A CONTINUOUS CULTURE STUDY OF PHOSPHATE UPTAKE,GROWTH RATE AND POLYPHOSPHATE IN SCENEDESMUS SP.1

The kinetics of phosphate uptake and growth in Scenedesmus sp. have been studied in continuous culture with particular reference to the shifts in the cellular P compounds as a function of growth rate. Uptake velocity is a function of both internal and external substrate concentrations and can be described by the kinetics of noncompetitive enzyme inhibition. The concentrations of polyphosphates (alkali-extractable or 7-min) can he substituted as inhibitors in the kinetic equation. The apparent half-saturation constant of uptake. K_m, is 0.6 μM. The apparent half-saturation concentration for growth is less than K_m, by 1 order of magnitude. Growth is a function of cellular P concentrations, and the polyphosphates (alkali-extractable or 7-min) appear to regulate growth rate directly or indirectly. To understand P limitation, therefore, it is necessary to measure both external P and internal polyphosphate levels. Evidence indicates that alkali-extractable polyphosphates, which can be quantitatively determined by a simple method of measuring surplus P, are involved in cell division process find that a maintenance concentration of functional phosphate exists in the form of poly phosphates. Alkaline phosphatase activity has an inversely linear relationship to growth rate and to the reciprocals of both polyphosphates and surplus P. Changes in lipid P, RNA P, and presumably all other forms except DNA are related to changes in growth rate.     

IN SITU UPTAKE KINETICS OF AMMONIUM AND PHOSPHATE AND CHEMICAL COMPOSITION OF THE RED SEAWEED GRACILARIA TIKVAHIAE1

The uptake kinetics of ammonium and phosphate by Gracilaria tikvahiae McLachlan were studied under field conditions. Seaweeds, pulse fed once a week for 6 h over a 4-week period, had maximum uptake rates of 19 μmol·g fwt^?1·h^?1 for ammonium and 0.28 μmol·g fwt^?1·h^?1 for phosphate. For both nutrients there was a positive linear correlation between uptake rate (v) and concentration (S) over the entire range of concentration tested. In a nutrient depletion experiment, the phosphate uptake curve determined over a wide range of concentrations consisted of two stages of saturation at low concentrations, and a linear phase at high concentrations. Ash free dry weight, chlorophyll a, phycoerythrin, and protein content were higher in pulse fed plants than in control plants receiving no nutrient additions, while the reverse held true for carbohydrate contents and the C/N ratios. The C/N ratio inversely correlated with ammonium and phosphate uptake rate as well as protein and phycoerythrin content, and positively with carbohydrate content.     

PHOSPHATE UPTAKE UNDER NITRATE LIMITATION BY SCENEDESMUS SP. AND ITS ECOLOGICAL IMPLICATIONS1

Under nitrogen limitation the phosphate content of Scenedesmus sp. shows little variation regardless of growth rate and the N/P atomic ratio of the medium. P uptake therefore can be calculated as the product of P content and N-dependent growth rate. The maximum rate of P uptake in N limitation is lower by a factor of about 8 than the rate in P limitation. As reported earlier, P uptake by this alga under P limitation is described by the kinetics resembling non-competitive enzyme inhibition, with one or several intracellular P fractions as inhibitors. These fractions include surplus P (water extractable) and inorganic polyphosphate fractions A (acid soluble) and B, C, and D (acid insoluble). In N limitation, the ratios of fractions A, B, C, and D are quite different from the ratios of P limitation at comparable growth rates. The concentrations of polyphosphate fraction A in N-limited cells are much, higher than the levels in P-limited cells, and this fraction becomes more predominant at low growth rates in N limitation. This fraction, if introduced as the inhibitor into the noncompetitive scheme, explains the uptake kinetics in both N- and P-limited cells and the low maximum uptake rate in N limitation. This finding may have two significant ecological implications: (1) A nutrient imbalance which brings about changes in the internal, level or the metabolism, of fraction A would affect P uptake. (2) Nitrogen sufficiency would cause a competitive advantage in P uptake. This advantage would be shared by N₂ fixers and algae with low optimum N/P ratios. In Scenedesmus sp. P limitation switches to N limitation and vice versa when the cell N/P atomic ratio is about 30.     

Phosphate‐limited growth of the marine diatom Thalassiosira weissflogii (Bacillariophyceae): evidence of non‐monod growth kinetics1

The marine diatom Thalassiosira weissflogii (Grunow) G. A. Fryxell & Hasle was grown in a chemostat over a series of phosphate‐limited growth rates. Ambient substrate concentrations were determined from bioassays involving picomolar spikes of ³³P‐labeled phosphate, and maximum uptake rates were determined from analogous bioassays that included the addition of micromolar concentrations of unlabeled phosphate and tracer concentrations of ³³P. The relationship between cell phosphorus quotas and growth rates was well described by the Droop equation. Maximum uptake rates of phosphate spikes were several orders of magnitude higher than steady state uptake rates. Despite the large size of the T. weissflogii cells, diffusion of phosphate through the boundary layer around the cells had little effect on growth kinetics, in part because the cellular N:P ratios exceeded the Redfield ratio at all growth rates. Fitting the Monod equation to the experimental data produced an estimate of the nutrient‐saturated growth rate that was ~50% greater than the maximum growth rate observed in batch culture. A modified hyperbolic equation with a curvature that is a maximum in magnitude at positive growth rates gave a better fit to the data and an estimate of the maximum growth rate that was consistent with observations. The failure of the Monod equation to describe the data may reflect a transition from substrate to co‐substrate limitation and/or the presence of an inducible uptake system.     

PRODUCTION OF HYALINE HAIRS BY INTERTIDAL SPECIES OF FUCUS (FUCALES) AND THEIR ROLE IN PHOSPHATE UPTAKE1

A field study to determine the precise times of year at which three intertidal species of Fucus start to produce hyaline hairs and cease producing such hairs was conducted on the Isle of Man, U.K. Hairs were first observed during February, and within 6 days of their initial appearance, all tagged plants of all species at all tidal heights on the shore possessed hairs. Hair production continued until the beginning of October, at which time Fucus plants growing at the lowest stations (+ 3.0 m) had glabrous apical growth. Hair production continued later into the year for plants growing higher on the shore, and it was not until mid-November that glabrous apical growth was observed in all plants. Phosphate uptake rates of pilose (hairy) and glabrous (hairless) apical sections were measured in November 1988 for F. spiralis L. and in January 1989 for F. spiralis and F. serratus L., at phosphate concentrations ranging from 0.8 μM (ambient seawater) to 9.0 μM. In ambient seawater, pilose plants of F. spiralis removed phosphate 2–3 times faster than glabrous plants, whereas the uptake rates of pilose plants of F. serratus were about 50% greater than those of glabrous plants. The differences between uptake rates of pilose and glabrous plants of both species were smaller or nonsignificant at higher phosphate concentrations. The field and laboratory data are consistent with the hypothesis that hairs are formed in Fucus as a response to increased nutrient demand and that hairs facilitate the uptake of nutrients from seawater at concentrations typical of natural situations.     

PHOSPHATE AND SILICATE GROWTH AND UPTAKE KINETICS OF THE DIATOMS ASTERIONELLA FORMOSA AND CYCLOTELLA MENEGHINIANA IN BATCH AND SEMICONTINUOUS CULTURE1

Information on the nutrient kinetics of Asterionella formosa Hass. and Cyclotella meneghiniana Kutz. under either phosphate or silicate limitation was obtained for use in a Monod model and in a variable internal stores model of growth. Short-term batch culture growth experiments were fit to the Monod model and long-term semicontinuous culture experiments and short-term uptake experiments were fit to the variable internal stores model. Mathematical analysis indicates that the parameters of the 2 models may be expressed in terms of each other at steady state. The qualitative results of both batch and steady state culture methods agree. For limiting phosphate experiments. A. formosa is better able to grow at low PO₄-P concentrations than C. meneghiniana, as shown by its lower K for PO₄-P limited growth. The k_Q of A. formosa compared to C. meneghiniana found in long-term semicontinuous culture indicates that A. formosa is almost an order of magnitude more efficient at using internal phosphate for growth. The qualitative results under silicate-limited growth of C. meneghiniana is less than that of A. formosa. The k_Q from semicontinuous culture experiments indicates that C. meneghiniana is the more efficient at using internal silicate for growth. Nutrient uptake experiments showed more variability from a Michaelis-Menten relationship than short-term growth experiments. There were no significant differences between the 2 species in half saturation constants for either phosphate or silicate uptake. We observed a marked dependence of the coefficient of luxury consumption (R) of phosphate on the steady state growth rate. A. formosa has a higher R than C. meneghiniana.     

Multiphasic uptake of phosphate by corn roots

Abstract The concentration dependence of phosphate uptake was studied using root sections of corn (Zea mays L. cv. Ganga 5). Detailed and wide-range (57 concentrations in the range 1 μmol m^?3-75 mol m^?3), precise (average SEM < 2.5%, n= 6) and reproducible (similar patterns in three independent experiments and for 5, 10, 15, 20, 25 and 30°C) data revealed six (or seven) concentration-dependent phases separated by ‘jumps’ or sharp breaks. These transitions were independent of temperature and occurred over relatively narrow concentration ranges (0.0001–0.0004, 0.08–0.31, 1.0–3.5, (7.5–10), 18–20 and 57–59 mol m^?3). The intermediate phases obeyed Michaelis-Menten kinetics, whereas sigmoidal kinetics were observed at lower concentrations. Uptake within each of the two highest phases increased more rapidly with increasing external phosphate concentration than predicted from Michaelis-Menten kinetics but also saturated more rapidly. The latter finding is not consistent with free diffusion across the plasmalemma at high external phosphate concentrations. Kinetic models yielding continuous isotherms, e.g. the sum of one or two Michaelis-Menten terms and a diffusion term, cannot account for the data.     

PHOSPHATE UPTAKE KINETICS IN EUGLENA GRACILIS (Z) (EUGLENOPHYCEAE) GROWN ON LIGHT/DARK CYCLES.' I. SYNCHRONIZED BATCH CULTURES1

The characteristics of phosphate uptake in synchronized populations of Euglena gracilis Klebs (Z) were studied. The cells were grown autotrophically in batch culture and synchronized with a cycle of 14:10 LD. Incorporation of P was nonlinear with time for the first 2 h of incubation over a wide range of P concentrations and completely inhibited by darkness. The kinetics of P uptake as a function of P concentration were triphasic between 0 and 100 μM PO₄, obeying Michaelis-Menten kinetics over the 0–3 μM PO₄ range-only. Uptake velocity increased linearly with, concentration above 3 μM PO₄. The kinetics of P uptake varied with stage in the cell cycle. The half-saturation constant for uptake at the lower concentrations oscillated between 0.7 and 2.8 μM PO₄, reaching a peak immediately before the onset of cell division (beginning of the dark period). V_max was largest in the middle of the light period, as was the slope of the linear portion of the kinetic pattern. Further analysis of the kinetics suggests that changes in this slope are responsible for the oscillation in K_s values calculated for the lower concentrations. This analysis assumes 2 uptake mechanisms, one which saturates at low concentrations of phosphate, and one which is nonsaturable over the entire concentration range examined.     

Succinate transport in Bacillus subtilis. Dependence on inorganic anions

Cations were generally ineffective in stimulating succinate transport in a succinate dehydrogenase mutant of Bacillus subtilis unless accompanied by polyvalent anions; phosphate and sulfate being particularly active. The K_m values for the phosphate or sulfate requirement were approx. 3 mM.Biphasic kinetics were characteristic of both the succinate (K_m values 0.1 and 1 mM), and inorganic phosphate (K_m values 0.1 and 3 mM) transport system(s). The phosphate transport system(s) was repressed by high inorganic phosphate and a coordinate increase in the transport of phosphate, arsenate, and phosphate-stimulated succinate transport accompanied growth in low phosphate media.A class of arsenate resistant mutants were simultaneously defective in the transport of arsenate, phosphate and succinate when cells were repressed for phosphate transport, however, the transport of these ions was regained in these mutants when grown in low phosphate media. Organic phosphate esters did not stimulate succinate transport in arsenate resistant mutants but were effective after growth in low phosphate media. Growth under phosphate limitation permitted the simultaneous regain of both phosphate and sulfate dependent succinate transport activities whereas sulfate limitation alone was ineffective.Succinate was not transported by an anion exchange diffusion mechanism since phosphate efflux was low or absent during succinate transport.The transport of C₄-dicarboxylates in B. subtilis is strongly stimulated by intracellular polyvalent anions. The absence of an anion permeability mechanism precludes succinate transport but partial escape from this restriction is mediated by the derepression of a phosphate transport system.     

ELEMENTAL AND BIOCHEMICAL COMPOSITION OF RHINOMONAS RETICULATA (CRYPTOPHYTA) IN RELATION TO LIGHT AND NITRATE‐TO‐PHOSPHATE SUPPLY RATIOS1

The biochemical basis for variations in the critical nitrogen‐to‐phosphorus (N:P) ratio, which defines the transition between N‐ and P‐limitation of growth rate, is currently not well understood. To assess this issue, we cultured the cryptophyte Rhinomonas reticulata NOVARINO in chemostats with inflow nitrate‐to‐phosphate ratios ranging from 5 to 60 mol N·(mol P)^?1 at two light intensities. The nitrate‐to‐phosphate ratio marking the transition between N‐ and P‐limitation was independent of light intensity and was between 30 and 45 mol N/mol P. In N‐limited cells, the particulate N:P ratio was stable at around 23 mol N/mol P over a range of inflow nitrate‐to‐phosphate from 5 to 30, whereas in P‐limited cells this ratio was around 90 mol N/mol P at inflow nitrate‐to‐phosphate ratios of 45 and 60. Cell phosphorus decreased with increasing nitrate‐to‐phosphate ratio up to the critical nitrate‐to‐phosphate ratio for each light intensity, above which they remained stable. The C:P of R. reticulata cells increased with increasing inflow nitrate‐to‐phosphate from around the Redfield value (106 mol C/mol P) to around 700. There was a significant effect of light on C:P in the N‐ limited cells, with higher C:P under high light conditions that was not observed in the P‐limited chemostats. Cellular RNA was not influenced by light but was greatly influenced by the type of nutrient limitation. In contrast, chl a, C, N, and protein were not influenced by the nitrate‐to‐phosphate in the inflow medium. Total protein per RNA was independent of light intensity but exhibited a maximum at inflow nitrate‐to‐phosphate of 30. Our results suggest a strong “two‐level” homeostatic mechanism of cellular N and P content in R. reticulata with two distinct states that are determined by the type of nutrient limitation and not by light.     

INFLUENCE OF FLUCTUATING PHOSPHATE SUPPLY ON THE REGULATION OF PHOSPHATE UPTAKE BY THE BLUE-GREEN ALGA ANACYSTZS NIDULANS1

The blue-green alga Anacystis nidulans Drouet (Synechococcus leopoliensis Raciborski) cultivated under phosphate-limited conditions adopts a threshold value in the nanomolar range below which uptake ceases. In this study, we investigated the influence of phosphate pulses on the regulation of uptake behavior during reestablishment of the threshold value. Short-term pulses had only a slight effect on uptake kinetics and, hence, on the threshold value, even if the population had been exposed several times to elevated concentrations above the steady-state level in the growth medium. The threshold value was also practically insensitive to the amount of phosphate stored during these short-term fluctuations. Long-term phosphate pulses resulted in a transition to a metastable state that was characterized by a severalfold higher threshold value. This transition, apparently an adaptation to the transiently elevated phosphate concentrations, was further studied by following the influx of ³²P-phosphate at constant external concentrations and was shown to be complete after a period of 10–20 min. After adaptation to pulses, the uptake behavior followed a linear flow-force relation over a wide range of external concentrations. This behavior was explained by the simultaneous operation of at least two uptake systems with different, but coordinated kinetic parameters. This linear flow-force relation facilitated a direct determination of the threshold value from uptake measurements. For applicability in the field the force-flow relation can be a diagnostic tool to assay for fluctuating phosphate and to establish threshold values below the normal measurable range .     

EFFECTS OF ARSENIC SPECIATION AND PHOSPHATE CONCENTRATION ON ARSENIC INHIBITION OF SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1

Arsenate is taken up readily by Skeletonema costatum (Greville) Cleve due to its chemical similarity to phosphate, and it inhibits primary productivity at concentrations as low as 67 nM when the phosphate concentration is low. A phosphate enrichment of greater than 0.3 μM alleviates this inhibition; however, the arsenate stress causes an increase in the cell's requirement for phosphorus. Arsenite is also toxic to Skeletonema at similar concentrations. Methylated species, such as dimethylarsinic acid, did not affect cell productivity at the levels examined. Thus, the reduction and methylation of arsenate to dimethylarsinic acid by the cell produces a stable, non-toxic compound.     

NITRATE AND PHOSPHATE UPTAKE INTERACTIONS IN A MARINE PRYMNESIOPHYTE1

The short- and long-term uptake of nitrate and phosphate ions, and their interactions, were studied as functions of the preconditioning of Pavlova lutheri (Droop) Green. Populations were preconditioned in continuous culture at a variety of growth rates and N:P supply ratios. The maximum uptake rates cell^?1 for nitrate and phosphate were of similar magnitudes, in spite of the forty-fold smaller requirement for phosphorus. Short-term phosphate uptake was independent of the nitrate concentration, but the short-term nitrate uptake rate was reduced in the presence of phosphate. The severity of inhibition of nitrate uptake by phosphate was positively correlated with the preconditioning N:P supply ratio and the preconditioning growth rate. In response to large additions of nutrients, P. lutheri was able to increase its phosphorus content sixty-fold, but was only able to take up enough nitrate to double its nitrogen content. The high rate of phosphate uptake relative to its requirement, the inhibition of nitrate uptake by phosphate, and the large capacity for phosphorus storage relative to its requirement, all of which were observed even under N limitation, may imply that even where nitrogen is limiting there can be interspecific competition for available phosphate.     

The Cyanelles (Organelles of a Low Evolutionary Scale) Possess a Phosphate-Translocator and a Glucose-Carrier in Cyanophora paradoxa

Cyanelles from Cyanophora paradoxa can easily be isolated and assayed for their carrier composition by the silicone oil filtering technique. The present investigation demonstrates a P_i-translocator transferring phosphate, dihydroxyacetone phosphate and 3-phosphoglycerate in a counter exchange mode in cyanelles as in chloroplasts of higher plants. The uptake of P_i is inhibited by dihydroxyacetone phosphate, phosphoglycerate and glucose-6-P, only poorly by phosphoenolpyruvate and not by 2-phosphoglycerate. The inhibitors pyridoxalphosphate and 4,4′diisothiocyanostilbene-2,2K'disulfonic acid at low concentration also affect P_i-uptake. Cyanelles probably transport photosynthate (reductant and ATP) by triosephosphates. This is the first demonstration of a phosphate translocator in an organism of a low evolutionary scale. Cyanelles also transport glucose which proceeds in two phases. In the lower concentration range (≤ 2.5 mM), glucose penetrates by facilitated diffusion, whereas transport follows first-order kinetics at higher amounts (> 2.5 mM). In the low concentration range, glucose-transport is affected by high concentrations of 3-O-methylglucose and fructose. The physiological role of the glucose-transport carrier in Cyanophora is doubtful. It may function in transporting glucose into cyanelles if the carbon level inside them becomes limiting, e.g. in dark periods.     

THE EFFECT OF PHOSPHATE STATUS ON THE KINETICS OF CYANOPHAGE INFECTION IN THE OCEANIC CYANOBACTERIUM SYNECHOCOCCUS SP. WH78031

Phycoerythrin-containing Synechococcus species are considered to be major primary producers in nutrient-limited gyres of subtropical and tropical oceanic provinces, and the cyanophages that infect them are thought to influence marine biogeochemical cycles. This study begins an examination of the effects of nutrient limitation on the dynamics of cyanophage/Synechococcus interactions in oligotrophic environments by analyzing the infection kinetics of cyanophage strain S-PM2 (Cyanomyoviridae isolated from coastal water off Plymouth, UK) propagated on Synechococcus sp. WH7803 grown in either phosphate-deplete or phosphate-replete conditions. When the growth of Synechococcus sp. WH7803 in phosphate-deplete medium was followed after infection with cyanophage, an 18-h delay in cell lysis was observed when compared to a phosphate-replete control. Synechococcus sp. WH7803 cultures grown at two different rates (in the same nutritional conditions) both lysed 24 h postinfection, ruling out growth rate itself as a factor in the delay of cell lysis. One-step growth kinetics of S-PM2 propagated on host Synechococcus sp. WH7803, grown in phosphate-deplete and-replete media, revealed an apparent 80% decrease in burst size in phosphate-deplete growth conditions, but phage adsorption kinetics ofS-PM2 under these conditions showed no differences. These results suggested that the cyanophages established lysogeny in response to phosphate-deplete growth of host cells. This suggestion was supported by comparison of the proportion of infected cells that lysed under phosphate-replete and-deplete conditions, which revealed that only 9.3% of phosphate-deplete infected cells lysed in contrast to 100% of infected phosphate-replete cells. Further studies with two independent cyanophage strains also revealed that only approximately 10% of infected phosphate-deplete host cells released progeny cyanophages. These data strongly support the concept that the phosphate status of the Synechococcus cell will have a profound effect on the eventual outcome of phage-host interactions and will therefore exert a similarly extensive effect on the dynamics of carbon flow in the marine environment.     

PHYSIOLOGICAL RESPONSES TO PHOSPHORUS LIMITATION IN BATCH AND STEADY-STATE CULTURES OF DUNALIELLA TERTIOLECTA (CHLOROPHYTA): A UNIQUE STRESS PROTEIN AS AN INDICATOR OF PHOSPHATE DEFICIENCY1

A protein unique to phosphorus stress observed in Dunaliella tertiolecta Butcher was studied in the context of phosphate-limited cell physiology and is a potential diagnostic indicator of phosphate deficiency in this alga. Cells were grown over a range of limited, steady-state growth rates and at maximum (replete) and zero (phosphate-starved) growth rates. The stress protein, absent in nutrient-replete cells, was produced under all steady-state phosphate-limited conditions and increased in abundance with increasing limitation (decreasing growth rate). Cellular carbon: phosphorus ratios and the maximum uptake rate of phosphate (V_m) increased with increasing limitation, whereas the ratio of chlorophyll a: carbon decreased. Alkaline phosphatase activity did not respond to limitation but was measurable in starved, stationary-phase cells. F_v/F_m, a measure of photochemical efficiency, was a nonlinear, saturating function of p, as commonly observed under N limitation. The maximum F_v/F_m of 0.64 was measured in nutrient-replete cells growing at μ_max, and a value of zero was measured in stationary-phase starved cells. When physiological parameters were compared, the P-stress protein abundance and F_v/F_m were the most sensitive indicators of the level of deficiency. The stress protein was not produced under N- or Fe-limited conditions. It is of high molecular weight (>200) and is associated with internal cell membranes. The stress protein has several characteristics that make it a potential diagnostic indicator: it is 1) unique to phosphorus limitation (i.e. absent under all other conditions), 2) present under limiting as well as starved conditions, 3) sensitive to the level of limitation, and 4) observable without time-course incubation of live samples.     

甘油醛-3-磷酸脱氢酶与辅酶类似物ATP及底物磷酸结合特性的晶体学研究

气相扩散共晶生长法培养出Ｐ．ｖｅｒｓｉｃｏｌｏｒ龙虾肌ＡＴＰ－Ｄ－甘油醛－３－磷酸脱氢酶（ＡＴＰ－ＧＡＰＤＨ）的晶体。用同步辐射Ｘ光源－磷光储屏－Ｗｅｉｓｓｅｎｂｅｒｇ照相机系统收集了一套２．０分辨率的衍射数据。用同晶差值傅立叶法解析了其结构。精化后的结构模型最终Ｒ因子为０．１９７,与标准键长、键角的均方根偏差为０．０１６°和３．２０°。ＰｖＡＴＰ－ＧＡＰＤＨ结构总体上和Ｐｖａｐｏ－ＧＡＰＤＨ相似。ＡＴＰ分子的占有率较低,并表现出一定程度的无序性,提示ＡＴＰ与酶蛋白结合的稳定性较低,表明ＮＡＤ＋的尼克酰胺核苷部分与蛋白质分子的作用在辅酶与蛋白质的稳定结合中起关键作用。ＡＴＰ－ＧＡＰＤＨ中每个亚基只有一个磷酸结合位点（Ｐｉ）。认为无机磷酸结合位点Ｐｉ的形成不依赖于ＮＡＤ＋,而底物磷酸结合位点ＰＳ的形成则依赖于ＮＡＤ＋的存在。     

Shape and dynamics of thermoregulating honey bee clusters

Bacterial transport systems are traditionally treated as enzymes exhibiting a saturable binding site giving rise to an apparent K(m)of transport, whereas the maximal rate of transport is regarded equivalent to the V(max)of enzymatic reactions. Thus, the Michaelis-Menten theory is usually applied in the analysis of transport data and K(m)and V(max)are derived from the treatment of data obtained from the rate of transport at varying substrate concentrations. Such an analysis tacitly assumes that the substrate recognition site of the transport system is freely accessible to substrate. However, this is not always the case. In systems endowed with high affinity in the micro M range or those recognizing large substrates or those exhibiting high V(max), the diffusion through the outer membrane may become rate determining, particularly at low external substrate concentrations. In such a situation the dependence of the overall rate of transport (from the medium into the cytoplasm) on the substrate concentration in the medium will no longer follow Michaelis-Menten kinetics. By analysing the deviation of transport data from the corresponding ideal Michaelis-Menten plot we developed a method that allows us to determine diffusion limitation through the outer membrane. The method allows us to find the correct K(m)of the transport system functioning at the inner membrane even under conditions of strong diffusion limitation through the outer membrane. The model was tested and validified with the Escherichia coli binding protein-dependent ABC transporter for maltose. The corresponding systems for sn -glycerol-3-phospate of Escherichia coli and the alpha -cyclodextrin transport of Klebsiella oxitoca were used as test systems.     

COPPER‐UPTAKE KINETICS OF COASTAL AND OCEANIC DIATOMS1

We investigated copper (Cu) acquisition mechanisms and uptake kinetics of the marine diatoms Thalassiosira oceanica Hasle, an oceanic strain, and Thalassiosira pseudonana Hasle et Heimdal, a coastal strain, grown under replete and limiting iron (Fe) and Cu availabilities. The Cu‐uptake kinetics of these two diatoms followed classical Michaelis–Menten kinetics. Biphasic uptake kinetics as a function of Cu concentration were observed, suggesting the presence of both high‐ and low‐affinity Cu‐transport systems. The half‐saturation constants (K_m) and the maximum Cu‐uptake rates (V_max) of the high‐affinity Cu‐transport systems (～7–350 nM and 1.5–17 zmol · μm^?2 · h^?1, respectively) were significantly lower than those of the low‐affinity systems (>800 nM and 30–250 zmol · μm^?2 · h^?1, respectively). The two Cu‐transport systems were controlled differently by low Fe and/or Cu. The high‐affinity Cu‐transport system of both diatoms was down‐regulated under Fe limitation. Under optimal‐Fe and low‐Cu growth conditions, the K_m of the high‐affinity transport system of T. oceanica was lower (7.3 nM) than that of T. pseudonana (373 nM), indicating that T. oceanica had a better ability to acquire Cu at subsaturating concentrations. When Fe was sufficient, the low‐affinity Cu‐transport system of T. oceanica saturated at 2,000 nM Cu, while that of T. pseudonana did not saturate, indicating different Cu‐transport regulation by these two diatoms. Using CuEDTA as a model organic complex, our results also suggest that diatoms might be able to access Cu bound within organic Cu complexes.     

STUDY ON THE INHIBITION OF BRAIN GLUTAMATE DECARBOXYLASE BY PYRIDOXAL PHOSPHATE OXIME-O-ACETIC ACID

The kinetics of the inhibition of mouse brain glutamate decarboxylase by pyri-doxaI-5′-phosphate oxime-O-acetic acid (PLPOAA) was studied. The inhibition was noncompetitive with regard to glutamic acid; it could be partially reversed by pyridoxal phosphate, but only when the concentration of the latter in the incubation medium was higher than that of pyridoxal-5′-phosphate oxime-O-acetic acid. The inhibition produced by aminooxyacetic acid, which is remarkably greater than that produced by PLPOAA, was also partially reversed only when an excess of pyridoxal phosphate was added. Both in the presence and in the absence of a saturating concentration of pyridoxal phosphate, the activity of the enzyme was decreased by PLPOAA at a 10^?4m concentration to a value of about 50 per cent of the control value obtained without added coenzyme. This activity could not be further reduced even when PLPOAA concentration was increased to 5 × 10^?3m . This same minimal activity of glutamate decarboxylase was obtained after dialysis of the enzymic preparation, or after incubation with glutamic acid in the cold followed by filtration through Sephadex G-25. The addition of pyridoxal phosphate to the dialysed or glutamic acid-treated enzyme restored the activity to almost the control values. PLPOAA did not affect the activity of glutamate decarboxylase from E. coli or that of DOPA decarboxylase and GABA transaminase from mouse brain. To account for the results obtained it is postulated that brain glutamate decarboxylase has two types of active site, one with firmly bound, non-dialysable pyridoxal phosphate and the other with loosely bound, dialysable coenzyme; PLPOAA behaves as a weak inhibitor probably because it can combine mainly with the loosely bound coenzyme site, while aminooxyacetic acid is a potent inhibitor probably because it can block both the ‘loosely bound coenzyme’ and the ‘firmly bound coenzyme’ sites.