degrees C. The metal substituted Con A derivatives consisted of Co2+, Ni2+, and Cd2+ substituted for the Mn2+ ion in the S1 site of Con A which is about 12.8 A away from the center of the carbohydrate binding site of Con A. The thermodynamic quantities determined from the ITC measurements were the same for most of the binding reactions indicating that the structure of the binding site in solution is the same for all the Con A derivatives in solution and that the presence of different 2+ metal ions in the S1 site has little effect on the binding reactions. Differential scanning calorimetry scans of solutions of the metal ion derivatives of Con A show that the thermodynamics of the unfolding transition for the cobalt and nickel substituted derivatives are the same as for Con A: they dissociate from tetramers into monomers as they unfold around 85 degrees C. The cadmium substituted Con A derivative, however, exhibits an additional transition around 93 degrees C which also appears following the addition of Cd2+ to the Con A solutions. This transition results from the unfolding of a species of Con A with Cd2+ substituted into a third binding site at the monomeric interface of the Con A tetramer. The higher stability of this species is not only exemplified by the higher thermal transition temperature but also by the lack of dissociation as it unfolds. Cd2+ is released from the S3 site upon decreasing the pH from 6.9 to 6.4. ITC measurements on the binding reaction of Cd2+ to Con A show that the binding enthalpy is 40.2 +/- 0.4 kJ mol-1 at 23.4 +/- 0.2 degrees C and the binding reaction exhibits a large heat capacity change of 1.43 +/- 0.41 kJ mol-1 K-1.     

Cadmium binding and sodium-dependent solute transport in renal brush-border membrane vesicles

Exposure to cadmium (Cd) impairs renal transport systems for glucose, amino acids, phosphate, and dicarboxylates. To investigate if these changes are directly related to a Cd binding to the renal brush-border membrane, Cd binding and the Na+-dependent uptakes of d-glucose, l-alanine, phosphate, and succinate were determined in rat renal brush-border membrane vesicles (BBMV) exposed to CdCl2. Cd uptake by BBMV showed time and concentration dependence. Changes in medium osmolality had no effect on Cd uptake, indicating that the process primarily involves binding of Cd to the membrane. Scatchard analysis indicated the presence of two types of Cd binding sites, differing in affinity and number. Increasing the medium Cd concentration from 50 to 200 microM resulted in a progressive increase in Cd binding to the membrane and decrease in Na+-dependent transport of d-glucose, l-alanine, inorganic phosphate, and succinate. In all cases, the inhibition of transport was directly proportional to the total amount of Cd binding to the membrane. These results suggest that, during chronic exposure to Cd, free Cd ions liberated in renal tubular cells may directly interact with brush-border membranes and impair Na+-dependent solute transports.     

Tolerance development to cadmium-induced alteration of drug action

Cadmium administration potentiates the duration of hexobarbital-induced hypnosis and inhibits the rate of hepatic microsomal metabolism of this drug in the male rat. The threshold dose of cadmium required to produce these alterations in drug action is 0.84 mg Ck/kg. If subthreshold doses of cadmium (0.21 or 0.42 mg Cd/kg) are administered prior to the 0.84 mg Cd/kg dose, the cadmium-induced alterations in drug action are no longer observed.     

Regulation of mesangial cell ion channels by insulin and angiotensin II. Possible role in diabetic glomerular hyperfiltration

We used patch clamp methodology to investigate how glomerular mesangial cells (GMC) depolarize, thus stimulating voltage-dependent Ca2+ channels and GMC contraction. In rat GMC cultures grown in 100 mU/ml insulin, 12% of cell-attached patches contained a Ca(2+)-dependent, 4-picosiemens Cl- channel. Basal NPo (number of channels times open probability) was < 0.1 at resting membrane potential. Acute application of 1-100 nM angiotensin II (AII) or 0.25 microM thapsigargin (to release [Ca2+]i stores) increased NPo. In GMC grown without insulin, Cl- channels were rare (4%) and unresponsive to AII or thapsigargin in cell-attached patches, and less sensitive to [Ca2+]i in excised patches. GMC also contained 27-pS nonselective cation channels (NSCC) stimulated by AII, thapsigargin, or [Ca2+]i, but again only when insulin was present. In GMC grown without insulin, 15 min of insulin exposure increased NPo (insulin > or = 100 microU/ml) and restored AII and [Ca2+]i responsiveness (insulin > or = 1 microU/ml) to both Cl- and NSCC. GMC AII receptor binding studies showed a Bmax (binding sites) of 2.44 +/- 0.58 fmol/mg protein and a Kd (binding dissociation constant) of 3.02 +/- 2.01 nM in the absence of insulin. Bmax increased by 86% and Kd was unchanged after chronic (days) insulin exposure. In contrast, neither Kd nor Bmax was significantly affected by acute (15-min) exposure. Therefore, we concluded that: (a) rat GMC cultures contain Ca(2+)-dependent Cl- and NSCC, both stimulated by AII. (b) Cl- efflux and cation influx, respectively, would promote GMC depolarization, leading to voltage-dependent Ca2+ channel activation and GMC contraction. (c) Responsiveness of Cl- and NSCC to AII is dependent on insulin exposure; AII receptor density increases with chronic, but not acute insulin, and channel sensitivity to [Ca2+]i increases with both acute and chronic insulin. (d) Decreased GMC contractility may contribute to the glomerular hyperfiltration seen in insulinopenic or insulin-resistant diabetic patients.     

Isolated perfused rabbit colon crypts: stimulation of Cl- secretion by forskolin

The aim of this study was to characterize ion conductances and carrier mechanisms of isolated in vitro perfused rabbit colonic crypts. Crypts were isolated from rabbit colon mucosa and mounted on a pipette system which allowed controlled perfusion of the lumen. In non-stimulated conditions basolateral membrane voltage (Vbl) was -65 +/- 1 mV (n = 240). Bath Ba2+ (1 mmol/l) and verapamil (0.1 mmol/l) depolarized Vbl by 21 +/- 2 mV (n = 7) and 31 +/- 1 (n = 4), respectively. Lowering of bath Cl- concentration hyperpolarized Vbl from -69 +/- 3 to -75 +/- 3 mV (n = 9). Lowering of luminal Cl- concentration did not change Vbl. Basolateral application of loop diuretics (furosemide, piretanide, bumetanide) had no influence on Vbl in non-stimulated crypts. Forskolin (10(-6) mol/l) in the bath depolarized Vbl by 29 +/- 2 mV (n = 54) and decreased luminal membrane resistance. In one-third of the experiments a spontaneous partial repolarization of Vbl was seen in the presence of forskolin. During forskolin-induced depolarization basolateral application of loop diuretics hyperpolarized Vbl significantly and concentration dependently with a potency sequence of bumetanide > piretanide > or = furosemide. Lowering bath Cl- concentration hyperpolarized Vbl. Lowering of luminal Cl- concentration from 120 to 32 mmol/l during forskolin-induced depolarization led to a further depolarization of Vbl by 7 +/- 2 mV (n = 10). We conclude that Vbl of rabbit colonic crypt cells is dominated by a K+ conductance. Stimulation of the cells by forskolin opens a luminal Cl- conductance. Basolateral uptake of Cl- occurs via a basolateral Na+:2Cl-:K+ cotransport system.     

Effect of cadmium on purified hepatic flavokinase: involvement of reactive -SH group(s) in the inactivation of flavokinase by cadmium

The effect of cadmium (Cd2+) was studied in vitro on the flavokinase (ATP : riboflavin 5'-phosphotransferase, EC 2.7.1.26) activity purified from rat liver. Cadmium inhibited flavokinase activity in a concentration-dependent manner and the effect was completely reversed by increasing concentration of zinc (Zn2+), indicating a competition between Zn2+ and Cd2+ for binding with the enzyme. Further, a competition between riboflavin and Cd2+ hints at the possibility that Zn2+ and Cd2+ probably compete for the same site on the enzyme where riboflavin binds. Our studies further reveal that hepatic flavokinase contains essential, accessible and functional thiol group(s) as evidenced by a concentration-dependent inhibition by sulfhydryl reagents and protection by thiol protectors like glutathione or dithiothreitol. Furthermore, the enzyme could also be protected from the inhibitory effect of Cd2+ and Hg2+ by glutathione and dithiothreitol suggesting that Cd2+ probably interacts with reactive thiol group at or near the active site of the enzyme to cause inhibition.     

Differential membrane-binding and activation mechanisms of protein kinase C-alpha and -epsilon

To elucidate the mechanisms of membrane binding and activation of conventional and novel protein kinase C (PKC), we measured the interactions of rat PKC-alpha and -epsilon with phospholipid monolayers and vesicles of various compositions. Besides the established difference in calcium requirement, the two isoforms showed major differences in their membrane-binding and activation mechanisms. For PKC-alpha, diacylglycerol (DG) specifically enhanced the binding of PKC-alpha to phosphatidylserine (PS)-containing vesicles by 2 orders of magnitude, allowing PKC-alpha high specificity for PS. Also, PKC-alpha could penetrate into the phospholipid monolayer with a packing density comparable to that of the cell membrane only in the presence of Ca2+ and PS. When compared to PKC-alpha, PKC-epsilon had lower binding affinity for PS-containing vesicles both in the presence and in the absence of DG. As a result, PKC-epsilon did not show pronounced specificity for PS. Also, PKC-epsilon showed reduced penetration into PS-containing monolayers, which was comparable to the Ca2+-independent penetration of PKC-alpha into the same monolayers. Taken together, these results suggest the following: (1) The role of Ca2+ in the membrane binding of PKC-alpha is to expose a specific PS-binding site. (2) Once bound to membrane surfaces, PS specifically induces the partial membrane penetration of PKC-alpha that allows its optimal interactions with DG, hence the enhanced membrane binding and activation. (3) PKC-epsilon, due to the lack of Ca2+ binding, cannot specifically interact with PS and DG, which implies the presence of other physiological activator(s) for this isoform.     

Analysis of the reaction coordinate of photosynthetic water oxidation by kinetic measurements of 355 nm absorption changes at different temperatures in photosystem II preparations suspended in either H2O or D2O

Flash-induced absorption changes at 355 nm were measured at different temperatures within the range of 2 degrees C S2) = 14 kJ/mol, EA(S2-->S3) = 35 kJ/mol, and EA(S3-->-->S0 + O2) = 21 kJ/mol for theta > 11 degrees C, 67 kJ/mol for theta < 11 degrees C in PS II core complexes dissolved in H2O; (b) replacement of exchangeable protons by deuterons causes only minor changes ( S2, S2 --> S3, and S3 -->--> S0 + O2, respectively. The corresponding values of PS II membrane fragments are 1.3, 1.3, and 1. 4. Based on these results and corresponding EA data reported in the literature for PS II membrane fragments from spinach [Renger, G., & Hanssum, B. (1992) FEBS Lett. 299, 28-32] and PS II particles from the thermophilic cyanobacterium Synechococcus vulcanus Copeland [Koike, H., Hanssum, B., Inoue, Y., & Renger, G. (1987) Biochim. Biophys. Acta 893, 524-533], the reaction coordinate of the redox sequence in the WOC is inferred to be almost invariant to the evolutionary development from cyanobacteria to higher plants. Furthermore, the rather high activation energy of the S2 --> S3 transition provides evidence for a significant structural change coupled with this reaction. Implications for the mechanism of photosynthetic water oxidation are discussed.     

A micromechanical model of lung tissue rheology

We evaluated the acute effects of ibuprofen and salicylic acid on cAMP-mediated Cl- secretion (Isc) in both colonic and airway epithelia. In T84 cells, ibuprofen inhibited the forskolin-dependent Isc in a concentration-dependent manner, having an apparent Ki of 142 microM. Salicylic acid inhibited Isc with an apparent Ki of 646 microM. We determined whether ibuprofen would also inhibit the forskolin-stimulated Isc in primary cultures of mouse trachea epithelia (MTE) and human bronchial epithelia (HBE). Similar to our results in T84 cells, ibuprofen (500 microM) inhibited the forskolin-induced Isc in MTEs and HBEs by 59+/-4% (n = 11) and 39+/-6% (n = 8), respectively. Nystatin was employed to selectively permeabilize the basolateral or apical membrane to determine the effect of ibuprofen on apical Cl- (ICl) and basolateral K+ (IK) currents after stimulation by forskolin. After forskolin stimulation, ibuprofen (500 microM) reduced both the ICl and IK; reducing ICl and IK by 60 and 15%, respectively. To determine whether this inhibition of ICl was due to the inhibition of CFTR, the effects of ibuprofen and salicylic acid on CFTR Cl- channels in excised, inside-out patches from L-cells were evaluated. Ibuprofen (300 microM) reduced CFTR Cl- current by 60+/-16% and this was explained by a short-lived block (approximately 1.2 ms) which causes an apparent reduction in single channel amplitude from 1.07+/-0.04 pA to 0.59+/-0.04 pA (n = 3). Similarly, salicylic acid (3 mM) reduced CFTR Cl- current by 50+/-8% with an apparent reduction in single channel amplitude from 1.08+/-0.03 pA to 0.48+/-0.06 pA (n = 4). Based on these results, we conclude that the NSAIDs ibuprofen and salicylic acid inhibit cAMP-mediated Cl- secretion in human colonic and airway epithelia via a direct inhibition of CFTR Cl- channels as well as basolateral membrane K+ channels. This may reduce their efficacy in conjunction with other therapeutic strategies designed to increase CFTR expression and/or function in secretory epithelia.     

Interaction of nonachlazine and noradrenaline on a model phospholipid membrane

The binding of nonachlazine (NCL), imipramine (IMP), and noradrenaline (NA) with the model phospholipid membrane vesicles--liposomes--was studied. The binding was determined by the qunching effect of these substances on the fluorescence of 3-methoxybenzantrone (MBA) present in the membrane of the fluorescent probe. A method rendering possible calculation of the binding of the preparations under study with the membrane of the basis of the fluorescence changes was developed. The binding constant of the NCL, IMP, and NA interaction with the membrane was equal to (4.3 +/- 0.3)-10(3)M-1, (2.7 +/- 0.2)-10(3)M-1, and (0.7 +/- 0.15)-10(3)M-1, respectively. It was shown that NCL and IMP could compete with NA for the membrane binding centers. Such competitive interactions could be regarded as a probable mechanism of the block of the reverse NA transport through the synaptic and the vesicular membranes characteristic of NCL and IMP.     

Genetic basis of the MbrC "ploidy" phenotype in Escherichia coli

We measured the spin-lattice relaxation times (T1) of water protons and intermolecular cross-relaxation times (T(IS)) from irradiated protein protons (f2-irradiation at 1.95 or -4.00 ppm) of rabbit normal and monoiodoacetate-induced degenerated knee articular cartilages to observed water protons. The mean values of T1 (T1) for control and degenerated rabbit knee cartilages were 1.87+/-0.15 (mean+/-SD, n=29) and 1.82+/-0.13 s (n=34), respectively. The mean values of water content (W(H2O)) for control and degenerated rabbit knee cartilages were 82.9+/-2.09 (n=26) and 83.1+/-2.57% (n=28), respectively. These values were not statistically different from each other. However, the mean values of T(IS) (T(IS)) for normal knee articular cartilage were significantly different from those for degenerated cartilage: (normal), T(IS) (f2=1.95 ppm)=2.46+/-0.62 s (n=28), T(IS) (f2=-4.00 ppm)=4.25+/-1.26 s (n=26); (degenerated), T(IS) (f2=1.95 ppm)=1.99+/-0.76s (n=34), T(IS) (f2=-4.00 ppm)=3.33+/-0.76 s (n=31). Obtained results may be attributed to the reported switchover from type II to type I collagen syntheses in osteoarthritic cartilage, resulting in broad collagen fibers. This specificity of cross-relaxation effect may prove useful in the noninvasive and pathophysiological evaluation of cartilage tissues in vivo.     

Selective interaction of homophtalazine derivatives with morphine

Binding of ATP to bovine serum albumin was shown by ultrafiltration and NMR. The binding was pH dependent. Scatchard analysis revealed that at pH 5.4, 6.4 and 7.4, dissociation constant Kd was 13, 40 and 120 microM, respectively, and no binding was observed at pH 8.4. The binding stoichiometry was 1:1 for all pH. Dimer of BSA did not bind ATP. From chemical shifts of 31P-NMR, Kd was estimated to be 15 microM at pH 5.4, which is very close to that determined by ultrafiltration. While adenosine did not interfere with the binding. GTP, dCTP, ADP, UTP, AMP, phosphate and pyrophosphate were competitive inhibitors and their inhibition constants Ki were 25, 32, 36, 50, 130, 1000 and 186 microM, respectively. Fatty acids such as lauric acid and palmitic acid did not interfere with the binding. Warfarin was a non-competitive inhibitor. Cl- competitively inhibited the binding, and the inhibition constant was 20 mM. The dissociation constants of the Cl- binding were reported to be 0.42 mM for the first binding site, 10-5 mM for the second and 303-143 mM for the third [G. Scatchard, W.T. Yap, J. Am. Chem. Soc., 86 (1964) 3434; G. Scatchard et al., J. Am. Chem. Soc. 79 (1957) 12]. This suggests that the ATP binding site may be the second Cl- binding site.     

Endoscopic treatment of vesicorenal reflux in children. Five-year assessment of the use of Macroplastique

Cadmium, unlike zinc, selenium and copper, has no known biological importance, and therefore, it is classified as a carcinogen in humans, as well as in animals. The effect(s) of levels of dermally-administered cadmium on cadmium genotoxicity and cytotoxicity was investigated in Harlan Sprague-Dawley rats for 14, 21, 28, 35 and 42 days at concentrations of 14 and 28 mg/kg/day. Exposure of rats to cadmium via dermal application caused lesions on the skin (hyperkeratosis, acanthosis and scabbing, alopecia and erythema) and tumors in the scrotum. Anatomical changes, such as distention of the stomach, atrophy of kidney and liver and loss of body weight were also observed in these rats. The toxic effects of cadmium on cell ultrastructure were nuclear membrane damage, chromatin condensation, regression of mitochondrial cristae and ultimately cell death. Analyses of the brain, kidney and liver cells of rats exposed to cadmium, clearly showed DNA damage. Of the three organs examined, DNA from kidney cells sustained the most damage followed by DNA in liver cells. There is a positive correlation between Cd dose(s) and duration of exposure and the extent of DNA damage.     

S-state dependence of chloride binding affinities and exchange dynamics in the intact and polypeptide-depleted O2 evolving complex of photosystem II

The Cl- binding properties in the successive oxidation states of the O2 evolving complex of photosystem II were investigated by measurements of UV absorbance changes, induced by a series of saturating flashes, that monitor manganese oxidation state transitions. In dark-adapted, intact photosystem II, Cl- can be replaced by NO3- in minutes, in an exchange reaction that depends on the NO3- concentration and that is not rate-limited by dissociation of Cl- from its binding site. Preillumination of dark-adapted photosystem II by one or two flashes accelerated the NO3- substitution reaction by an order of magnitude. A quantitative analysis of the Cl- concentration dependence of UV absorbance changes, measured in photosystem II preparations depleted of extrinsic 17 and 23 kDa polypeptides, shows that the Cl- binding properties of photosystem II change with the oxidation state of the oxygen evolving complex. Although the affinity for the individual S-states could not be determined with precision, it is shown that the affinity is an order of magnitude lower in the S2 state than in the S1 state. Comparison of the results obtained using intact photosystem II and preparations depleted of the 17 and 23 kDa extrinsic polypeptides suggests that these proteins constitute a diffusion barrier, which prevents fast equilibration of the Cl- binding site with the medium, but does not change the Cl- affinity of the binding site.     

Oxidative decarboxylation of 6-phosphogluconate by 6-phosphogluconate dehydrogenase proceeds by a stepwise mechanism with NADP and APADP as oxidants

Primary kinetic deuterium, 13C, and multiple deuterium/13C-isotope effects on V/K6PG have been measured for the Candida utilis (cu) and sheep liver (sl) 6-phosphogluconate dehydrogenases (6PGDH). With NADP as the dinucleotide substrate, the following values of D(V/K6PG), 13(V/K6PG)H, and 13(V/K6PG)D were measured at pH 8 for cu6PGDH (sl6PGDH): 1.57 +/- 0.08 (1.87 +/- 0.10), 1.0209 +/- 0.0005 (1.0059 +/- 0.000 10), 1.0158 +/- 0.0001 (1.0036 +/- 0.0008). With APADP as the dinucleotide substrate, values for the above isotope effects at pH 8 are as follows: 2.98 +/- 0.08 (2.47 +/- 0.06), 1. 0106 +/- 0.0002 (1.0086 +/- 0.000 09), and 0.9934 +/- 0.0003 (0.9950 +/- 0.0003). Results indicate the oxidative decarboxylation of 6PG to the 1,2-enediol of ribulose 5-phosphate proceeds via a stepwise mechanism with hydride transfer preceding decarboxylation in all cases. The inverse 13C-isotope effect observed with APADP and 6PG-3d may reflect a preequlibrium isotope effect on the binding of 6PG preceding hydride transfer. Deuterium-isotope effects on V, V/KNADP, and V/K6PG are identical at all pHs and for both enzymes. The primary deuterium-isotope effect on V/K6PG for both enzymes is constant at pH values below the pK in the pH profile for V/K6PG, and decreases as the pH increases. Data suggest the development of rate limitation by a step or steps other than the hydride-transfer step as the pH is increased.