Inhibition of PAF-acether effects on isolated guinea pig hearts by zinc ions (Zn2+)

PAF-acether is a phospholipid synthesized by most animal tissues and exerting a strong decrease on the heart’s contractile force and coronary flow. PAF-acether (10⁻⁹ and 10⁻¹⁰ M) was administered to isolated guinea pig hearts perfused via the Langendorff apparatus with Chenoweth solution. Zinc (1.5 μM) is known to benefit heart function thus, Zn²⁺ (1.5, 7.5, and 30 μM) was added in the perfusing solution before or after PAF-acether administration. Contractile force, coronary flow, and heart rate were recorded by means of a Narco MK-IV Physiograph throughout all modes of perfusion. Calcium inhibitor (Verapamil 10⁻¹⁰ M) and Pb²⁺ Co²⁺ (1.5×10⁻⁶ M) were used subsequently in the perfusing solutions in order to elucidate some of the Zn and PAF interactions observed. All hearts were analyzed for their Zn and Ca content by means of an Atomic Absorption Spectrophotometry (AAS). Our data suggest that low concentrations of zinc (1.5 μM) can strongly inhibit PAF-induced decrease of contractile force and coronary flow. Zinc-inhibiting effects on PAF's negative inotropic action (myocytic level) is not exerted through Zn−Ca antagonism. Nevertheless, a Zn−Ca antagonism in the arteriolar level cannot be excluded. Zinc inhibits PAF selectively only if it is administered before PAF injection and this strongly suggests a receptor interaction between the metal and the phospholipid at the heart level.     

Electrocardiographic alterations induced by zinc ions on isolated guinea pig heart preparations

On guinea pig isolated hearts perfused according to the Langendorff technique, by Chenoweth's solution, and by various concentration of ZnCl₂, surface E.C.G. was recorded for 65 min, after the equilibration period. Concentrations of zinc corresponding to plasma free and exchangable fractions (1.5 μM) exerted only a nonstatistically significant sinus bradycardia. Bradycardia was prominent between the 25th and 35th min of perfusion, with higher concentrations of zinc (7.5 and 15 μM). In even higher (toxic) concentrations of zinc (30 μM), various types of arrhythmias were recorded, such as first degree AV block, idioventricular rhythms, atrial, and ventricular extrasystoles, and asystolia, evoked since the 15th min of perfusion. The above-mentioned findings in relation to low plasma zinc concentrations found in coronary patients and experimental myocardial infarction as well as myocardial injury related to metal fume fever, brought our attention to the involvement of this divalent cation to cardiac physiology and pathophysiology.     

Effects of glyburide (glibenclamide) on myocardial function in Langendorff perfused rabbit heart and on myocardial contractility and slow calcium current in guinea-pig single myocytes

Glyburide, also known as glibenclamide, was shown to have positive inotropic effect in human and animal hearts. The objectives of the present study was to investigate the effects of glyburide on developed left ventricular pressure (DLVP), coronary flow (CF), and heart rate (HR), in isolated rabbit heart as well as its effects on myocardial contractility and L-type calcium current, i_Ca, in guinea pig myocytes. Rabbit hearts were mounted on Langendorff apparatus and perfused with an oxygenated Krebs for 30 min until reaching steady state to be followed by 20 min of experimental perfusion divided into 5 min of control perfusion and 15 min of perfusion with Glyburide (10 M). Ventricular myocytes were isolated by enzymatic dispersion technique and superfused in an oxygenated Tyrode solution. Cells were voltage-clamped at holding potential –40 mV to inactivate Na⁺ current and a step depolarizations, 200 msec duration, to 0 mV was applied to elicit i_Ca. The contractions of the myocytes were measured by optical methods. Glyburide significantly increased DLVP by 30% and CF by 36% but had no effect on HR. Glyburide increased cell contractility by 7 ± 6, 18 ± 7, 28 ± 9 and 54 ± 15% for 0.1, 1, 10 and 100 M respectively, p < 0.001. Meanwhile it depressed i_Ca by 9 ± 6 and 19 ± 8% for 1 and 10 M respectively. In conclusion, glyburide increased contractility of guinea pig single myocytes and of isolated rabbit heart, as indicated by increased developed left ventricular pressure while it depressed i_Ca. It is hypothesized that an elevation in intracellular calcium, which caused increased myocardial contractility, could be attributed to an increase in intracellular Na⁺ that could increase intracellular calcium via Na⁺/Ca²⁺ exchange.     

Atrial natriuretic peptide relation to plasma and heart zinc concentrations of wistar rats exposed to cold and hot ambients

Plasma atrial natriuretic peptide (ANP) and zinc levels, as well as heart tissue zinc concentrations were determined, in male Wistar rats after the exposure of 114 rats at low temperature (4°C) and 95 rats at high temperature (35–36°C) for 28 d. Plasma ANP was estimated by radioimmunoassay and Zn²⁺ concentrations by atomic absorption spectrometry. Values were compared to a control group exposed at 20–22°C (76 rats). Plasma ANP and Zn²⁺ levels, as well as heart tissue Zn²⁺ concentrations of control rats did not show statistically significant variations during the study, whereas rats exposed to cold and hot ambients showed significant variations of the parameters. A significant increase of plasma ANP and plasma zinc and heart tissue Zn²⁺ concentrations developed during cold exposure, whereas a gradual decrease of plasma ANP and Zn²⁺ levels was revealed during hot adaptation. Results also indicate that plasma ANP and zinc levels are proportionally related, whereas there is an inverse relationship between plasma ANP levels and heart Zn²⁺ concentrations, in both cold and hot exposed rats. In conclusion, our results show that ANP in relation to zinc probably play an important role in cold and hot acclimatization of rats.     

Choline Modulates Cardiac Membrane Repolarization by Activating an M3 Muscarinic Receptor and its Coupled K+ Channel

Choline is a necessary substrate of the lipid membrane and for acetylcholine synthesis. Accumulating evidence indicates that besides being a structural component, choline is also a functional modulator of the membrane. It has been shown to be a muscarinic acetylcholine receptor (mAChR) agonist and can induce a novel K⁺ current in cardiac cells. However, the potential role of choline in modulating cardiac functions remained unstudied despite that mAChRs are known to be important in regulating heart functions. With microelectrode techniques, we found that choline produced concentration-dependent (0.1∼10 mm) decreases in sinus rhythm and action potential duration in isolated guinea pig atria. The effects were reversed by 2 nm 4DAMP (an M₃-selective antagonist). Whole-cell patch-clamp recordings in dispersed myocytes from guinea pig and canine atria revealed that choline is able to induce a K⁺ current with delayed rectifying properties. The choline-induced current was suppressed by low concentrations of 4DAMP (2∼10 nm). Antagonists toward other subtypes (M₁, M₂ or M₄) all failed to alter the current. The affinity of choline (K _d ) at mAChRs derived from displacement binding of [³H]-NMS in the homogenates from dog atria was 0.9 mm, consistent with the concentration needed for the current induction and for the HR and APD modulation. Our data indicate that choline modulates the cellular electrical properties of the hearts, likely by activating a K⁺ current via stimulation of M₃ receptors. Received: 1 December 1998/Revised: 12 February 1999     

Fluctuation in recoverable nickel and zinc resistant copiotrophic bacteria explained by the varying zinc ion content of Torsa River in different months

Heavy metal content analysis of River Torsa in India did not indicate any alarming level of toxicity for human consumption but revealed variation at the ppb level in different months. The variation in recoverable nickel and zinc resistant copiotrophic (or eutrophic) bacterial counts was explained by the variation of the zinc content (34.0–691.3 ppb) of the river water in different sampling months. Growth studies conducted with some purified nickel and/or zinc resistant strains revealed that pre-exposure of the cells to ppb levels of Zn²⁺, comparable to the indigenous zinc ion concentration of the river, could induce the nickel or zinc resistance. A minimum concentration of 5–10 μM Zn²⁺ (325–650 ppb) was found effective in inducing the Nickel resistance of the isolates. Zinc resistance of the isolates was tested by pre-exposing the cells to 4 μM Zn²⁺ (260 ppb). The lag phase was reduced by 6–8 h in all the cases. Biochemical characteristics and phylogenetic analysis based on 16S rDNA sequence indicated that some of the Torsa River isolates, having inducible nickel and zinc resistance, are members of the genus Pseudomonas, Acinetobacter, Bacillus, Enterobacter, Serratia and Moraxella.     

Evidence for the role of zinc on the performance of dibenzothiophene desulfurization by <Emphasis Type="Italic">Gordonia alkanivorans</Emphasis> strain 1B

Gordonia alkanivorans strain 1B is able to desulfurize dibenzothiophene (DBT) to 2-hydroxybiphenyl (2-HBP), the final product of the 4S pathway. However, both the cell growth and the rate of desulfurization can be largely affected by the nutrient composition of the growth medium due to cofactor requirements of many enzymes involved in the biochemical pathways. In this work, the effect of several metal ions on the growth and DBT desulfurization by G. alkanivorans was studied. From all the metal ions tested, only the absence of zinc significantly affected the cell growth and the desulfurization rate. By increasing the concentration of Zn from 1 to 10 mg L⁻¹, 2-HBP productivity was improved by 26%. The absence of Zn²⁺, when sulfate was also used as the only sulfur source, did not cause any difference in the bacterial growth. Resting cells grown in the presence of Zn²⁺ exhibited a 2-HBP specific productivity of 2.29 μmol g⁻¹ (DCW) h⁻¹, 7.6-fold higher than the specific productivity obtained by resting cells grown in the absence of Zn²⁺ (0.30 μmol g⁻¹ (DCW) h⁻¹). These data suggests that zinc might have a key physiological role in the metabolism of DBT desulfurization.     

Effects of carbenoxolone on heart rhythm, contractility and intracellular calcium in streptozotocin-induced diabetic rat

Cardiac dysfunction is a frequently reported complication of clinical and experimental diabetes mellitus. Streptozotocin (STZ) – induced diabetes in rat is associated with a variety of cardiac defects including disturbances to heart rhythm and prolonged time-course of cardiac muscle contraction and/or relaxation. The effects of carbenoxolone (CBX), a selective gap junction inhibitor, on heart rhythm and contractility in STZ-induced diabetic rat have been investigated. Heart rate was significantly (P < 0.05) reduced in Langendorff perfused spontaneously beating diabetic rat heart (171±12 BPM) compared to age-matched controls (229± 9 BPM) and further reduced by 10⁻⁵ M CBX in diabetic (20%) and in control (17%) hearts. Action potential durations (APDs), recorded on the epicardial surface of the left ventricle, were prolonged in paced (6 Hz) diabetic compared to control hearts. Perfusion of hearts with CBX caused further prolongation of APDs and to a greater extent in control compared to diabetic heart. Percentage prolongation at 70% from the peak of the action potential amplitude after CBX was 18% in diabetic compared to 48% in control heart. CBX had no significant effect on resting cell length or amplitude of ventricular myocyte shortening in diabetic or control rats. However, resting fura-2 ratio (indicator for intracellular Ca²⁺ concentration) and amplitude of the Ca²⁺ transient were significantly (P < 0.05) reduced by CBX in diabetic rats but not in controls. In conclusion the larger effects of CBX on APD in control ventricle and the normalizing effects of CBX on intracellular Ca²⁺ in ventricular myocytes from diabetic rat suggest that there may be alterations in gap junction electrophysiology in STZ-induced diabetic rat heart.     

Protective effect of vanadate on oxyradical-induced changes in isolated perfused heart

In order to examine the mechanisms of the beneficial effects of vanadate on cardiac dysfunction in chronic diabetes, rat hearts were perfused with xanthine plus xanthine oxidase, an oxyradical generating system in the absence or presence of vanadate. The heart failed to generate contractile force and increased the resting tension markedly within 5 min of perfusion with xanthine plus xanthine oxidase. These changes were prevented by the addition of 4 M vanadate in the perfusion medium. The protective effects of vanadate on the loss of developed tension and increased resting tension due to xanthine plus xanthine oxidase were dose-dependent (0.1–5 M). Perfusion of the hearts with glucose-free medium did not abolish the protective actions of vanadate. The sarcolemmal Ca²⁺-pump (ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase) and Na⁺-dependent Ca²⁺ uptake activities were decreased upon perfusing the hearts with a medium containing xanthine plus xanthine oxidase for 5 min; these effects were prevented by the addition of 2–4 M vanadate in the perfusion medium. The signals for superoxide radicals produced by xanthine plus xanthine oxidase, as detected by electron paramagnetic resonance spectroscopic technique, were inhibited by 5–100 M vanadate. These results suggest that vanadate is an oxyradical scavenger and thus may prevent heart dysfunction under some pathological conditions by its antioxidant action.     

Effect of extraneous zinc on calf intestinal alkaline phosphatase

The effect of extraneous zinc on calf intestinal alkaline phosphatase was studied for quick reversible binding and slow irreversible binding of zinc ions at various concentrations. Under the conditions of slow binding of zinc to CIP increasing Zn²⁺ (less than 1.0 mM, n_M/n_E 1.0 × 10⁶) inhibited enzymatic activity, and further increasing Zn²⁺ resulted in an increase of activity. For quick reversible binding of Zn²⁺, the effect on CIP activity changed at lower concentrations of substrate, indicating a complex cooperativity between Zn²⁺ and pNPP. Both protein intrinsic emission fluorescence and ANS-bound protein fluorescence, as well as circular dichroism spectra have shown that the binding of zinc ions changed the enzyme conformation, which was the reason for the changes in enzyme activity induced by extraneous zinc.     

Mercury (Hg2+) and zinc (Zn2+): Two divalent cations with different actions on voltage-activated calcium channel currents

Summary 1. We examined the actions of mercury (Hg²⁺) and zinc (Zn²⁺) on voltage-activated calcium channel currents of cultured rat dorsal root ganglion (DRG) neurons, using the whole-cell patch clamp technique.2. Micromolar concentrations of both cations reduced voltage-activated calcium channel currents. Calcium channel currents elicited by voltage jumps from a holding potential of –80 to 0 mV (mainly L- and N-currents) were reduced by Hg²⁺ and Zn²⁺. The threshold concentration for Hg²⁺ effects was 0.1 µM and that for Zn²⁺ was 10µM. Voltage-activated calcium channel currents were abolished (>80%) with 5µM Hg²⁺ or 200µM Zn²⁺. The peak calcium current was reduced to 50% (IC₅₀) by 1.1µM Hg²⁺ or 69µM Zn²⁺. While Zn²⁺ was much more effective in reducing the T-type calcium channel current—activated by jumping from –80 to –35 mV—Hg²⁺ showed some increased effectiveness in reducing this current.3. The effects of both cations occurred rapidly and a steady state was reached within 1–3 min. While the action of Zn²⁺ was not dependent on an open channel state, Hg²⁺ effects depended partially on channel activation.4. While both metal cations reduced the calcium channel currents over the whole voltage range, some charge screening effects were detected with Hg²⁺ and with higher concentrations (>100µM) of Zn²⁺.5. As Zn²⁺ in the concentration range used had no influence on resting membrane currents, Hg²⁺ caused a clear inward current at concentrations µM.6. In the present study we discuss whether the actions of both metals on voltage-activated calcium channel currents are mediated through the same binding site and how they may be related to their neurotoxic effects.     

The effects of low concentrations of aluminium on the growth and uptake of nitrate-N by white clover

Summary The effects of aluminium (Al³⁺) at concentrations of 0, 25, 50 and 100 μM on the growth of white clover, dependent upon N supplied as NO ₃ ⁻ , were examined in flowing solution culture. Plants were established with a normal nutrient supply for 7 weeks and then grown with carefully controlled pH (at 4.5) and P concentrations, and with 0, 25, 50 or 100 μM Al³⁺ for a further three weeks. There were rapid visual effects (i.e. symptoms of P deficiency and reduction in root extension) and the dry weights of shoots and roots were reduced at 50 and 100 μM. Less than 10% of Al absorbed from solution was transported to the shoots. The uptake of P, and its transport between roots and shoots, were reduced in plants grown with Al. The uptake of NO ₃ ⁻ stopped immediately after the introduction of 50 or 100 μM Al, and was significantly reduced at 25 μM after three weeks. During a second phase of the experiment, plants previously grown at 0, 25, 50 and 100 μM Al, were grown for a further 2 weeks either with NO ₃ ⁻ (with and without 50 μM Al³⁺) or without NO ₃ ⁻ but with inoculation by Rhizobia (and with or without 50 μM Al³⁺). The effects of the previous treatments with Al on N uptake were small during the second phase, but uptake by all plants was restricted when Al was present. Inoculation did not result in nodulation in the second phase when Al³⁺ was present in the solution, but Al already in the plant from the first phase did not prevent nodulation in the absence of Al during the second phase.     

Effects of zinc on the mineralization of bone nodules from human osteoblast-like cells

Zinc is an important mineral that is required for normal bone development. However, the direct effects of zinc on the mineralization of bone cells of human origin are not clear. The objective of this study was to determine the effects of zinc on the differentiation of SaOS-2 human osteoblast-like cells and the formation of mineralized bone nodules. Cells were cultured for 8 d and then transferred to zinc-free medium and treated with varying concentrations (0–50 μM) of zinc. Alkaline phosphatase (ALP) activity was used as a measure of osteoblast differentiation, and bone nodules were detected by von Kossa staining. After 4, 6, and 8 d of treatment, zinc increased ALP activity at 1 and 10 μM, but decreased activity at 50 μM. After 9 d of treatment, zinc increased both the number and area of mineralized bone nodules at low concentrations (1 and 10 μM), but decreased both at higher concentrations (25 and 50 μM). These findings demonstrate that zinc has biphasic effects on the differentiation and mineralization of human osteoblast-like cells.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Eucalyptus phylacis</Emphasis> L. Johnson and K. Hill., A critically endangered relict from Western Australia

Summary In vitro methods were applied to the only remaining plant of the Meelup Mallee (Eucalyptus phylacis), a critically endangered species from the southwest of Western Australia. Shoot explants were initiated into culture using a 1/2 MS [Murashige and Skoog basal medium (BM) for all experiments] liquid medium supplemented with 1% (w/v) activated charcoal, which was replenished twice daily, followed by transfer of explants to agar medium supplemented with 0.5 μM zeatin. Explants were cultured under low intensity lighting (PPFD of 5–10 μmol m⁻²s⁻¹) to minimize blackening of tissues, and some explants were induced to produce nodular green calluses in response to BM supplemented with 5 μM thidiazuron. Nodular green calluses were induced to form adventitious shoots following transfer to medium supplemented with 0.5 μM zeatin and 1 μM gibberellic acid, A₄ isomer (GA₄). Development of shoots was completed on 1 μM zeatin + 0.1 μM 6-benzylaminopurine (BA) in vented culture tubes. Regenerated shoots were sequentially cultured on medium containing 0.5 μM zeatin + 0.2 μM indoleacetic acid (IAA) followed by either 0.5 μM zeatin + 1μM GA₄ for shoot elongation or 1 μM zeatin + 0.5 μM IAA to optimize shoot growth. Rooted microshoots were produced after 4 weeks on 5 μM indolebutyric acid (IBA) and survived acclimatization and transfer to potting mixture.     

Synergistic or Antagonistic Effect of MTE plus TF or Icariin from <Emphasis Type="Italic">Epimedium koreanum</Emphasis> on the Proliferation and Differentiation of Primary Osteoblasts In Vitro

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test and alkaline phosphatase activity assay were employed to assess the effects of mixed trace elements including Zn²⁺, Ca²⁺, and Mn²⁺ plus total flavonoids or icariin from Epimedium koreanum on the proliferation and differentiation of primary osteoblasts in vitro. The results indicated that icariin (0.1, 1, and 10 μmol/L) and total flavonoids (0.06, 0.6, and 6 μg/mL) inhibited the proliferation and promoted the differentiation of primary osteoblasts. Mixed trace elements including Zn²⁺, Ca²⁺, and Mn²⁺ (0.1, 1, and 10 μmol/L) inhibited the proliferation and promoted the differentiation at 0.1 and 1 μmol/L, but inhibited the differentiation at 10 μmol/L. The effects of mixed trace elements including Zn²⁺, Ca²⁺, and Mn²⁺ plus total flavonoids or icariin from E. koreanum on the proliferation and differentiation of primary osteoblasts in vitro are complicated, and both synergistic and antagonistic effects are generated. The results suggest that there may be a potential cooperative action between flavonoids and trace metal elements on the proliferation and differentiation of primary osteoblasts by forming metal complexes. The combination model between flavonoids and trace metal elements is a pivotal factor for switching the biological effects from toxicity to activity, from damage to protection.     

Thio-NADP is not an antagonist of NAADP

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP, which can release Ca²⁺ from stores that are distinct from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP₃). It has previously been suggested that thio-NADP is a specific antagonist of NAADP (Chini et al. [1995]J. Biol. Chem. 270, 3216–3223). Its effects in sea-urchin egg homogenates were investigated. At 50 μM, thio-NADP activates partial Ca²⁺ release and totally inhibits subsequent challenge with a saturating concentration of NAADP. Purification by HPLC eliminates the Ca²⁺ releasing activity of 50 μM thio-NADP and reduces the subsequent inhibition by 73.7±1.3%. The residual inhibitory effect is no more than that exerted by 50 μM of either NADP itself or nicotinic acid adenine dinucleotide (NAAD). These results are confirmed by³²P-NAADP binding studies. Unpurified thio-NADP inhibits the specific³²P-NAADP binding to egg microsomes with an IC₅₀ of 40 μM. After HPLC purification, only 20% inhibition is seen at a concentration as high as 50 μM, similar to the extent of inhibition effected by 40 μM NADP. These results indicate the inhibitory substance in thio-NADP is a contaminant. The partial Ca²⁺ release activity of unpurified thio-NADP suggests the contaminant is NAADP itself. This is supported by the fact that pretreatment with a subthreshold concentration of only 2 nM NAADP totally desensitizes the egg homogenates such that no Ca²⁺ response is seen with saturating NAADP. Estimation from the binding studies shows that a contamination of 0.012% of NAADP in the unpurified thio-NADP samples is sufficient to account for the inhibitory effects. These results indicate thio-NADP is not an antagonist of NAADP.     

Effects of cadmium and copper on zinc transport kinetics by isolated renal proximal cells

Zinc, cadmium, and copper are known to interact in many transport processes, but the mechanism of inhibition is widely debated, being either competitive or noncompetitive according to the experimental model employed. We investigated the mechanisms of inhibition of zinc transport by cadmium and copper using renal proximal cells isolated from rabbit kidney. Initial rates of⁶⁵Zn uptake were assessed after 0.5 min of incubation. The kinetics parameters of zinc uptake obtained at 20°C were a J_max of 208.0±8.4 pmol· min⁻¹·(mg protein)⁻¹, aK _m of 15.0±1.5 μM and an unsaturable constant of 0.259±0.104 (n=8). Cadmium at 15 μM competitively inhibited zinc uptake. In the presence of 50 μM cadmium, or copper at both 15 and 50 μM, there was evidence of noncompetitive inhibition. These data suggest that zinc and cadmium enter renal proximal cells via a common, saturable, carrier-mediated process. The mechanisms of the noncompetitive inhibition observed at higher concentrations of cadmium or with copper require further investigation, but may involve a toxic effect on the cytoskeleton.     

<Emphasis Type="Italic">In vitro</Emphasis> selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (<Emphasis Type="Italic">Dendrocalamus strictus</Emphasis> Nees)

Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l⁻¹ (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l⁻¹ (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus had high levels of Na⁺, sugar, free amino acids, and proline but a slight decline was recorded in K⁺ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0 mg l⁻¹ (9.0 μM) 2,4-D+0.5 mg l⁻¹ (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l⁻¹ (0.1 μM) α-naphthaleneacetic acid +0.1 mg l⁻¹ (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing 0.2% (w/w) NaCl.     

Purification and Characterization of an Esterase from <Emphasis Type="Italic">Acinetobacter lwoffii</Emphasis> I6C-1

EstA was purified from the supernatant by A. lwoffii 16C-1. Its molecular mass was determined to be 45 kDa, and the optimal activity occurred when the pH level was 8.0 at a temperature of 37°C. The activation energies for the hydrolysis of p-nitrophenyl butyrate was determined to be 11.25 kcal/mol in the temperature range of 10–37°C. The enzyme was unstable at temperatures higher than 50°C. The Michaelis constant (K _m ) and V _max for p-nitrophenyl butyrate were 11 μM and 131.6 μM min⁻¹ mg of protein-1, respectively. The enzyme was strongly inhibited by Hg²⁻, Ca²⁺, Mg²⁺, Fe²⁺, Cu²⁺, Zn²⁺, Mn²⁺, Co²⁺, ethylemediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), and diisopropyl fluorophosphate (DFP). Received: 20 August 2001 / Accepted: 20 September 2001     

Effects of metal ions on activity of plasmin

The effects of some metal ions on amidolytic and fibrinogenolytic activities of highly purified human plasmin were investigated in vitro. In the presence of Zn²⁺, Cu²⁺, Cd²⁺, and Au⁺ in the incubation mixture at the concentrations of 1×10⁻⁵−1×10⁻³ M, the anidolytic plasmin activity was strongly inhibited, whereas Ca²⁺ and Mg²⁺ at the same concentrations were not effective. The analysis of the kinetic study has shown that Zn²⁺ or Cu²⁺ acts as mixed-type inhibitors of plasmin activity. The inhibition of amidolytic plasmin activity by Zn²⁺ and Cu²⁺ was reduced in the presence of EDTA, histidine, or albumin. Incubation of plasmin with Zn²⁺ or Cu²⁺ (at the concentration of 5×10⁻⁴ M) resulted in complete loss of its proteolytic action on fibrinogen, whereas Cd²⁺ and Au⁺ under the same conditions only partially inhibited this process.