REABSORPTION OF ORGANIC SOLUTES IN SOME MARINE AND FRESHWATER PROSOBRANCH GASTROPODS

Influx in vitro of glucose to the heart, kidney and ureter,where present, in Monodonta, Pomacea, and Viviparus, and alsoof leucine to these tissues in Viviparus, was measured using[³H]-labelledtracers. Phloridizin-sensitive, Na⁺-dependent [³H]D-glucose uptake wasevident in the papillary sac of Monodonta, the ventricle ofViviparus, and the kidney of Pomacea. Viviparus ventricle alsoshowed Na⁺-dependent uptake of [³H]L-leucine. Nevertheless Viviparusappears to excrete some amino acids (by undefined routes) andmay therefore lack the carriers for their resorption. In Viviparusand Pomacea the site of organic solute resorption (taken tobe where Na⁺-dependent tracer uptake predominates) is proximalto the sites of Na⁺resorption. The ultrastructure of the resorptivesites has been examined in the three genera and compared withthat of the nephridial gland and dorsal wall of the kidney ofLittorina. The ciliated cells comprising the epithelium of thepapillary sac in Monodonta and their homologues over the nephridialgland and dorsal wall of Littorina and dorsal wall of Pomaceashow common features typical of transporting epithelia in othermolluscs. The ventricular epicardium of Viviparus shows secondaryspecializations for resorption but lacks an endocytotic canalsystem. The site of organic solute resorption (the most highlyspecialized part of which is the nephridial gland in marinespecies), has been correlated with the anatomy of the renalveins in the four genera, and with other specializations inthe freshwater genera.The difficulty of quantifying rates oftransport of solutes from studies in vitro is discussed. ^*Present address: Dept. of Physiology, The University, DundeeDD1 4HN (Received 31 January 1989; accepted 24 March 1989)     

Fine structure,mechanism of heart function and haemodynamics in the prosobranch gastropod molluscLittorina littorea (L.)

Summary The heart, main blood vessels, and associated structures ofLittorina littorea were examined by scanning and transmission electron microscopy. The auricle is subdivided into two compartments, one receiving blood from the gill and opening to the nephridial gland vein, the other connecting with the latter anteriorly and the ventricle posteriorly.Video recordings were made of the beating heart in vivo and revealed that the auricle expelled blood not only to the ventricle, but also the nephridial gland vein at systole and provided further evidence of tidal flow of blood in the vein. There is clear indication that the constant volume mechanism of auricular re-filling is not strictly true inLittorina.Blood pressure in the heart and major vessels was measured using a servo-nulling micropressure system. The rate of formation of urine (derived by filtration of blood through the auricular wall) was measured using [⁵¹Cr] EDTA as a blood marker.Basal blood pressure was slightly above ambient (0.7 cm H₂O). Peak systolic pressure in the ventricle (3.8 cm H₂O) was synchronised with a subambient trough in pericardial pressure (–1.0 cm H₂O); these pressure pulses were out of phase with that of the auricle (2.3 cm H₂O) at systole. The observations are consistent in broad terms with a constant volume mechanism, but this does not take into account urine formation or filling of the nephridial gland vein.A filtration pressure of 1.5 cm H₂O has been demonstrated across the auricular wall throughout the cardiac cycle. Colloidal back pressure appears to be negligible. The mean rate of urine formation is 0.26 l g^–1 min^–1.     

Factors influencing glucose flux and the effect of insulin in cultured human cells

Uptake of glucose-³H into cultured HLM cells was measured. Equilibration of intracellular and extracellular pools occurred after 25 min. Glucose influx was determined subsequently by measuring the glucose-³H entering in precisely 1 min. Although saturation kinetics were demonstrated these were not of the simple Michaelis-Menten type. The K_m of the glucose carrier system is probably about 60 mM glucose. Galactose did not compete with glucose. Insulin stimulated glucose flux without increasing the value of V _max. The stimulation was fully demonstrable after 10 min, could be elicited at concentrations of 10^-4 units/ml, and was absent 2–4 hr after removal. Increasing pH had little or no effect in stimulating glucose flux. Increasing osmotic pressure caused a marked increase and reduced the effect of insulin. Glucose influx was unaffected by anoxia. Glucose influx was increased and the effect of insulin abolished in the absence of K⁺. Glucose influx was increased by mercuric chloride, iodoacetate, and fluoride which abolished the effect of insulin. Dinitrophenol decreased the rate of glucose uptake but did not alter the effect of insulin. Phlorizin reduced the rate of glucose uptake and abolished the effect of insulin. ATP and AMP enhanced the rate of glucose uptake. These findings are discussed in relation to the mode of action of insulin.     

Evidence for a sodium ion exchange carrier linked with glucose transport across the brush border of a flatworm (Hymenolepis diminuta, Cestoda)

The manner in which the flatworm, Hymenolepis diminuta (Cestoda), regulates the transport of glucose and Na⁺ across the brush border was examined. While the presence of an unstirred region in the brush border may favor the reabsorption of leaked glucose, some leaked glucose was lost to the ambient medium. This loss was markedly enhanced by preloading the worms with glucose and by removing Na⁺ from the incubation medium. Since glucose and Na⁺ influxes are coupled, glucose leakage stimulated the influx of ²²Na⁺. However, this ²²Na⁺ influx was balanced by a simultaneous increased ²²Na⁺ efflux. The presence of phlorizin inhibited both unidirectional fluxes of ²²Na⁺ indicating that efflux of ²²Na⁺ occurred by counter-transport; countertransport of [¹⁴C]glucose appeared to be negligible. A model has been proposed in which the transport of glucose and compensating transfers of Na⁺ across the membrane occur via the same carrier.     

Metabolism and excretion of carnitine and acylcarnitines in the perfused rat kidney

Rat kidneys were perfused for 30 min with a Krebs-Henseleit bicarbonate buffer with 5 mM glucose. Albumin proved superior to pluronic polyols as oncotic agent with regard to carnitine reabsorption in the perfused kidney. The reabsorption of 30 μM (−)-[methyl-³H]carnitine was approx. 96% during the first 10 min. At 750 μM the reabsorption decreased to 40%. The tubular reabsorptive maximum (T_max) was approx. 170 nmol/min per kidney. The fractional reabsorption and clearance of (+)-carnitine, γ-butyrobetaine, and carnitine esters did not deviate significantly from that of (−)-carnitine. (+)-Carnitine was not metabolized by the perfused kidney. In perfusions with (−)-carnitine or (−)-carnitine plus 10 mM α-ketoisocaproate or α-ketoisovalerate increased amounts of acetylcarnitine, isovalerylcarnitine and isobutyrylcarnitine were found. Propionate (5 mM) inhibited acetylcarnitine formation. Isovalerylcarnitine, isobutyrylcarnitine and propionylcarnitine were actively degraded to free (−)-carnitine. In urine, we found a disproportionally high excretion of carnitine or carnitine esters formed in the kidney, compared to the same derivatives when ultrafiltrated. Leakage of metabolites formed in the kidney into preurine may explain this phenomenon.     

A Highlights from MBoC Selection: Regulation of the epithelial Ca2+ channel TRPV5 by reversible histidine phosphorylation mediated by NDPK-B and PHPT1

The kidney, together with bone and intestine, plays a crucial role in maintaining whole-body calcium (Ca²⁺) homoeostasis, which is primarily mediated by altering the reabsorption of Ca²⁺ filtered by the glomerulus. The transient receptor potential-vanilloid-5 (TRPV5) channel protein forms a six- transmembrane Ca²⁺-permeable channel that regulates urinary Ca²⁺ excretion by mediating active Ca²⁺ reabsorption in the distal convoluted tubule of the kidney. Here we show that the histidine kinase, nucleoside diphosphate kinase B (NDPK-B), activates TRPV5 channel activity and Ca²⁺ flux, and this activation requires histidine 711 in the carboxy-terminal tail of TRPV5. In addition, the histidine phosphatase, protein histidine phosphatase 1, inhibits NDPK-B–activated TRPV5 in inside/out patch experiments. This is physiologically relevant to Ca²⁺ reabsorption in vivo, as short hairpin RNA knockdown of NDPK-B leads to decreased TRPV5 channel activity, and urinary Ca²⁺ excretion is increased in NDPK-B^−/− mice fed a high-Ca²⁺ diet. Thus these findings identify a novel mechanism by which TRPV5 and Ca²⁺ reabsorption is regulated by the kidney and support the idea that histidine phosphorylation plays other, yet-uncovered roles in mammalian biology.     

Hymenolepis diminuta: Chloride fluxes and membrane potentials associated with sodium-coupled glucose transport

Glucose transport by Hymenolepis diminuta was inhibited when Cl^? in the bathing medium was replaced with acetate (C₂H₃O₂^Post?), but was unaffected when Cl^? was replaced with SCN^?. The relative effectiveness of the anions to inhibit influx of 7.4 mM Cl^? in the presence of 1 mM glucose was SCN^? > Cl^? > C₂H₃O₂^Post?. Glucose stimulated the influxes of 120 mM Cl^? and SCN^?, but had little effect on 120 mM C₂H₃O₂^Post? influx. While the diffusion rates of the anions were C₂H₃O₂^Post? > SCN^? = Cl^?, the preference of the glucose transport system for the anions was SCN^? > Cl^? > C₂H₃O₂^Post?. Efflux of Cl^? was not affected by the rate of glucose influx. Finally, microelectrode recordings of worms anesthetized with 2 mM arecoline revealed a transmembrane potential (TMP) of ?45 ± 3.6 mV (inside negative). Three to four minutes after addition of glucose (5 mM) there was a progressive hyperpolarization of the TMP to ?58 mV. A revised model of the glucose transport system that is consistent with previous observations on this organism is proposed.     

Glucose Availability to Individual Cerebral Structures Is Correlated to Glucose Metabolism

Regional cerebral glucose influx was measured using quantitative autoradiography after the intravenous infusion of [2-¹⁴C]glucose for a period of 10 or 20 s. Glucose influx varied considerably among structures over an almost threefold range. When compared with rates of regional glucose utilization, a significant correlation by region was found between glucose influx and utilization, demonstrating that the glucose supply to individual cere bral structures is closely matched to their metabolic needs.     

Modeling the Pancreatic α-Cell: Dual Mechanisms of Glucose Suppression of Glucagon Secretion

The mechanism by which glucose induces insulin secretion in β-cells is fairly well understood. Despite years of research, however, the mechanism of glucagon secretion in α-cells is still not well established. It has been proposed that glucose regulates glucagon secretion by decreasing the conductance of either outward ATP-dependent potassium channels (K(ATP)) or an inward store-operated current (SOC). We have developed a mathematical model based on mouse data to test these hypotheses and found that both mechanisms are possible. Glucose metabolism closes K(ATP) channels, which depolarizes the cell but paradoxically reduces calcium influx by inactivating voltage-dependent calcium and sodium channels and decreases secretion. Glucose metabolism also activates SERCA pumps, which fills the endoplasmic reticulum and hyperpolarizes the cells by reducing the inward current through SOC channels and again suppresses glucagon secretion. We find further that the two mechanisms can combine to account for the nonmonotonic dependence of secretion on glucose observed in some studies, an effect that cannot be obtained with either mechanism alone.     

Enzymes of glucose metabolism in normal mouse pancreatic islets

1. Glucose-phosphorylating and glucose 6-phosphatase activities, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP⁺-linked isocitrate dehydrogenase, `malic' enzyme and pyruvate carboxylase were assayed in homogenates of normal mouse islets. 2. Two glucose-phosphorylating activities were detected; the major activity had K<sub>m</sub> 0.075mm for glucose and was inhibited by glucose 6-phosphate (non-competitive with glucose) and mannoheptulose (competitive with glucose). The other (minor) activity had a high K<sub>m</sub> for glucose (mean value 16mm) and was apparently not inhibited by glucose 6-phosphate. 3. Glucose 6-phosphatase activity was present in amounts comparable with the total glucose-phosphorylating activity, with K<sub>m</sub> 1mm for glucose 6-phosphate. Glucose was an inhibitor and the inhibition showed mixed kinetics. No inhibition of glucose 6-phosphate hydrolysis was observed with mannose, citrate or tolbutamide. The inhibition by glucose was not reversed by mannoheptulose. 4. 6-Phosphogluconate dehydrogenase had K<sub>m</sub> values of 2.5 and 21μm for NADP<sup>+</sup> and 6-phosphogluconate respectively. 5. Glucose 6-phosphate dehydrogenase had K<sub>m</sub> values of 4 and 22μm for NADP<sup>+</sup> and glucose 6-phosphate. The K<sub>m</sub> for glucose 6-phosphate was considerably below the intra-islet concentration of glucose 6-phosphate at physiological extracellular glucose concentrations. The enzyme had no apparent requirement for cations. Of a number of possible modifiers of glucose 6-phosphate dehydrogenase, only NADPH was inhibitory. The inhibition by NADPH was competitive with NADP<sup>+</sup> and apparently mixed with respect to glucose 6-phosphate. 6. NADP<sup>+</sup>–isocitrate dehydrogenase was present but the islet homogenate contained little, if any, `malic' enzyme. The presence of pyruvate carboxylase was also demonstrated. 7. The results obtained are discussed with reference to glucose phosphorylation and glucose 6-phosphate oxidation in the intact mouse islet, and the possible nature of the β-cell glucoreceptor mechanism.     

Specificity of d-glucose transport by the apical membrane of Nereis diversicolor epidermis

(1) The specificity of d-[6-³H]glucose influx by a Na⁺-dependent and phlorizin-sensitive transport system in the apical epidermal membrane of the polychaete worm, Nereis diversicolor, was investigated in vivo. (2) The inhibitory effect of eleven d-glucose analogues on d-[6-³H]glucose influx from a 5 μM external concentration was recorded. The inhibitors (each tested at 5, 50, 500 and 5000 μM) were selected to illuminate the configurational requirements for interaction with the d-glucose transport system. (3) The following compounds were found to be significant inhibitors: methyl α-d-glucoside, methyl β-d-glucoside, d-galactose, $\text{3-O-}\text{methyl-}\text{d}\text{-glucose}$, 2-deoxy-d-glucose, d-xylose, myo-inositol, β-d-fructose; the effect was graded according to inhibitor concentration. l-Glucose also inhibited d-glucose influx but to the same extent at all four concentrations tested, suggesting transport site heterogeneity. d-Mannose and l-arabinose did not inhibit influx. (4) The most potent inhibitor, methyl-α-d-glucoside, was itself a substrate, and its transport was inhibited by phlorizin and d-glucose, as well as by substitution of Na⁺ in the incubation medium with Li⁺ or choline⁺. (5) We conclude that the specificity of the Na⁺-dependent d-glucose transporter in the apical epidermal membrane of Nereis is similar to that in the apical membrane of vertebrate small intestinal and proximal tubular epithelium, and in the tapeworm integument.     

Lithium Transport in the Kidney of the Herring Gull Larus argentatus: Induction of Renal Li+ Net Secretion by a Salt Loading

To find out whether salt loading can induce Li⁺ net secretion in the kidney of the birds that have an extrarenal organ (the salt gland) for excretion of NaCl excess (earlier, such effect was revealed in the pigeon and chicken, the birds that do not have the salt gland), the effect of intravenous NaCl injections (4–5 injections of 14–20 mmol/kg at 20–30-min intervals) on Li⁺ transport in the gull kidney was studied. Prior to the salt loading, above 99% of the filtered Li⁺ were reabsorbed in the kidney: fractional excretion of Li⁺ (FE_Li) was 0.0024 ± 0.0007 (mean ± SD), the Li⁺ reabsorption occurring not only in the proximal, but also in the distal tubule. Under conditions of the salt loading, two essentially different regimes of the kidney functioning were observed: net Li⁺ reabsorption (FE_Li = 0.63 ± 0.26) and net Li⁺ secretion (FE_Li = 1.26 ± 0.12). In the absence of the salt loading, Li⁺ (due to its distal reabsorption) does not fit requirements of an indicator of proximal reabsorption of Na⁺ and water. However, in the regimes of the salt net reabsorption and the salt net secretion, FE_Li probably can serve as an indicator of delivery of these substances to the end of the proximal tubule (the lithium clearance method). If this suggestion is correct, transition from the net Li⁺ reabsorption (FE_Li < 1) to its net secretion (FE_Li > 1) with rise of the salt loading indicates the appearance of net Na⁺ secretion in the proximal tubule under these conditions. A comparison of the results for the gull and the chicken has shown that although the presence of the salt gland did not prevent transition of the kidney to the net Li⁺ secretion, its duration in the gull was significantly shorter. The comparative data are presented on parameters of renal function in the regimes of net Li⁺ secretion and net Li⁺ reabsorption. In both regimes, a linear correlation was observed between FE_Li and FE_Na; however, regression coefficients considerably differed. An abrupt break of the curve occurred at FE_Li 1.     

Physiological significance of the concentration of human milk glucose

Summary The concentration of glucose in human milk, [Glucose]_milk, was directly related to the volume of milk secreted not only during lactogenesis and weaning but also during full lactation. To investigate the mechanism for this observation we first established that glucose equilibrates across the apical membrane of the mammary alveolar cell, using infusion of stable isotopically labelled glucose into lactating women. Our results indicate that [Glucose]_milk can be used to measure the glucose concentration in the mammary alveolar cell, [Glucose]_cell. We then investigated the regulation of glucose transport into the mammary alveolar cell using glucose clamp methodology in fully lactating and weaning women. Maintenance of high plasma insulin concentrations for four hours under euglycemic conditions had no effect on [Glucose]_milk, demonstrating that insulin does not regulate glucose transport into the mammary gland. On the other hand, maintaining the [Glucose]_plasma at twice the fasting level resulted in a 3-fold increase in the steady state [Glucose]_milk in fully lactating women and a 5-fold increase in [Glucose]_milk in weaning women. Kinetic analysis of the data showed that the V_max for glucose transport into the mammary alveolar cell across the basolateral membrane is regulated by the level of synthetic activity in the mammary alveolar cell.Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

The effects of phlorizin,phloretin and ouabain on the reabsorption of glucose by the Malpighian tubules of Locusta migratoria migratorioides

Isolated Malpighian tubules of Locusta reabsorb significant levels of glucose from their lumen back into the bathing fluid (haemolymph). This reabsorption is inhibited by phlorizin, phloretin and ouabain. Both phlorizin and phloretin are found to accumulate in the secreted fluid of the tubules against concentration gradients. Ouabain inhibition is explained in terms of its effect on intracellular Na⁺ concentrations. A hypothetical model of the role that Na⁺ may play in glucose reabsorption is presented as a possible explanation of these observations.     

Glycogen synthesis in the fungus Neurospora crassa

An enzymic activity, obtained from Neurospora crassa, catalyzing the incorporation of [¹⁴C]glucose from ADP-[¹⁴C]glucose into a glucan of the glycogen type, is described. The properties of the ADPglucose: glycogen glucosyltransferase as compared with those of the already known UDP glucose: glycogen glucosyltransferase were studied. The radioactive products obtained with UDP-[¹⁴C]glucose or ADP-[¹⁴C]glucose released all the radioactivity as maltose after α or β amylase treatment. Glucose 6-phosphate stimulated the synthetase when UDP-[¹⁴C]glucose was the substrate but the stimulation was much greater with ADP-[¹⁴C]glucose as glucosyl donor. Glucose 6-phosphate plus EGTA gave maximal stimulation. The system was completely dependent on the presence of a ‘primer’ of the α 1 → 4 glucan type.     

Regulation of canine renal vesicle Pi transport by growth hormone and parathyroid hormone

Renal phosphate (P_i) reabsorption is increased by growth hormone (GH) and decreased by parathyroid hormone (PTH). Na⁺-stimulated P_i transport across the brush border membrane of the proximal tubule is the initial step in the process of P_i reabsorption. To determine whether changes in P_i reabsorption induced by GH or PTH are accompanied by changes in brush border membrane Na⁺-gradient-stimulated P_i transport, we examined the effect of in vivo GH and PTH administration and thyroparathyroidectomy on P_i transport by isolated brush border membrane vesicles prepared from canine kidney. In experiments in which the effect of PTH administration was examined, the same animal provided the control kidney (before PTH administration) and the experimental kidney (after PTH administration). The Na⁺-gradient P_i overshoot in vesicles isolated from normal, GH-treated and thyroparathyroidectomized dogs was increased after in vivo PTH administration. GH administration and thyroparathyroidectomy increased the height of the overshoot compared to normal. PTH administration decreased the apparent $\text{V}$ value by 44% in vesicles from normal animals. The apparent $\text{V}$ value was increased, compared to normal, by GH (34%) and thyroparathyroidectomy (57%). PTH administration decreased the apparent $\text{V}$ in both the latter groups. GH administration to thyroparathyroidectomized dogs further increased the apparent $\text{V}$. Changes in the apparent $\text{V}$ paralleled changes in P_i reabsorption in vivo induced by experimental manipulations. We conclude that changes in renal P_i reabsorption induced by GH were like those induced by PTH, accompanied by changes in the Na⁺-stimulated P_i transport system in the renal brush border membrane, and that the effect of PTH on vesicular P_i transport in GH-treated dogs did not differ from the effect on vesicles from normal animals.     

Glucose inhibits 45Ca efflux from pancreatic β-cells also in the absence of Na+-Ca2+ countertransport

During perifusion with medium deprived of Ca²⁺, addition of glucose or omission of Na⁺ resulted in prompt and quantitatively similar inhibitions of ⁴⁵Ca efflux from β-cell rich pancreatic islets microdissected from ob / ob mice. Glucose had no additional inhibitory effect when Na⁺ was isoosmotically replaced by sucrose or choline⁺. When K⁺ was used as a substitute for Na⁺, the inhibitory effect of Na⁺ removal on ⁴⁵Ca efflux became additive to that of glucose. The observation that glucose can be equally effective in inhibiting ⁴⁵Ca efflux in the presence or absence of Na⁺ is difficult to reconcile with the postulate that the Na⁺-Ca²⁺ countertransport mechanism is a primary site of action for glucose.     

Untersuchungen über den Glucosestoffwechsel keimender Uredosporen von Puccinia graminis var. tritici

Zusammenfassung ¹⁴C-Glucose wurde an Uredosporen von Puccinia graminis appliziert, und es wurde die Aufnahme der Glucose durch die Sporen und die Inkorporation des Glucosekohlenstoffs in das Sporenmaterial untersucht. Das Studium der Abhängigkeit der Glucoseaufnahme von der Konzentration im Medium ergab: Bei höheren Glucosekonzentrationen werden größere Glucosemengen aufgenommen. Die bei einer Konzentration von 5×10^-2 mol aufgenommene Glucose reicht aus, um den gesamten Kohlenhydratbedarf für die Bildung der Keimschlauchwände zu decken. Die Glucoseaufnahme nimmt im Verlauf der Inkubationszeit zu. Die aufgenommene Glucose wird nur in geringem Umfang veratmet. Sie wird statt dessen bevorzugt zur Synthese von in 40% igem Äthanol unlöslichen Verbindungen verwendet.Weiterhin wurden die Stoffwechselwege verfolgt, auf denen Glucose umgesetzt wird. Wir untersuchten, in welche Verbindungen des Sporenmaterials Glucose-kohlenstoff eingebaut wird. Der größte Teil der löslichen ¹⁴C-Aktivität fand sich in der Fraktion der Kohlenhydrate und der freien Aminosäuren. Mannit, Arabit und Glycerin hatten die höchsten spezifischen Aktivitäten. Unter den freien Aminosäuren zeigten Alanin, Tyrosin und Glutaminsäure relativ hohe spezifische ¹⁴C-Aktivität. Hieraus kann man folgern, daß Glucose sowohl über den PPC wie über den EMP und TCA-Cyclus umgesetzt wird. Es wurde festgestellt, in welchem Ausmaß die aufgenommene Glucose zur Synthese der Keimschlauchwand verwendet wird. Eine Analyse der Keimschlauchwände ergab: Fast 90% des inkorporierten ¹⁴C fand sich in den unlöslichen Verbindungen der Keimschlauchwände. Während der Inkubation gelangte keine ¹⁴C-Aktivität in das Material der Sporenwände.

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Investigation of glucose metabolism in germinating uredospores of Puccinia graminis var. tritici

Summary Uredospores of Puccinia graminis were incubated in a medium containing ¹⁴C-glucose. The uptake of glucose by the spores and the incorporation of glucose carbon into the spore material was investigated. With higher glucose concentrations in the medium larger amounts of glucose were taken up by the spores. At a concentration of 5×10^-2 mol the glucose withdrawn from the medium is sufficient to supply all carbohydrate material necessary for the synthesis of germ tube walls. The glucose taken up increased during the incubation time and was respired only to a small extent. It was used predominantly for the synthesis of compounds insoluble in 40% ethanol.Fractionation of the soluble ¹⁴C-activity showed that most of it had been incorporated into carbohydrates and free amino acids. Mannitol, arabitol and glycerol had higher specific activities than all other compounds. The amino acids alanine, tyrosine and glutamic acid showed relatively high ¹⁴C incorporation. The labelling pattern is in agreement with the view that glucose is metabolized via the pentose phosphate pathway as well as the Embden-Meyerhof pathway and the tricarboxylic acid cycle.However, only 12% of the ¹⁴C incorporated could be accounted for by soluble products. An analysis of the germ tube wall revealed that it contained the bulk of the incorporated ¹⁴C-activity. Most of the activity was found in the carbohydrate constitutents of the germ tube wall, namely glucose, mannose, galactose and glucosamine. This indicates that the glucose taken up from the medium by the spores is predominantly used for the synthesis of the germ tube wall. The spore wall material did not contain ¹⁴C.

     

Measurement of Glucose Utilization by Pseudomonas fluorescens That Are Free-Living and That Are Attached to Surfaces

The assimilation and respiration of glucose by attached and free-living Pseudomonas fluorescens were compared. The attachment surfaces were polyvinylidene fluoride, polyethylene, and glass. Specific uptake of [¹⁴C]glucose was determined after bacterial biomass was measured by (i) microscopic counts or (ii) prelabeling of cells by providing [³H]leucine as substrate, followed by dual-labeling scintillation counting. The glucose concentration was 1.4, 3.5, 5.5, 7.6, or 9.7 μM. Glucose assimilation by cells which became detached from the surfaces during incubation with glucose was also measured after the detached cells were collected by filtration. The composition of the substratum had no effect on the amount of glucose assimilated by attached cells. Glucose assimilation by attached cells exceeded that by free-living cells by a factor of between 2 and 5 or more, and respiration of glucose by surface-associated cells was greater than that by free-living bacteria. Glucose assimilation by detached cells was greater than that by attached bacteria. Measurements of biomass by microscopic counts gave more consistent results that those obtained with dual-labeling, but in general, results obtained by both methods were corroborative.