Chilling-induced potassium leakage of cultured citrus cells

Callus of 'Marsh' grapefruit ( Citrus paradisi Macf.) albedo tissue was used to investigate the effect of preconditioning temperature on the rate of chilling - stimulated K⁺ leakage. Callus grew most rapidly at 30°C and attained a weight of about 1 g after 30 days. The rate of K.⁺ leakage from nonchilled callus tissue decreased as temperature decreased from 20 to 7.5°C, but no measurable change in rate was observed between 7.5 and 0°C. When calli were held for 40 days at 0¹ 2.5 or 5°C, K⁺ leakage increased 200%, 60% or 0%) respectively. Holding callus for 5 days at 10 or 15°C prior to chilling for 40 days at 0°C prevented the increase in K⁺ leakage observed in callus receiving no preconditioning treatment. Preconditioning at 7.5 and 20°C was less effective in reducing chilling - induced leakage. Preconditioning at 10°C for 5, 2 or 1 day reduced chilling – induced leakage after 40 days at 0°C by 50%, 33% and 15%. respectively.     

The influence of thermal parameters on the acclimation responses of pinfish Lagodon rhomboides exposed to static and decreasing low temperatures

Pinfish Lagodon rhomboides acclimation rates were determined by modelling changes in critical thermal minimum ( T _{crit min}, ° C) estimates at set intervals following a temperature decrease of 3–4° C. The results showed that pinfish gained a total of 3·7° C of cold tolerance over a range of acclimation temperatures ( T _acc, ° C) from (23–12° C), that cold tolerance increased with exposure time to the reduced temperature at all T _acc, but that the rate of cold tolerance accruement (mean 0·14° C day⁻¹) was independent of T _acc. A highly significant ( P < 0·001) multivariate predictive model was generated that described the acclimation rates and thermal tolerance of pinfish exposed to reduction in water temperature: log₁₀ T _{crit min}= 0·41597 − 0·01704 T _acc+ 0·04320 T _plunge− 0·08376[log₁₀ ( t + 1)], where T _plunge is plunge temperature (° C) and t is the time (days). A comparison of the present data, with acclimation rate data for other species, suggests that factors such as latitude or geographic range may play a more important role than ambient temperature in determining cold acclimation rates in fishes.     

Differential effects of chilling on the activity of C4 enzymes in two ecotypes of Echinochloa crus-galli from sites of contrasting climates

Five-week-old plants of Echinochloa crusgalli (L.) Beauv. from Mississippi and from Québec grown under controlled conditions were subjected to dark chilling for 10 h at 5°C or light chilling treatments for 14 h at 7°C under hight light (1 000 μmol m⁻² s⁻¹). The activities of four C₄ enzymes of Québec plants, measured 4 h after the completion of the cold treatment, were not affected by the chilling treatment in the dark. The activities of pyruvate, P_i dikinase (PPDK; EC 2.7.9.1) and NADP⁺-malic enzyme (NADP⁺-ME; EC 1.1.1.40), were significantly reduced in dark-chilled Mississippi plants. Chilling under high light conditions elicited significant levels of reduction in the activities of the four enzymes from both ecotypes but the reductions were significantly less severe for Québec plants. The recovery of activities of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and PPDK for both ecotypes was completed within 36 to 60 hours following the chilling treatment, but NADP⁺-malate dehydro-genase (NADP⁺-MDH; EC 1.1.1.82) and NADP⁺-ME activities of chilled Mississippi plants remained below that of control plants at the end of the 5-day monitoring period. PPDK was inactivated in vitro at 0 and 10°C and the rates of cold inactivation were significantly higher for PPDK extracted from Mississippi plants. The activity of PEPC of Mississippi extracts was slightly, but significantly reduced by a 60 min treatment at 0°C.     

β-Amylase from Clostridium thermocellum SS8 - a thermophilic, anaerobic, cellulolytic bacterium

The extracellular amylase produced by Clostridium thermocellum strain SS8 on starch was characterized as a β-amylase based on blue value reduction test and the production of maltose from starch. The enzyme had a temperature and pH optima of 60°C and 6.0, respectively. Of the metal ions tested, Ca^{2 +} and Mg^{2 +} had little effect on enzyme activity, but their presence increased its thermal stability. Ca^{2 +} displayed a higher stabilizing effect and at 10 mmol 1^-1 Ca^{2 +}, the enzyme retained 86% activity even after exposure at 70°C for 30 min. The amylase was induced on starch or maltose but was repressed strongly by glucose.     

Biochemical characterization of pectate lyases produced by fluorescent pseudomonads associated with spoilage of fresh fruits and vegetables

An improved method for purification of pectate lyases (PLI and PLII) from culture fluids of Pseudomonas fluorescens CY091 and Ps. viridiflava PJ-08-6 by using a phosphocellulose cation exchanger was described. Analysis of purified PLI and PLII by sodium dodecyl sulphate-polyacrylamide and isoelectric focusing gel electrophoresis revealed that both enzymes had been purified to near homogeneity. Optimal Ca²⁺ concentration required for PLI and PLII activity was determined to be 0·5 mmol l⁻¹. The Ca²⁺ requirement could not be replaced by other metal cations such as Mg²⁺, Cu²⁺, Zn²⁺, Fe³⁺ and Co²⁺. Optimal pH for activity was determined to be between 8·5 and 9·0. The K _m values for sodium polygalacturonate were 1·28 and 1·11 mg ml⁻¹ for PLI and PLII, respectively. Both PLI and PLII were stable at low temperatures (25°C or below) for at least 1 month. However, at 37°C, the activity decreased 50% in 36 h. Optimal temperatures for activity were estimated to be 46° and 52°C for PLI and PLII, respectively. Thermal stability of both enzymes at elevated temperatures (48°C or higher) increased when CaCl₂ or a positively charged molecule such as polylysine was present, but decreased when polygalacturonate or a negatively charged molecule such as heparin was present. PLI and PLII exhibit differential degrees of sensitivity to group-specific inhibitors, including iodoacetic acid and diethylpyrocarbonate. This result suggests that both sulphydryl and imidazole groups are important for the catalytic function of PLI and PLII.     

The interaction of temperature and fish size on growth of juvenile halibut

Growth rate of individually tagged juvenile halibut was influenced significantly by the interaction of temperature and fish size. The results suggest an optimum temperature for growth of juvenile halibut in the size range 5–70 g between 12 and 15° C. Overall growth rate was highest at 13° C (1·62% day ⁻¹). At c. 5 g at the beginning of the experiment, fish at 16° C had the highest growth rate (3·2% day ⁻¹), but reduced this rate as they grew bigger. At 9 and 11°p C, growth rates were equal or only slightly lower during the later stages of the experiment, while the fish at 6° C showed significantly lower overall growth rate (0·87% day⁻¹). Optimal temperature for growth decreased rapidly with increasing size, indicating an ontogenetic reduction in optimum temperature for growth. Moreover, a more flattened parabolic regression curve between growth and temperature as size increased indicated reduced temperature dependence with size. Although individual growth rates varied significantly at all times within the experimental temperatures, significant size rank correlations were maintained during the experiment. This indicated an early establishment of a stable size hierarchy within the fish groups. Haematocrit was highest at the highest temperature while Na⁺/K⁺-ATPase activity was inversely related to temperature. There was no difference in plasma Na⁺, Cl⁻ and K⁺ concentrations among the temperature groups.     

THE COMBINED EFFECTS OF LOW TEMPERATURE AND SO2+NO2 POLLUTION ON THE NEW SEASON'S GROWTH AND WATER RELATIONS OF PICEA SITCHENSIS

Picea sitchensis (Bong.) Carr. seedlings were exposed to SO₂, NO₂ and SO₂+ NO₂ during dormancy in controlled environments, and were taken to night temperatures of 4, 0, −5, −10 and −15 °C in a freezer. Conditions in the freezer were carefully monitored during the low–temperature treatments. In two experiments, different photoenvironments and temperature regimes were imposed prior to the cold treatments, and different effects were observed. In the first, only limited frost hardiness was achieved and night temperatures of −15 °C were lethal. Temperatures of −5 and − 10 °C led to poor survival of lateral buds, particularly in plants exposed to 45 ppb SO₂. The poor bud break in plants exposed to SO₂ and to − 5 °C resulted in a loss of the effectiveness of this temperature as a chill requirement. Pressure-volume analysis showed that the shoots of plants exposed to NO₂ had greater elasticity (lower elastic moduli, e), so that loss of turgor occurred at lower relative water contents. In contrast, a hardening period (2 weeks in night/day temperatures of 3/10 °C and 8 h days at 50 μmol m⁻² s⁻¹ PAR) gave decreased elasticity and lower solute potentials of spruce shoots. In the second experiment, exposure to 30 ppb SO₂ and SO₂+ NO₂ led to slight, but consistent, increases in frost injury to the needles of plants frozen to − 5 and − 10 °C. The results suggest that the main interaction of low temperatures and winter pollutants may be on bud survival rather than on needle damage, but that effects are subtle, only occurring with certain combinations of pollutant dose and cold treatment.     

Studies on the Production of Extracellular Proteinases by a Non-pigmented Strain of Chromobacterium lividum Isolated from Abattoir Effluent

A highly proteolytic bacterium isolated from abattoir effluent was identified as a non-pigmented strain of Chromobacterium lividum. Ferrous or ferric ions at concentrations between 1·8 × 10^-5 and 9 × 10^-4 g ions/1, which is 2–3 orders of magnitude greater than that required for growth, were essential for extracellular proteinase production in aerated but not in static culture. Co²⁺, Ni²⁺, Mn²⁺, Cu²⁺ or Zn²⁺ ions could not replace iron. Four proteinases (I-IV) were produced in static culture, but only proteinase I was formed in significant quantities in aerated culture. With both forms of culture amino nitrogen was essential for proteinase production; glucose inhibited formation in aerated, but not static, cultures. Growth occurred over the range 1–33 °C, whereas proteinase production ceased at 27 °C, with maximum activity at 13 °C. Proteinase production appeared to be controlled by an interaction between iron, oxygen tension and glucose.     

Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion

Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn²⁺, Mg²⁺, Hg²⁺, Li⁺, K⁺, and Na⁺ were assessed on the binding of [³H]mazindol and [³H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn²⁺ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [³H]mazindol; Mg²⁺ (0.1–100 µ M ) had no effect; Hg²⁺ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [³H]mazindol than [³H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li⁺ and K⁺ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na⁺ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn²⁺ at 0 and 21°C and Hg²⁺ at 0°C.     

Effect of temperature and dissolved oxygen concentration on the metabolic rate of the turbot and the relationship between metabolic scope and feeding demand

Turbot Scophthalmus maximus maximum oxygen uptake following feeding and exhaustive exercise increased from 107 mg O2 kg⁻¹ h⁻¹ at 6° C to c . 218 mg O2 kg⁻¹ h⁻¹ at 18° C, then increased slightly from 18 to 22° C to 224 mg O2 kg⁻¹ h⁻¹. Standard oxygen uptake increased exponentially as a function of temperature from 11 mg O2 kg ⁻¹ h⁻¹ at 6° C to 66 mg O2 kg⁻¹ h⁻¹ at 22° C. Gradual reduction in oxygen concentration to 87–90% air saturation at 6, 10. 18° C and <80% at 14 and 22° C limited the maximum metabolic rate but, supersaturation (>100% saturation) had little effect. Metabolic scope attained a maximum of 176 mg O₂ kg⁻¹ h⁻¹ at 18° C. Interpolation of the results showed that this value changed little between 16 and 20° C. It is suggested that this temperature range is optimal for turbot of c . 500 g. A comparison with a previous study on feeding demand in intensive farming conditions showed a linear relationship between appetite and metabolic scope. It is concluded that the ability of a fish to supply energy (including the energy requirement of digestive metabolism) above a standard level is a limiting factor in the manifestation of its feeding demand.     

Properties and significance of an α-amylase produced by Myxococcus coralloides D

M.E.FÁREZ-VIDAL, A. FERNÁNDEZ-VIVAS, F. GONZÁLEZ AND J.M. ARIAS. 1995. The extracellular amylase activity from Myxococcus coralloides D was purified by Sephacryl S-200 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-25. The molecular weight was estimated by SDS-PAGE and by gel filtration as 22.5 kDa. The optimum temperature was 45°C. The pH range of high activity was between 6.5 and 8.5, with an optimum at pH 8.0. Activity was strongly inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Ag⁺, Pb²⁺, Fe²⁺ and Fe³⁺, EDTA and glutardialdehyde, but was less affected by Ni²⁺ and Cd²⁺. Li⁺, Mg²⁺, Ba²⁺, Ca²⁺, N -ethylmaleimide, carbodiimide and phenyl methyl sulphonyl fluoride had almost no affect. The K _m (45°C, pH 8) for starch hydrolysis was 2.0 times 10^-3 gl^-1. Comparison of the blue value-reducing curves with the time of appearance of maltose identified the enzyme produced by M. coralloides D as an α-amylase.     

Nickel and rubidium uptake by whole oat plants in solution culture

Nickel and rubidium uptake by oat plants ( Avena sativa L. cv. Victory) were examined in relation to solution temperature, solution concentrations, metabolic inhibitors, anaerobic root conditions, transpiration and time. Over a 4-h period, uptake rates for both Ni²⁺ and Rb⁺ remained constant at 23°C. Decreasing temperatures to 2°C, 20 μ M concentrations of 2,4-dinitrophenol (DNP), or anaerobic root conditions decreased Ni²⁺ and Rb⁺ uptake rates by 97 to 86% in whole plants. Treatment of excised roots with 20 μ M DNP decreased Ni²⁺ uptake by 93%. Nickel and Rb⁺ uptake rates measured as a function of the external solution concentration followed a typical parabolic curve. K_m (0.012 m M ) and V_max [2.72 μmol (g dry weight)^-1 h^-1] values for Ni²⁺ were nearly 7 times lower than those for Rb⁺ [0.09 m M and 19.2 μmol (g dry weight)^-1 h^-1]. In all experiments, Ni²⁺ and Rb⁺ showed qualitatively similar uptake patterns, but Rb⁺ uptake was quantitatively more sensitive than Ni²⁺ to experimental manipulations.     

Temperature Dependence of Catecholamine Secretion from Cultured Bovine Chromaffin Cells

Abstract: Secretion of both epinephrine and norepinephrine by cultured chromaffin cells was studied at temperatures ranging from 0°C to 37°C. The percentage of epinephrine secreted was always lower than that of norepinephrine when the cells were stimulated with either acetylcholine or high K⁺ at any temperature. When the cells were stimulated with acetylcholine or carbachol the percentage of catecholamine secreted at 10 min increased with temperature from 4°C to 24°C and then decreased from 24°C to 37°C. Potassium-stimulated cells secreted increasing amounts of catecholamine as the temperature was increased to 37°C. We found, however, that the initial rates of secretion increased continuously as temperature increased throughout the range for both carbachol-and K⁺-stimulated cells. The temperature maximum of acetylcholine-stimulated secretion is caused by a faster shut-off of secretion at higher temperature. The Arrhenius plots of initial rates show an inflection point at approximately 17°C for carbachol-stimulated cells. The plot for K⁺-stimulated cells is a straight line over the entire temperature range. The transition could be caused by a conformational change in the cholinergic receptor/ion channel molecule.     

PHYSIOLOGICAL RESPONSES TO TEMPERATURE AND IRRADIANCE IN SPIROGYRA (ZYGNEMATALES, CHAROPHYCEAE)1

Spirogyra Link (1820) is an anabranched filamentous green alga that forms free-floating mats in shallow waters. It occurs widely in static waters such as ponds and ditches, sheltered littoral areas of lakes, and stow-flowing streams. Field observations of its seasonal distribution suggest that the 70-μm-wide filament form of Spirogyra should have a cool temperature and high irradiance optimum for net photosynthesis. Measurements of net photosynthesis and respiration were marie at 58 combinations of tight and temperature in a controlled environment facility. Optimum conditions were 25°C and 1500 μmol photons m⁻² s⁻¹, at which net photosynthesis averaged 75.7 mg O₂ gdm⁻¹ h⁻¹. Net photosynthesis was positive at temperatures from 5° to 35°C at most irradiances except at combinations of extremely low irradiances and high temperatures (7 and 23 μmol photons m⁻² s⁻¹ at 30°C and 7, 23, and 35 μmol photons m⁻² s⁻¹ at 35°C). Respiration rates increased with both temperature and prior irradiance. Light-enhanced respiration rates were significantly greater than dark respiration rates following irradiances of 750 μmol photons m⁻² s⁻¹ or greater. Polynomials were fitted to the data to generate response surfaces; such response surfaces can be used to represent net photosynthesis and respiration in ecological models. The data indicate that the alga can tolerate the cool water and high irradiances characteristic of early spring but cannot maintain positive net photosynthesis under conditions of high temperature and low light (e.g. when exposed to self-shading ).     

Reappraisal of the effect of temperature on the growth kinetics of Aeromonas salmonicida

The effect of temperature (1–34 °C ) on the maximum specific growth rate of Aeromonas salmonicida could not be described by the classical growth models ; for some strains, two optimal temperatures at 23 °C and 30 °C were observed, as well as an unexpected increase in the pseudolag time above 27 °C. This could be explained by the presence of two subsets, notably S-layer⁺ and S-layer⁻ sub-populations. The A⁻ cells had higher growth parameters (T_opt and μ_opt) than the A⁺ cells and were selected by subcultures above 30 °C. Yet the relative proportion of A⁺ cells did not explain all the variation of μ_max versus temperature, and the growth kinetics of an Aer. salmonicida isolate remained unpredictable.     

Effect of long-term and rapid cold hardening on the cold torpor temperature of an aphid

Abstract.  The effect of long-term (seasonal) acclimation and rapid cold hardening is investigated on the cold torpor temperature ( CT _min) of adult grain aphids, Sitobion avenae, reared at 20 or 10 °C for more than 6 months before experimentation. Rapid cold hardening is induced by exposing aphids reared at 20 to 0 °C for 3 h and aphids reared at 10 to 0 °C for 30 min (acclimation regimes previously found to induce maximum rapid cold hardening). The effect of cooling aphids from the same rearing regimes from 10 to −10 °C at 1, 0.5 and 0.1 °C min⁻¹ is also investigated. In the 20 °C acclimated population, rapid cold hardening and cooling at 0.1 °C min⁻¹ both produce a significant decrease in CT _min from 1.5 ± 0.3 to –0.9 ± 0.3 and –1.3 ± 0.3 °C, respectively. Rapid cold hardening also results in a significant reduction in CT _min of the population reared at 10 °C from 0.8 ± 0.1 to –0.9 ± 0.2 °C. However, none of the cooling regimes tested reduces the CT _min of the winter-acclimated (10 °C) population. The present study demonstrates that rapid cold-hardening induced during the cooling phase of natural diurnal temperature cycles could lower the movement threshold of S. avenae , allowing insects to move and continue feeding at lower temperatures than would otherwise be possible.     

The protective effect of some food ingredients on Staphylococcus aureus MF31

The upper limiting temperature of growth of Staphylococcus aureus MF31 in heart infusion broth (HI) was about 44°C but addition of monosodium glutamate (MSG) and soy sauce permitted the organism to grow above this temperature. This effect is similar to that of NaCl. Tomato ketchup, Worcestershire and HP sauces added to HI did not allow growth at the non-permissive temperature of 46°C but death was delayed. Staphylococcus aureus died in unsupplemented chicken meat slurry at 46°C but grew at 48°C in slurry supplemented with 5.8% NaCl and survived incubation for 18 h at 50°C in slurry supplemented with 5.8% NaCl and 5% MSG. Cultures grown at 37°C had a D ₆₀ value of 2 min in 50 mmol/l Tris (pH 7.2) buffer. Cultures grown at 46°C in HI containing 5.8% NaCl had a D ₆₀ value of 8 min in Tris buffer. Addition of 5.8% NaCl plus 5% MSG to the buffer increased the D ₆₀ by a factor of about 7 for both cultures. In storage experiments at room temperature, the culture grown at 37°C and at 46°C plus 5.8% NaCl died at about the same rate in salami. In milk powder, however, the count of 37°C culture decreased from 10⁹/g to 10⁶/g in 5 weeks while the count of 46°C culture remained unchanged. In cottage cheese, freeze-dried rice and macaroni, the 37°C cultures also died more rapidly. It is suggested that cultures grown at 46°C plus 5.8% NaCl may be suitable for experiments with artificially contaminated foods.     

C4 photosynthesis in Cyperus longus L., a species occurring in temperate climates

Abstract. Cyperus longus L. , which has a widespread but disjunct distribution throughout Europe and extends northwards into Britain, was found to be a C₄ species based upon its Kranz leaf anatomy, low CO₂ compensation point and the labelling of malate as an early product of ¹⁴CO₂ fixation. The photosynthetic characteristics of C. longus are similar to many other C₄ species with a high maximum rate of photosynthesis (> 1.5 mg CO₂ m ⁻² s ⁻¹) and a relatively high temperature optimum (30–35°C), but unlike many C₄ species the rate of photosynthesis does not decline rapidly below the optimum temperature and a substantial rate (0.6 mgCO₂ m⁻²s⁻¹)occursat 15°C. Leaf extension is very slow at 15°C and shows a curvilinear response to temperatures between 15 and 25°C. Leaves extend at a rate of almost 4 cm d⁻¹ at 25°C.     

Purification and characterization of the major β-1,4-endoglucanase from Thermomonospora curvata

The major β-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata , contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose. The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC. This monomeric enzyme had a specific activity of 750 IU mg⁻¹ when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70°C, pH 6.0. Highest activity was observed on CMC with a degree of polymerization of 3200. The EG was stable for 48 h at 60°C, pH 6.0 and had a half-life of 30 min at 80°C; temperature and pH optima were 70–73°C and 6.0–6.5, respectively. The mol. wt was 100000 and the pI was 4.0. The K _m and V _max values were 7.33 mg ml⁻¹ and 833 μmol min⁻¹, respectively. EG activity was inhibited by Fe^{2 +}, Hg^{2 +}, Ag⁺ and Pb^{2 +}, and enhanced by dithiothreitol and Zn^{2 +}. The first 12 amino acid residues at the N -terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser. Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found.     

Deoxyribonuclease activities in Myxococcus coralloides D

Myxococcus coralloides D produced cell-bound deoxyribonucleases (DNases) during the exponential phase of growth in liquid medium. DNase activity was much higher than that detected in other myxobacterial strains and was fractionated into three different peaks by filtration through Sephadex G-200. The DNases were named G, M and P. The optimum temperatures were 37°C, 33°C and 25°C respectively, although high activities were recorded over the temperature range 20–45°C. The pH range of high activity was between 6·0 and 9·0, with an optimum for each DNase at 8·0. DNases M and P were strongly inhibited by low concentrations of NaCl, but activity of DNase G was less affected by NaCl. The three activities required divalent metal ions as cofactors (especially Mg²⁺ and Mn²⁺); however, other metal ions (Fe²⁺, Ni²⁺, Zn²⁺) were inhibitors. The molecular weights were estimated by gel filtration chromatography and SDS-PAGE as 44 kDa (DNase G), 49 kDa (DNase M) and 39 kDa (DNase P).