Biotin Uptake by Cold-Shocked Cells, Spheroplasts, and Repressed Cells of Saccharomyces cerevisiae: Lack of Feedback Control

Cold-osmotic-shocked cells and spheroplasts of Saccharomyces cerevisiae (ATCC 9896) display a biotin uptake system similar to that observed in intact cells. 2-Mercaptoethanol was found to inhibit biotin transport. Cells repressed for biotin uptake by growth in excess biotin (25 ng/ml) possess an energy-dependent transport system that has a K(m) for biotin of 6.6 x 10(-7) M and a V(max) equal to 39 pmol per mg (dry weight) per min. A similar K(m) (6.4 x 10(-7) M) but a considerably higher V(max) (530 pmol per mg (dry weight) per min) was determined for biotin uptake by cells grown in sufficient biotin (0.25 ng/ml). The V(max) rates of biotin uptake by both repressed and derepressed cells were increased approximately 35-fold in the presence of glucose. These yeast cells appear to regulate their biotin uptake by two mechanisms. An exit system provides for immediate adjustments, whereas turnover of the transport system and repression of new synthesis establishes a slower adaptation to changes in the environment. Feedback inhibition was ruled out as a mechanism of regulation of transport.     

Manganese Active Transport in Escherichia coli

Manganese was accumulated by cells of Escherichia coli by means of an active transport system quite independent of the magnesium transport system. When the radioisotope (54)Mn was used, manganese transport showed saturation kinetics with a K(m) of 2 x 10(-7)m and a V(max) of 1 to 4 nmoles/min per 10(12) cells at 25 C. The manganese transport system is highly specific; magnesium and calcium did not stimulate, inhibit, or compete with manganese for cellular uptake. Cobalt and iron specifically interfered with (54)Mn uptake, but only when added at concentrations 100 times higher than the K(m) for manganese. Active transport of manganese is temperature- and energy-dependent: uptake of (54)Mn was inhibited by cyanide, dinitrophenol, and m-chlorophenyl carbonylcyanide hydrazone (CCCP). Furthermore, the turnover or exit of manganese from intact cells was inhibited by energy poisons such as dinitrophenol and CCCP.     

Active transport of D-alanine and related amino acids by whole cells of Bacillus subtilis

Whole cells of Bacillus subtilis transported d-alanine and l-alanine by two different systems. The high-affinity system (K(m) of 1 muM and V(max) of 0.6 to 0.8 nmol/min per mg of protein) was specific for the two stereoisomers of alanine. The low-affinity system (K(m) of 10 muM for l-alanine and 20 muM for d-alanine and glycine) had a V(max) of 5 to 12 nmol/min per mg of protein. This system transported glycine, d-cycloserine, and d-serine, in addition to d- and l-alanine. Azide inhibited the uptake of these amino acids and caused the efflux of d-alanine from preloaded cells. These data suggest that transport of these amino acids is energized by the electron transport chain.     

Uptake and utilization of glutamic acid by Cryptococcus albidus

Cryptococcus albidus utilizes glutamate as a sole carbon source. The kinetics of uptake of this amino acid were studied. l-Glutamic acid was taken up by two saturable systems: a high affinity system with a Michaelis constant (K(m)) of 1.15 x 10(-5) M and a V(max) of 0.049 mumol per mg per h and a low affinity system with a K(m) of 2.5 x 10(-3) M and a V(max) of 3.61 mumol per mg per h. Both systems possessed characteristics of active transport which were dependent on temperature and pH and which required metabolic energy. Uptake was inhibited at 37 C but the temperature-sensitive step was reversible. Chemical fractionation of cells with 5% trichloroacetic acid showed that glutamic acid initially entered a soluble pool which decreased after 1 h as the amino acid was incorporated into the protein and nucleic acid fractions of the yeast. Some of the glutamate was completely oxidized and could be recovered as (14)CO(2). Therefore, the amino acid was also used as an energy source.     

Properties of the inducible hydroxyproline transport system of Pseudomonas putida

Features of the transport system for hydroxyproline in a strain of Pseudomonas putida were studied. A mutant, lacking hydroxyproline-2 epimerase and unable to metabolize hydroxy-l-proline, was shown to transport and accumulate this compound after induction. Both entry and exit rates were examined, and kinetic constants for the reaction were determined. Increasing the induction time from 0.5 to 3 hr increased the entry rate three- to fourfold but had only a small and variable effect on the exit rate. Entry followed saturation kinetics. For hydroxy-l-proline, the K(m) and V(max) values were found to be 3 x 10(-5)m and 6 mumoles per g (dry weight) per min, respectively. The K(m) and V(max) for the epimer allohydroxy-d-proline were 10(-3)m and 0.1 mumole per g (dry weight) per min. Entry rates into "loaded" and "unloaded" cells were found to be the same. Exit was shown to be first order over the range of internal substrate concentrations measured. Exit rates were measured by several different methods and found to be independent of external substrate concentration. The first-order exit rate constant was computed to be 0.23 min(-1). Several metabolic inhibitors were examined for their effect on transport. The inhibitory action of N-ethyl maleimide was shown to be greatly reduced if cells were allowed to accumulate hydroxy-l-proline before exposure to the inhibitor. A number of other amino acids interfered with the transport of hydroxy-l-proline; the greatest effect was produced by l-alanine and l-proline.     

Manganese Transport in Bacillus subtilis W23 During Growth and Sporulation

Manganese is accumulated in Bacillus subtilis by a highly specific active transport system. This trace element "pump" is insensitive to added magnesium or calcium and preferentially accumulates manganese in the presence of cobalt, iron, and copper. Manganese uptake in B. subtilis is inhibited by cyanide, azide, pentachlorophenol, and m-chlorophenyl carbonylcyanide hydrazone. The uptake of manganese follows Michaelis-Menten kinetics, and the net accumulation of manganese is regulated by increasing the V(max) after exposure to manganese-starvation conditions and by decreasing the V(max) for manganese uptake during growth in excess manganese. The K(m) remains constant during these regulatory changes in V(max). Manganese accumulated during growth is exchangeable for exogenous manganese and can be released from the cells by toluene (which causes leakage but not lysis) or by lysis with lysozyme. Two stages can be distinguished with regard to intracellular manganese during the process of growth and sporulation. During logarithmic growth, B. subtilis maintains a relatively constant internal manganese content, which is a function of the external manganese concentration following approximately a Langmuir adsorption isotherm. At the end of log phase, net accumulation of manganese slows. A second phase of net manganese accumulation begins at about the same time during sporulation as the accumulation of calcium begins. The manganese accumulated during growth and early sporulation is exchangeable and therefore relatively "free"; intracellular manganese is converted later during sporulation into a bound form that cannot be released by toluene or lysozyme.     

Amino Acid Transport into Cultured Tobacco Cells: I. LYSINE TRANSPORT

Lysine transport into suspension-cultured Wisconsin-38 tobacco cells was observed. Uptake was linear (up to 90 minutes) with respect to time and amount of tissue only after 4 to 6 hours preincubation in calcium-containing medium. The observed cellular accumulation of lysine was against a concentration gradient and not due to exchange diffusion. Transport was stimulated by low pH and characterized by a biphasic uptake isotherm with two K(m) values for lysine. System I (K(m) approximately 5 x 10(-6) molar; V(max) approximately 180 nanomoles per gram fresh weight per hour) and system II (K(m) approximately 10(-4) molar; V(max) approximately 1900 nanomoles per gram fresh weight per hour) were inhibited by N-ethylmaleimide and a variety of respiratory inhibitors. This inhibition was not due to increased efflux. In antagonism experiments, system I was inhibited most effectively by basic amino acids, followed by the sulfur amino acids. System I was only slightly inhibited by the neutral and aromatic amino acids and was not inhibited by the acidic amino acids aspartic and glutamic acids. Transport by system II was inhibited by all of the tested amino acids (including aspartic and glutamic acids) and analogs; however, this system was not inhibited by d-arginine. Neither system was strongly inhibited by d-lysine or the lysine analog S-2-aminoethyl-l-cysteine. Arginine was shown to be a competitive inhibitor of both systems with values for K(i) similar to the respective K(m) values.These studies suggest the presence of at least two amino acid permeases in W-38 tobacco cells.     

Active Transport of Biotin in Escherichia coli K-12

The transport of [(14)C]biotin into cells of a biotin prototroph, Escherichia coli K-12 strain Y10-1, was investigated. The vitamin taken up by the cells in this strain existed primarily in the free form. Addition of glucose enhanced the rate of uptake six- to eightfold and the steady level was reached in 2 to 3 min resulting in accumulation of biotin against a concentration gradient. The uptake showed marked dependence on temperature (Q(10), 2.3; optimum, 37 C) and pH (optimum 6.6) and was inhibited by iodoacetate. Energy of activation for glucose-dependent uptake was calculated to be 16,200 cal per mol. The rate of biotin uptake with increasing biotin concentrations showed saturation kinetics with an apparent K(m) and V(max) values of 1.4 x 10(-7) M and 6.6 pmol per mg of dry cells per min respectively. The cells also accumulated biotin against a concentration gradient in the absence of added glucose, although at a much lower rate. This accumulation was much more susceptible to inhibition by azide and uncouplers of oxidative phosphorylation suggesting that the energy source was supplied through the electron-transport chain. Inhibition studies with a number of biotin analogues indicated the requirement for an intact ureido ring. The biotin uptake was inhibited in cells grown in biotin-containing medium and was shown to be the result of repression of the transport system, suggesting the control of the biotin transport.     

Characterization and reconstitution of a 4Fe-4S adenylyl sulfate/phosphoadenylyl sulfate reductase from Bacillus subtilis

CysH1 from Bacillus subtilis encodes a 3'-phospho/adenosine-phosphosulfate-sulfonucleotide reductase (SNR) of 27 kDa. Recombinant B. subtilis SNR is a homodimer, which is bispecific and reduces adenylylsulfate (APS) and 3'-phosphoadenylylsulfate (PAPS) alike with thioredoxin 1 or with glutaredoxin 1 as reductants. The enzyme has a higher affinity for PAPS (K(m)PAPS 6.4 microm Trx-saturating, 10.7 microm Grx-saturating) than for APS (K(m) APS 28.7 microm Trx-saturating, 105 microm Grx-saturating) at a V(max) ranging from 280 to 780 nmol sulfite mg(-1) min(-1). The catalytic efficiency with PAPS as substrate is higher by a factor of 10 (K(cat)/K(m) 2.7 x 10(4)-3.6 x 10(4) liter mol(-1) s(-1). B. subtilis SNR contains one 4Fe-4S cluster per polypeptide chain. SNR activity and color were lost rapidly upon exposure to air or upon dilution. M?ssbauer and absorption spectroscopy revealed that the enzyme contained a 4Fe-4S cluster when isolated, but degradation of the 4Fe-4S cluster produced an inactive intermediate with spectral properties of a 2Fe-2S cluster. Activity and spectral properties of the 4Fe-4S cluster were restored by preincubation of SNR with the iron-sulfur cluster-assembling proteins IscA1 and IscS. Reconstitution of the 4Fe-4S cluster of SNR did not affect the reductive capacity for PAPS or APS. The interconversion of the clusters is thought to serve as oxygen-sensitive switch that suppresses SO(3) formation under aerobiosis.     

Beta-D-phosphogalactoside galactohydrolase from Streptococcus cremoris HP: purification and enzyme properties

beta-d-phosphogalactoside galactohydrolase (beta-PGal) was isolated and purified from cell-free extracts of Streptococcus cremoris HP to apparent homogeneity to gel electrophoresis. Using the chromogenic o-nitrophenol-beta-d-galactopyranoside-6-phosphate as substrate, the purified enzyme exhibited a specific activity of 18.71 U/mg of protein and K(m) and V(max) values of 5.88 x 10(-4) M and 23.8 mumol of o-nitrophenol liberated per min per mg of protein, respectively. d-Galactose-6-phosphate was a weak competitive inhibitor of beta-PGal. Activity was relatively heat resistant and was maximal from pH 5.0 to 8.0 and over a temperature range of 45 to 52 C. Dithiothreitol, ethylenediaminetetraacetic acid, and citrate stimulated beta-PGal activity, whereas Mg(2+), Li(1+), and p-hydroxymercuribenzoate were inhibitory. Molecular weight of the enzyme was estimated at 6.76 x 10(4). Amino acid composition was similar to other beta-phosphogalactosidases previously investigated, with the exception that the S. cremoris enzyme contains a small amount of half cystine.     

Purification and kinetics of a raw starch-hydrolyzing,thermostable, and neutral glucoamylase of the thermophilic mold Thermomucor indicae-seudaticae

The purified glucoamylase of the thermophilic mold Thermomucor indicae-seudaticaehad a molecular mass of 42 kDa with a pI of 8.2. It is a glycoprotein with 9-10.5% carbohydrate content, which acted optimally at 60 degrees C and pH 7.0, with a t(1/2) of 12 h at 60 degrees C and 7 h at 80 degrees C. Its experimental activation energy was 43 KJ mol(-1) with temperature quotient (Q(10)) of 1.35, while the values predicted by response surface methodology (RSM) were 43 KJ mol(-1) and 1.28, respectively. The enzyme hydrolyzed soluble starch at 50 degrees C (K(m) 0.50 mg mL(-1) and V(max) 109 micromol mg(-1) protein min(-1)) and at 60 degrees C (K(m) 0.40 and V(max) 143 micromol mg(-1) protein min(-1)). The experimental K(m) and V(max) values are in agreement with the predicted values at 50 degrees C (K(m) 0.45 mg mL(-1) and V(max) 111.11 micromol mg(-1) protein min(-1)) and at 60 degrees C (K(m) 0.36 mg mL(-1)and V(max) 142.85 micromol mg(-1) protein min(-1)). An Arrhenius plot indicated thermal activation up to 60 degrees C, and thereafter, inactivation. The enzyme was strongly stimulated by Co(2+), Fe(2+), Ag(2+), and Ca(2+), slightly stimulated by Cu(2+) and Mg(2+), and inhibited by Hg(2+), Zn(2+), Ni(2+), and Mn(2+). Among additives, dextran and trehalose slightly enhanced the activity. Glucoamylase activity was inhibited by EDTA, beta-mercaptoethanol, dithiothreitol, and n-bromosuccinimide, and n-ethylmaleimide inhibited its activity completely. This suggested the involvement of tryptophan and cysteine in catalytic activity and the critical role of disulfide linkages in maintaining the conformation of the enzyme. The enzyme hydrolyzed around 82% of soluble starch and 65% of raw starch (K(m) 2.4 mg mL(-1), V(max) 50 micromol mg(-1) protein min(-1)), and it was remarkably insensitive to glucose, suggesting its applicability in starch saccharification.     

Characterization of cadmium uptake in Lactobacillus plantarum and isolation of cadmium and manganese uptake mutants.

Two different Cd(2+) uptake systems were identified in Lactobacillus plantarum. One is a high-affinity, high-velocity Mn(2+) uptake system which also takes up Cd(2+) and is induced by Mn(2+) starvation. The calculated K(m) and V(max) are 0.26 microM and 3.6 micromol g of dry cell(-1) min(-1), respectively. Unlike Mn(2+) uptake, which is facilitated by citrate and related tricarboxylic acids, Cd(2+) uptake is weakly inhibited by citrate. Cd(2+) and Mn(2+) are competitive inhibitors of each other, and the affinity of the system for Cd(2+) is higher than that for Mn(2+). The other Cd(2+) uptake system is expressed in Mn(2+)-sufficient cells, and no K(m) can be calculated for it because uptake is nonsaturable. Mn(2+) does not compete for transport through this system, nor does any other tested cation, i.e., Zn(2+), Cu(2+), Co(2+), Mg(2+), Ca(2+), Fe(2+), or Ni(2+). Both systems require energy, since uncouplers completely inhibit their activities. Two Mn(2+)-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn(2+) for growth as the parental strain. Mn(2+) starvation-induced Cd(2+) uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn(2+) or Cd(2+) accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn(2+) and Cd(2+) uptake system.     

Direct effect of temperature on the catalytic activity of nitric oxide synthases types I, II, and III.

The effect of temperature (between 5.0 and 45.0 degrees C) on the catalytic activity of nitric oxide synthases types I, II, and III (NOS-I, NOS-II, and NOS-III, respectively) has been investigated, at pH 7.5. The value of V(max) for NOS-I activity increases from 1.8 x 10(1) pmol min(-1) mg(-1), at 5.0 degrees C, to 1.8 x 10(2) pmol min(-1) mg(-1), at 45.0 degrees C; on the other hand, the value of K(m) (=4.0 x 10(-6) M) is temperature independent. Again, the value of V(max) for NOS-II activity increases from 8.0 pmol min(-1) mg(-1), at 7.0 degrees C, to 5.4 x 10(1) pmol min(-1) mg(-1), at 40.0 degrees C, the value of K(m) (=1.8 x 10(-5) M) being unaffected by temperature. Temperature exerts the same effect on NOS-I and NOS-II activity, as shown by the same values of DeltaH(V(max)) (=4.2 x 10(1) kJ mol(-1)), DeltaH(K(m)) (=0 kJ mol(-1)), and DeltaH((V(max))(/K(m))()) (=4.2 x 10(1) kJ mol(-1)). On the contrary, the value of K(m) for NOS-III activity decreases from 3.8 x 10(-5) M, at 10.0 degrees C, to 1.6 x 10(-5) M, at 40.0 degrees C, the value of V(max) (=6.8 x 10(1) pmol min(-1) mg(-1)) being temperature independent. Present results indicate that temperature influences directly NOS-I and NOS-II activity independently of the substrate concentration, the values of K(m) being temperature independent. However, when l-arginine level is higher than 2 x 10(-4) M, as observed under in vivo conditions, NOS-III activity is essentially unaffected by temperature, the substrate concentration exceeding the value of K(m). As a whole, although further studies in vivo are needed, these observations seem to have potential physiopathologic implications.     

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.     

Genetically alterable transport of amethopterin in Diplococcus pneumoniae. I. Physiological properties and kinetics of the wild-type system

A system of H(3)-amethopterin uptake, physiologically and kinetically resembling active transport, has been described in Diplococcus pneumoniae. Uptake by this system has a pH optimum near 6.0, is temperature-dependent, requires a readily available source of energy, and conforms to Michaelis-Menten kinetics. The system showed a K(m) of 0.9 x 10(-6)m and a V(max) of 1.9 x 10(-13) moles per min per mg (dry weight). Both folate and H(2)-folate compete with H(3)-amethopterin for the same system, but to a limited degree. The intracellular concentration of H(3)-amethopterin accumulated at equilibrium was 1.06 x 10(-9) moles/ml or fivefold the external concentration when the latter was limiting, but at least 60-fold the internal concentration attained solely by diffusion in the same time interval at 0 C.     

d-Glucose Transport System of Zymomonas mobilis

The properties of the d-glucose transport system of Zymomonas mobilis were determined by measuring the uptake of nonmetabolizable analogs (2-deoxy-d-glucose and d-xylose) by wild-type cells and the uptake of d-glucose itself by a mutant lacking glucokinase. d-Glucose was transported by a constitutive, stereospecific, carrier-mediated facilitated diffusion system, whereby its intracellular concentration quickly reached a plateau close to but not above the external concentration. d-Xylose was transported by the d-glucose system, as evidenced by inhibition of its uptake by d-glucose. d-Fructose was not an efficient competitive inhibitor of d-glucose uptake, indicating that it has a low affinity for the d-glucose transport system. The apparent K(m) of d-glucose transport was in the range of 5 to 15 mM, with a V(max) of 200 to 300 nmol min mg of protein. The K(m) of Z. mobilis glucokinase (0.25 to 0.4 mM) was 1 order of magnitude lower than the K(m) for d-glucose transport, although the V(max) values for transport and phosphorylation were similar. Thus, glucose transport cannot be expected to be rate limiting at concentrations of extracellular glucose normally used in fermentation processes, which greatly exceed the K(m) for the transport system. The low-affinity, high-velocity, nonconcentrative system for d-glucose transport described here is consistent with the natural occurrence of Z. mobilis in high-sugar environments and with the capacity of Z. mobilis for rapid conversion of glucose to metabolic products with low energetic yield.     

Correlation between Multi-Drug Resistance-Associated Membrane Transport in Clonal Cancer Cells and the Cell Cycle Phase

Multidrug resistance driven by ABC membrane transporters is one of the major reasons for treatment failure in human malignancy. Some limited evidence has previously been reported on the cell cycle dependence of ABC transporter expression. However, it has never been demonstrated that the functional activity of these transporters correlates with the cell cycle position. Here, we studied the rate of intrinsic ABC transport in different phases of the cell cycle in cultured MCF-7 breast cancer cells. The rate was characterized in terms of the efflux kinetics from cells loaded with an ABC transporter substrate. As averaging the kinetics over a cell population could lead to errors, we studied kinetics of ABC transport at the single-cell level. We found that the rate of ABC transport in MCF-7 cells could be described by Michaelis-Menten kinetics with two classical parameters, V(max) and K(M). Each of these parameters showed similar unimodal distributions with different positions of maxima for cell subpopulations in the 2c and 4c states. Compared to the 2c cells, the 4c cells exhibited greater V(max) values, indicating a higher activity of transport. They also exhibited a greater V(max)/K(M) ratio, indicating a higher efficiency of transport. Our findings suggest that cell cycle-related modulation of MDR may need to be taken into account when designing chemotherapy regimens which include cytostatic agents.