Metabolism of cytokinin: Phosphoribosylation of cytokinin bases by adenine phosphoribosyltransferase from wheat germ

Adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase EC 2.4.2.8) which catalyzes the phosphoribosylation of cytokinin bases and adenine to form the corresponding nucleotides were partially purified from the cytosol of wheat (Triticum aestivum) germ. This enzyme (molecular weight, 23,000 ± 500) phosphoribosylates the bases at an optimum Mg²⁺ concentration of 5 mm and optimum pH of 7.5 (50 mm Tris-HCl buffer). K_m values for N⁶-(Δ²-isopentenyl)adenine, N⁶-furfuryladenine, N⁶-benzyladenine, and adenine are 130, 110, 154, and 74 μm, respectively, in 50 mm Tris-HCl buffer (pH 7.5) at 37 °C. Hypoxanthine and guanine are not substrates for the enzyme. In concerting with other cytokinin metabolic enzymes, this enzyme may play a significant role in maintaining the supply of adequate levels of “active cytokinin.”     

Effect of Escherichia coli endotoxin on adenine nucleotide translocation in canine heart mitochondria

The in vitro effect of Escherichia coli endotoxin on the translocation of adenine nucleotides in dog heart mitochondria was studied. Mitochondrial adenine nucleotides were labeled with ¹⁴C by incubating mitochondrial preparations in the presence of [¹⁴C]ADP. The exchange reaction was initiated by addition of unlabeled ADP, proceeded for 5 to 60 s at 4 °C, and was terminated by addition of atractyloside. The results showed that preincubation of mitochondria with endotoxin (50 μg/mg protein) for 10 min at 23 °C decreased the exchange reaction by 21.2% (P < 0.05). The inhibitory effect of endotoxin was increased with increasing concentrations of endotoxin with an I₅₀ value of 45 μg/mg protein. The initial rate and the total extent of exchange were both affected. Double reciprocal plots showed that only the V but not the K_m for ADP was affected by endotoxin, indicating that the inhibition was noncompetitive in nature. The exchange of adenine nucleotide remained depressed by endotoxin in the presence of either oligomycin or antimycin A, indicating that the inhibitory effect of endotoxin was independent of the action of endotoxin on oxidative phosphorylation. The leakage of labeled adenine nucleotides from mitochondria at 23 °C was increased by 100% by endotoxin (100 μg/mg protein) in the absence of added unlabeled ADP, and this increase in the leakage could not be blocked by atractyloside. The endotoxin-induced changes in adenine nucleotide exchange and leakage were either partially or completely prevented by hydrocortisone, heparin, dibucaine, or EDTA. Since most of these agents have in common an effect on lipid metabolism, it is suggested that endotoxin-induced alterations in the exchange and leakage of adenine nucleotides in heart mitochondria are protected through a mechanism involving membrane lipid reorganization.     

Effect of dimethylsulphoxide on the functional characteristics of isolated perfused rat liver

Exposure of rat liver, perfused with 7% BSA in Krebs-Ringer bicarbonate buffer, to 1.4 m Me₂SO at 35 °C had no effect on the release of potassium from the livers, but the rate of urea synthesis fell from 0.6 to 0.1 μmol/min. Bile production also decreased and the total amount collected during perfusion was only half that produced by controls. After perfusion for 4 hr at 35 °C control livers and those exposed to Me₂SO started to release GOT into the perfusate but livers exposed to the cryoprotective compound released the enzyme at a faster rate.Exposure of livers to Me₂SO at 5 °C resulted in potassium being released at a slower rate (0.98 μmol/min) than from cooled controls (1.19 μmol/min) and urea synthesis was decreased from 0.8 to 0.2 μmol/min. Bile production also declined but, because bile flow normally ceases during hypothermia, the effect on this aspect of liver function was probably less than was found at 35 °C. Release of GOT from livers exposed to Me₂SO at 5 °C was quite different from that observed at 35 °C; the enzyme appeared in the perfusate after about 8 hr and it was present in much lower concentration than was found with appropriately cooled controls which started to release the enzyme after 6 hr.Thus, exposure of rat liver to Me₂SO at 5 °C appears to be slightly less damaging than exposure at 35 °C and it may even have a beneficial effect on some aspects of liver function in vitro.     

Regulation of the mitochondrial adenine nucleotide pool size

A mechanism by which normal adult rat liver mitochondria may regulate the matrix adenine nucleotide content was studied in vitro. If mitochondria were incubated with 1 mm ATP at 30 ° C in 225 mm sucrose, 2 mm K₂HPO₄, 5 mm MgCl₂, and 10 mm Tris-Cl (pH 7.4), the adenine nucleotide pool size increased at a rate of 0.44 ± 0.02 nmol/mg mitochondrial protein/min. The rate of adenine nucleotide accumulation under these conditions was concentration dependent and specific for ATP or ADP; AMP was not taken up. The rate of net ADP uptake was 50–75% slower than that for ATP. The K_m values for net uptake of ATP and ADP were 2.08 and 0.36 mm, respectively. Adenine nucleotide uptake was stoichiometrically dependent on Mg²⁺ and stimulated by inorganic phosphate. Net uptake was inhibited by n-ethylmaleimide, or mersalyl, but not by n-butylmalonate. Nigericin inhibited net uptake, but valinomycin did not. In the presence of uncouplers, net uptake was not only inhibited, but adenine nucleotide efflux was observed instead. Like uptake, uncoupler-induced efflux of adenine nucleotides was inhibited by mersalyl, indicating that a protein was required for net flux in either direction. Carboxyatractyloside, bongkrekic acid, or respiratory substrates reduced the rate of adenine nucleotide accumulation, however, this did not appear to be a direct inhibition of the transport process, but rather was probably related indirectly to an increase in the matrix $\text{ATP}\text{ADP}$ ratio. The collective properties of the transport mechanism(s) for adenine uptake and efflux were different from those which characterize any of the known transport systems. It is proposed that uptake and efflux operate to regulate the total matrix adenine nucleotide pool size: a constant pool size is maintained if the rates of uptake and efflux are equal. Transient alterations in the relative rates of uptake and efflux may occur in response to hormones or other metabolic signals, to bring about net changes in the pool size.     

Photosynthetic performance and resistance to photoinhibition of Zea mays L. leaves grown at sub-optimal temperature

The performance of the photosynthetic apparatus was examined in the third leaves of Zea mays L. seedlings grown at near-optimal (25 °C) or at suboptimal (15 °C) temperature by measuring chlorophyll (ChI) a fluorescence parameters and oxygen evolution in different temperature and light conditions. In leaf tissue grown at 25 and 15 °C, the quantum yield of PSII electron transport (ψ_PSII) and the rate of O₂ evolution decreased with decreasing temperature (from 25 to 4 °C) at a photon flux density of 125 μmol m^?2 s^?1. In leaves grown at 25 °C, the decrease of ψ_PSII correlated with a decrease of photochemical ChI fluorescence quenching (q_p), whereas in leaves crown at 15 °C q_p was largely insensitive to the temperature decrease. Compared with leaves grown at 25 °C, leaves grown at 15 °C were also able to maintain a higher fraction of oxidized to reduced Q_A (greater q_p) at high photon flux densities (up to 2000 μmol m^?2 s^?1), particularly when the measurements were performed at high temperature (25 °C). With decreasing temperature and/or increasing light intensity, leaves grown at 15 °C exhibited a substantial quenching of the dark level of fluorescence F₀ (q₀) whereas this type of quenching was virtually absent in leaves grown at 25 °C. Furthermore, leaves grown at 15 °C were able to recover faster from photo inhibition of photosynthesis after a photoinhibitory treatment (1200 μmol m^?2 s^?1 at 25, 15 or 6 °C for 8 h) than leaves grown at 25 °C. The results suggest that, in spite of having a low photosynthetic capacity, Z. mays leaves grown at sub optimal temperature possess efficient mechanisms of energy dissipation which enable them to cope better with photoinhibition than leaves grown at near-optimal temperature. It is suggested that the resistance of Z. mays leaves grown at 15 °C to photoinhibition is related to the higher content of carotenoids of the xanthophyll cycle (violaxanthin + antheraxanthin + zeaxanthin) measured in these leaves than in leaves grown at 25 °C.     

Effect of irradiance and temperature on the photosynthesis of a cultivated red alga,Pyropia tenera (= Porphyra tenera), at the southern limit of distribution in Japan

The effect of irradiance and temperature on the photosynthesis of the red alga, Pyropia tenera, was determined for maricultured gametophytes and sporophytes collected from a region that is known as one of the southern limits of its distribution in Japan. Macroscopic gametophytes were examined using both pulse‐amplitude modulated fluorometry and/or dissolved oxygen sensors. A model of the net photosynthesis–irradiance (P‐E) relationship of the gametophytes at 12°C revealed that the net photosynthetic rate quickly increased at irradiances below the estimated saturation irradiance of 46 μmol photons m^?2 s^?1, and the compensation irradiance was 9 μmol photons m^?2 s^?1. Gross photosynthesis and dark respiration for the gametophytes were also determined over a range of temperatures (8–34°C), revealing that the gross photosynthetic rates of 46.3 μmol O₂ mg_chl‐a^?1 min^?1 was highest at 9.3 (95% Bayesian credible interval (BCI): 2.3–14.5)°C, and the dark respiration rate increased at a rate of 0.93 μmol O₂ mg_chl‐a^?1 min^?1°C^?1. The measured dark respiration rates ranged from ?0.06 μmol O₂ mg_chl‐a^?1 min^?1 at 6°C to ?25.2 μmol O₂ mg_chl‐a^?1 min^?1 at 34°C. The highest value of the maximum quantum yield (Fv/Fm) for the gametophytes occurred at 22.4 (BCI: 21.5–23.3) °C and was 0.48 (BCI: 0.475–0.486), although those of the sporophyte occurred at 12.9 (BCI: 7.4–15.1) °C and was 0.52 (BCI: 0.506–0.544). This species may be considered well‐adapted to the current range of seawater temperatures in this region. However, since the gametophytes have such a low temperature requirement, they are most likely close to their tolerable temperatures in the natural environment.     

Purification and partial characterization of beef liver gluconolactonase.

Gluconolactonase is isolated and purified from beef liver. The molecular weight is estimated at 233,000 and that of its six similar subunits is 39,400. The pH maximum is 7.1 in 50 mm Tris-acetate buffer at 27 °C. K_m and V_m values of 9.1 mm and 1.62 mmol/min/ mg, respectively, were obtained at 27 °C in 50 mm Tris-HCl buffer. This enzyme requires a divalent metal for activity, with manganese being preferred over magnesium. A subcellular fractionation study indicates that gluconolactonase is located primarily in the cytosol, and its hepatic concentration is 2.3 μmol/kg of hepatic tissue.     

Approaches to the measurement of intracellular adenine and the discrimination between adenine transport and metabolism in L1210 leukemia cells

Incubation of L1210 leukemia cells with 10 μM [³H]adenine in the absence of energy substrate results in a very rapid accumulation of ³H within the cells. By 20 s intracellular adenine is near steady-state; beyond this the rate of accumulation of intracellular ³H reflects nucleotide synthesis, predominantly the rate of ATP accumulation within the cell as determined by liquid chromatography. Adenine incorporation into the nucleotides proceeds via adenine-phosphoribosyl transferase, which is rate-limiting to AMP formation and subsequently the formation of ADP and ATP. Acceleration of this pathway by the addition of glucose and phosphate decreases the intracellular adenine level far below equilibrium as metabolism is increased relative to transport. Assessment of methodology to evaluate intracellular adenine and its metabolites indicates that (i) a 4°C wash removes the major portion of intracellular adenine and (ii) at 4°C, transport of adenine remains rapid and while nucleotide synthesis is decreased, ATP still accumulates within the cell. Hence, measurement of cellular uptake of radioactive label at 4°C after cells are washed free of adenine cannot be used as a measurement of adenine surface binding since this radioactive label represents, at least in part, phosphorylated derivatives of adenine within the cell. Unlabeled adenine and structurally related compounds were found to inhibit [³H]adenine net uptake under conditions where metabolism of adenine was reduced, suggesting that base transport is mediated by a facilitated diffusion mechanism. This is consistent with other studies from this laboratory that demonstrate exchange diffusion between adenine and other bases.     

Purification and characterization of human lung calmodulin-independent cyclic AMP phosphodiesterase

A high-affinity calmodulin-independent cyclic AMP phosphodiesterase was purified to homogeneity from human lung tissue. This enzyme has a molecular weight of 60,000, a sedimentation coefficient of 3.2–3.4 S, and an isoelectric pH of 4.6–4.8. Neither Ca²⁺ nor calmodulin (in the presence or absence of added Ca²⁺) stimulates the enzymatic activity. This enzyme appears to be very similar to that described previously from dog kidney (W. J. Thompson, P. M. Epstein, and S. J. Strada, (1979) Biochemistry18, 5228–5237). Hydrolysis of cyclic AMP is greatly enhanced by Mg²⁺ (25–30× at 10 mm Mg²⁺) and Mn²⁺ (20× at 10 mm Mn²⁺). Zn²⁺, Cu²⁺, and Co²⁺ are ineffective at these concentrations. Cyclic AMP is the exclusive substrate with a K_m of 0.7–0.8 μm. The I₅₀ of cyclic GMP is 1 mm using 1 μm cyclic AMP as substrate. In contrast, aminophylline, MIX, and SQ 20009 have I₅₀s of 0.28, 0.021, and 0.001 mm, respectively). The purified enzyme is susceptible to temperature inactivation and protease degradation. Significant (10%) inhibition is seen at 37 °C for 20 min. Trypsin, at 0.1 μg/ml, destroys 50% of the activity in 30 min at 25 °C. Our observations concerning its lability to temperature and proteases coupled with its lack of response to calmodulin suggest this enzyme is a basic catalytic subunit of other cyclic AMP phosphodiesterases present within human lung tissue.     

Comparative effects of adenine analogs upon metabolic cooperation between Chinese hamster cells with different levels of adenine phosphoribosyltransferase activity

Colony formation by variant Chinese hamster cells highly resistant to adenine analogs and deficient in adenine phosphoribosyltransferase (APRT) activity was measured after co-cultivation with APRT⁺, CHO-K1 cells in medium containing one of three different adenine analogs. Depending upon the density of APRT⁺ cells and the specific adenine analog, large differences in the recovery of APRT^? colonies were observed. The particular adenine analog and APRT⁺ cell density were more significant factors in the recovery of APRT^? colonies than the concentration of the analog or the level of APRT activity. The number of wild-type cells (CHO-K1) required to inhibit formation of APRT^? colonies by 50% (mean lethal density; MLD₅₀) with 65 μg/ml 8-aza-adenine (AzA) as the selective drug was 8.0 × 10⁵ cells/100 mm dish (1.5 × 10⁴/cm²). With 100 μg/ml 2,6-diaminopurine (DAP) the MLD₅₀ for CHO-K1 was 4.0 × 10⁵ cells/100 mm dish (7.3 × 10³/cm²). The MLD₅₀ for CHO-K1 when the DAP concentration was decreased to 50 μg/ml was only slightly higher, 5 × 10⁵ cells/100 mm dish (9.1 × 10³/cm²). The most toxic effect was observed with 2-fluoroadenine (FA). The MLD₅₀ for CHO-K1 in 2 μg/ml FA was 4.5 × 10⁴ cells/100 mm dish (8.2 × 10²/cm²), a cell density which permits minimal direct contact between APRT⁺ and APRT^? cells. The toxic effects of FA on individually resistant, APRT^? cells were found to be mediated by metabolites released into the medium by dying APRT⁺ cells. This metabolite toxicity to APRT^? cells was also demonstrated in mixtures with cells having only 8% of wild-type APRT activity. The MLD₅₀ for these APRT⁺ (8%) cells in 2 μg/ml FA was 7.5 × 10⁴ cells/100 dish (1.4 × 10³/cm²), a small difference from the MLD₅₀ for cells with wild-type levels of APRT activity. The differences in the recovery of APRT^? colonies from mixtures with APRT⁺ cells in these three adenine analogs are critical to the design of procedures for the selection of APRT^? cells from populations of APRT⁺ cells and emphasize the importance of establishing the parameters of metabolic cooperation, not only in terms of cell density but also with regard to the particular selective agent, in any experiment designed to determine precise mutation rates or to test putative mutagens upon mammalian cells in culture.     

Characterization of phosphate absorption in maize root cortex segments

Uptake of phosphate ions by 1 mm segments of isolated maize root cortex layers was studied. Cortex segments (from roots of 8 days old maize plants) absorb phosphate ions from 1 mM KH₂PO₄ in 0.2 mM CaSCO₄ at the average rate of 34.3 ±3.2 μg P_i g^?1 (fr. m.) h^?1,i.e. 0.35± 0.02 μmol P_i g^?1 (fr. m.) h^?1. Phosphate uptake considerably increases after a certain period of “augmentation”,i.e. washing in aerated 0.2 mM CaSO₄. This increase is completely blocked by the presence of 10 μg ml^?1 cycloheximide. The relation of uptake rate to phosphate concentration in the medium was shown to have 3 phases in the concentration range of 0.02 - 40 mM. Transition points were found between 0.8–1 mM and 10–20 mM. Following K_m and V_max values were found: K_m[mM] : 0.37 - 3.82 - 27.67 V_max[μg P_i g^?1 (fr. m.) h^?1] : 3.33 - 39.40 - 66.67 We have found no sharp pH optimum for phosphate uptake. It proceeds at almost constant rate till pH 6.0 and then the uptake rate drops with increasing pH. At low phosphate concentrations (1 mM) the lowest uptake rate was found at 5 and 13 °C, while the uptake is higher at 5 °C than at 13 °C at phosphate concentrations higher than 1 mM. At these concentrations uptake rate at 35 °C is lower than at 25 °C. Phosphate uptake considerably decreased in anaerobic conditions. DNP and iodoacetate (0.1 mM) completely blocked phosphate uptake from 1 mM KH₂PO₄, while uptake from 5 and 10 mM KH₂PO₄ was left unaffected by these substances. The inhibitors of active - SH groups NEM and PCMB inhibited phosphate uptake: 10^?3 M NEM by 81.6%, 10⁴ M NEM by 42% and 10^?4 M PCMB by 42%.     

TEMPERATURE EFFECTS ON SILICON LIMITED GROWTH OF THE LAKE MICHIGAN DIATOM STEPHANODISCUS MINUTUS (BACILLARIOPHYCEAE)1

The effect of temperature on the silicon limited growth and nutrient kinetics of Stephanodiscus minutus Grun. was examined using batch and semicontinuous culture methods. Short-term batch culture methods gave maximum growth rates which were essentially constant over the temperature range of 10° to 20°C (μ₃= 0.71–0.80 d^?1). The half-saturation constant for growth (K_s) was significantly lowest at 10°C (K_s= 0.31 μM Si; 0.22–0.41), and higher at both 15°C (K_s= 1.03 μM Si; 0.68–1.47) and 20°C (K_s= 0.88 μM Si; 0.60–1.22). Two methods were used to evaluate the semicontinuous experiments. The Droop relationship showed that the minimum cell quota was about 1.50 × 10^?7 nmol Si cell^?1, but there was much overlap in the results at all three temperatures. The Monod growth relationship for the semicontinuous experiments gave estimates of K_s which were lowest at 15°C (K_s= 0.12 μM Si), and higher at 10°C (K_s= 0.68 μM Si) and 20°C (K_s= 1.24 μM Si), although 95% confidence intervals overlapped. The maximum growth rate estimates for the semicontinuous experiments were similar at 10° and 15°, and higher at 20°C, but the number of points used in making the calculations makes the results less reliable than those from batch cultures. Generally, there were no consistent significant differences in the silicon limited growth of S. minutus over the temperature range studied. Our values of K_s for S. minutus are the lowest recorded for a freshwater diatom, and are consistent with the distribution of this species in nature. Generally, this species becomes abundant in areas with high phosphorus loading and very low silicon levels (low Si:P loading rates). Stephanodiscus species are also fossil indicators of eutrophication in north temperate lakes.     

Differential precipitation with concanavalin a as a method for the purification of glycoproteins: Human alkaline phosphatase

The purification of a glycoprotein from a mixture by first precipitating with a lectin and fractional solution of the precipitate could be applicable as an important preparative procedure. Another possibility could be fractional precipitation with the lectin by varying the concentration of Ca²⁺ and Mn²⁺ required for binding. These principles have been tested in relation to the preparation of human liver alkaline phosphatase. Both approaches have led to the purification of alkaline phosphatase from single human livers with a high recovery (28 and 34%) and purification factors of 16 × 10³ and 13 × 10³ over the initial aqueous butanl-ol extract. The specific activities for the preparations were 1610 and 1500 units/mg of protein, respectively; a unit is defined as the amount of enzyme catalyzing the hydrolysis of 1.0 μmol of p-nitrophenyl phosphate/min at 35°C in 0.1 m 2-amino-2-methylpropan-1-ol/HCl buffer, pH 10.5, containing 10 mmp-nitrophenyl phosphate, 0.1 mm MgCl₂, and 1 μm ZnSO₄.     

The effects of development at sub-optimal growth temperatures on photosynthetic capacity and susceptibility to chilling-dependent photoinhibition in Zea mays

When plants of Zea mays L. cv. LG11 that have been grown at optimal temperatures are transferred to chilling temperatures (0–12°C) photoinhibition of photosynthetic CO₂ assimilation can occur. This study examines how growth at sub-optimal temperatures alters both photosynthetic capacity and resistance to chilling-dependent photoinhibition. Plants of Z. mays cv. LG11 were grown in controlled environments at 14, 17, 20 and 25°C. As a measure of the capacity for photosynthesis under light limiting conditions, the maximum quantum yields of CO₂ assimilation (φ^a.c) and O₂ evolution (φ^a.o) were determined for the laminae of the second leaves at photon fluxes of 50–150 μmol m^-2s^-1. To determine photosynthetic capacity at photon fluxes approaching light saturation, rates of CO₂ uptake (A₁₅₀₀) and O₂ evolution (A₁₅₀₀) were determined in a photon flux of 1500 μmol m^-2s^-1. In leaves developed at 14°C, φ and φ were 26 and 43%, respectively, of the values for leaves grown at 25°C. Leaves grown at 17°C showed intermediate reductions in φ and φ, whilst leaves developed at 20°C showed no significant differences from those grown at 25°C. Similar patterns of decrease were observed for A₁₅₀₀ and A_1500.0 with decreasing growth temperature. Leaves developed at 25°C showed higher rates of CO₂ assimilation at all light levels and measurement temperatures in comparison to leaves developed at 17 and 14°C. A greater reduction in A₁₅₀₀ relative to A_1500.0 with decreasing growth temperature was attributed to increased stomatal limitation. Exposure of leaves to 800–1000 μmol m^-2 s^-1 when plant temperature was depressed to ca 6.5°C produced a photoinhibition of photosynthetic CO₂ assimilation in all leaves. However, in leaves developed at 17°C the decrease in A₁₅₀₀ following this chilling treatment was only 25% compared to 90% in leaves developed at 25°C. Recovery following chilling was completed earlier in leaves developed at 17°C. The results suggest that growth at sub-optimal temperatures induces increased tolerance to exposure to high light at chilling temperatures. This is offset by the large loss in photosynthetic capacity imposed by leaf development at sub-optimal temperatures.     

Modification of liver mitochondrial lipids and of adenine nucleotide translocase and oxidative phosphorylation by cold adaptation

The decrease in respiration rate following thyroidectomy is preceded by changes in the lipid composition of the mitochondrial membrane (Hoch, F.L., Subramanian, C., Dhopeshwarkar, G.A. and Mead, J.F. (1981) Lipids 16, 328–334) and in concert, changes in the kinetic parameters of the adenine nucleotide translocase (Mak, I.T., Shrago, E. and Elson, C.E. (1981) Fed. Proc. 40, 398). To demonstrate that physiological adaptation also involves this sequence of events, rats were housed at 8°C for 3–4 weeks. Cold adaptation resulted in a modest (5%) increase in the unsaturation index for the mitochondrial fatty acids comprised of a significant increase in arachidonic acid and a reciprocal decrease in linoleic acid. Phospholipid analysis indicated that cold adaptation increased the mitochondrial phosphatidylethanolamine and reciprocally decreased the phosphatidylcholine content. Concomitantly, cold adaptation resulted in 25–30% increases in rat liver mitochondrial respiratory activities without changing the respiratory control or ADP/O ratios. The kinetic parameters of the adenine nucleotide translocase were determined by the back-exchange method (Pfaff, E. and Klingenberg, M. (1968) Eur. J. Biochem. 6, 66–79). At 0–4 and 10°C, the V_max and K_m of the cold-adapted rat liver adenine nucleotide translocase were not distinguishable from the control values. The K_i values determined by Dixon plot studies for atractylate and palmitoyl-CoA were also comparable between the two groups. However, at 25 and 37°C, cold-adapted rat liver adenine nucleotide translocase exhibited a 20% increase in V_max and a 20% decrease in K_m for external ADP. The results suggest that one adaption to a cold environment involves hormone-mediated changes in the lipid composition in the mitochondrial membranes which in turn modulate the adenine nucleotide translocase and subsequent respiratory activities.     

CYANOBACTERIAL DOMINANCE OF POLAR FRESHWATER ECOSYSTEMS: ARE HIGH-LATITUDE MAT-FORMERS ADAPTED TO LOW TEMPERATURE?1

Although it is generally believed that cyanobacteria have high temperature optima for growth (> 20° C), mat-foming cyanobacteria are dominant in many types of lakes, streams, and ponds in the Arctic and Antarctic. We studied the effect of temperature on growth (μ) and relative pigment composition of 27 isolates of cyanobacteria (mat-forming Oscillatoriaceae) from the Arctic, subarctic, and Antarctic to investigate whether they are a) adapted to the low temperature (i.e. psychrophilic) or b) tolerant of the low temperature of the polar regions (i.e. psychrotrophic). We also derived a parabolic function that describes both the rise and the decline of cyanobacterial growth rates with increasing temperature. The cyanobacteria were cultured at seven different temperatures (5°-35° C at 5° C intervals), with continuous illumination of 225 μmol photons.m⁻².s⁻¹. The parabolic function fits the μ-temperature data with 90% confidence for 75% of the isolates. Among the 27 isolates of cyanobacteria studied, the temperature optima (T_opt) for growth ranged from 15° to 35° C, with an average of 19.9° C. These results imply that most polar cyanobacteria are psychrotrophs, not psychrophiles. The cyanobacteria grew over a wide temperature range (typically 20° C) but growth rates were low men at T_opt (average μ_max of 0.23 ± 0.069 d⁻¹). Extremely slow growth rates at low temperature and the high temperature for optimal growth imply that the cyanobacteria are not adapted genetically to cold temperatures, which characterize their ambient environment. Other competitive advantages such as tolerance to desiccation, freeze—thaw cycles, and bright, continuous solar radiation may contribute to their dominance in polar aquatic ecosystems.     

Dicarboxylate transport in maize mesophyll chloroplasts

Evidence is presented for high rates of carrier-mediated dicarboxylate anion transport in maize mesophyll chloroplasts. Radioactively labeled malate is transported across the chloroplast envelope leading to accumulation in the stroma. Malate in the stroma will exchange for external malate, oxaloacetate, glutamate, aspartate, and oxoglutarate. At 4 °C the V of malate uptake is 50 μmol·h^?1·mg Chl^?1 and the K_m for malate is 0.5 mm. Oxaloacetate competitively inhibits malate uptake with a K_i estimated to be 0.3 mm. The temperature dependence of malate uptake indicates an activation energy of 12 kcal/mol, and extrapolation using this value gives a rate of transport at 30 °C of approximately 300 μmol·h^?1·mg Chl^?1. This rate approximates the rates of photosynthetic malate production by these chloroplasts.     

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XVIII platypus (Ornithorhynchus anatinus) and saltwater crocodile (Crocodylus porosus)

As part of a programme of comparative measurements of P _d (diffusional water permeability) the RBCs (red blood cells) from an aquatic monotreme, platypus (Ornithorhynchus anatinus), and an aquatic reptile, saltwater crocodile (Crocodylus porosus) were studied. The mean diameter of platypus RBCs was estimated by light microscopy and found to be ～6.3 μm. P _d was measured by using an Mn²⁺‐doping 1H NMR (nuclear magnetic resonance) technique. The P _d (cm/s) values were relatively low: ～2.1×10^?3 at 25°C, 2.5×10^?3 at 30°C, 3.4×10^?3 at 37°C and 4.5 at 42°C for the platypus RBCs and ～2.8×10^?3 at 25°C, 3.2×10^?3 at 30°C, 4.5×10^?3 at 37°C and 5.7×10^?3 at 42°C for the crocodile RBCs. In parallel with the low water permeability, the E _a,d (activation energy of water diffusion) was relatively high, ～35 kJ/mol. These results suggest that “conventional” WCPs (water channel proteins), or AQPs (aquaporins), are probably absent from the plasma membranes of RBCs from both the platypus and the saltwater crocodile.     

SHORT‐TERM EFFECT OF TEMPERATURE ON THE PHOTOKINETICS OF MICROALGAE FROM THE SURFACE LAYERS OF ANTARCTIC PACK ICE1

Microalgae growing within brine channels (85 psu salinity) of the surface ice layers of Antarctic pack ice showed considerable photosynthetic tolerance to the extreme environmental condition. Brine microalgae exposed to temperatures above ?5°C and at irradiances up to 350 μmol photons·m^?2·s^?1 showed no photosynthetic damage or limitations. Photosynthesis was limited (but not photoinhibited) when brine microalgae were exposed to ?10°C, provided the irradiance remained under 50 μmol photons·m^?2·s^?1. The highest level of photosynthetic activity (maximum relative electron transport rate [rETR_max]) in brine microalgae growing within the surface layer of sea ice was at approximately 18 μmol electrons·m^?2·s^?1, which occurred at ?1.8°C. Effective quantum yield of PSII and rETR_max of the halotolerant brine microalgae exhibited a temperature‐dependent pattern, where both parameters were higher at ?1.8°C and lower at ?10°C. Relative ETR_max at temperatures above ?5°C were stable across a wide range of irradiance.     

Suberin lamellae in the hypodermis of maize (Zea mays) roots; development and factors affecting the permeability of hypodermal layers

Abstract The development of suberin lamellae in the hypodermis of Zea mays cv. LG 11 was observed by electron microscopy and the presence of suberin inferred from autoliuorescence and by Sudan black B staining in nodal (adventitious) and primary (seminal) root axes. Suberin lamellae were evident at a distance of 30–50 mm from the tip of roots growing at 20°C and became more prominent with distance from the tip. Both oxygen deficiency and growth at 13°C produced shorter roots in which the hypodermis was suberized closer to the root tip. There were no suberin lamellae in epidermal cells or cortical collenchyma adjacent to the hypodermis. Plasmodesmata were not occluded by the suberin lamellae: there were twice as many of them in the inner tangential hypodermal wall (1,14 μn^?2) as in the junction between the epidermis and hypodermis (0.54 μm^?2). Water uptake by seminal axes (measured by micropotometry) was greater at distances more than 100 mm from the root lip than in the apical zone where the hypodermis was unsuberized. In the more mature zones of roots grown at 13°C rates of water uptake were greater than in roots grown at 20°C even though hypodermal suberization was more marked. Sleeves of epidermal/hypodermal cells (plus some accessory collenchyma) were isolated from the basal 60 mm of nodal axes by enzymatic digestion (drisclase). The roots were either kept totally immersed in culture solution or had the basal 50 mm exposed to moist air above the solution surface. In both treatments the permeabilities to tritiated water and ₈₆Rb were low (circa 10^?5mms^?1) in sleeves isolated from the extreme base. In roots grown totally immersed, however, the permeability of sleeves increased 10 to 50-fold over a distance of 40 mm. In roots exposed to moist air the permeability remained at a low level until the point where the root entered the culture solution and then increased rapidly (> 50-fold in a distance of 8 mm). Growth of roots in oxygen depleted (5% O₂) solutions promoted the development of extensive cortical aerenchymas. These developments were not associated with any reduction in permeability of sleeves isolated from the basal 40 mm of the axis. It was concluded that the presence of suberin lamellae in hypodermal walls does not necessarily indicate low permeability of cells or tissues to water or solutes. The properties of the walls (lamellae?) can be greatly changed by exposure to moist air, perhaps due to increased oxygen availability.