Kinetics of L-valine uptake in tobacco leaf discs. Comparison of wild-type,the digenic mutant Valr-2, and its monogenic derivatives

Uptake rates of L-valine in epidermis-free leaf discs of tobacco (Nicotiana tabacum L. cv. Xanthi) were measured over the concentration range 0.1 M to 50 mM. Wild-type tobacco was compared with the digenic mutant Val^r-2 (genotype vr2/vr2; vr3/vr3), and with the monogenic mutant strains h9 and h10 (genotype +/+; vr3/vr3) and h17 and h23 (genotype vr2/vr2; +/+). Rate equations consisting of one to three Michaelis-Menten terms, possibly in combination with a linear term were fitted to the kinetic data. These rate equations are equivalent to rational polynomials which may be regarded as the general type of mathematical function describing the kinetics of enzymes and carriers. Kinetic data of the four genotypes conformed to the sum of three Michaelis-Menten terms. Accordingly, three kinetic components could be distinguished. In the wild-type the approximate K_ms were 40 M, 1mM, and 40 mM, respectively. In Val^r-2 a component with a very low K_m (about 4 M) was found which may represent either the modified low-K_m component of the wild-type or a fourth component which is undetectable in the wild-type by kinetic analysis. The V_max of the low-K_m component in Val^r-2 was at least a 100-fold lower than in the wild-type. In the presence of one of the mutant genes the calculated V_max of the low-K_m component was 48% (strains h9 and h10) or 40% (strains h17 and h23) of the corresponding V_max in the wild-type. It is reasoned that the mutations have no effect on the activity of the other two kinetic components, though the evidence for this is circumstantial. Autoradiographs of leaf discs showed that in Val^r-2 the uptake of ¹⁴C-labelled valine in both mesophyll and minor veins was strongly reduced as compared with the wild-type.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DW dry weight - TPP⁺ tetraphenylphosphonium ion A preliminary account of part of this work has been presented (Borstlap 1986)     

Kinetic analyses of calcium movements in cell cultures. 3. Effects of calcium and parathyroid hormone in kidney cells

Calcium compartments and fluxes were measured by kinetic analyses in kidney cell suspensions in a three-compartment closed system. The fast phase influx and compartment size increase linearly with the medium calcium and the half-time of exchange is only 1.3 min which suggests that the fast component is extracellular. The slow phase compartment rises linearly from 0.1 to 0.5 mmole calcium/kg cell water when the medium calcium is raised from 0.02 to 2.5 mM. The slow phase calcium influx exhibits the pattern of saturation kinetics with a V _max of 0.065 µµmole cm^-2 sec^-1 and a K_m of 0.3 mM indicating that it is a carrier-mediated transport process. PTH has no effect on the fast phase of calcium influx, but increases both calcium influx and the calcium pool size of the slow component. The maximum effect is obtained at medium calcium concentration of 1.3 mM. Below 0.3 mM extracellular calcium, the effects of the hormone cannot be demonstrated. PTH increases the V _max of calcium influx from 0.065 to 0.128 µµmole cm^-2 sec^-1 while the K_m rises from 0.3 to 1.15 mM. These findings suggest that PTH increases the translocation of the calcium-carrier complex across the membrane and not the carrier concentration or its binding affinity for calcium.     

Enzymic Properties of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Purified from Rice Leaves

The enzymic properties of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase purified from rice (Oryza sativa L.) leaves were studied. Rice RuBPcarboxylase, activated by preincubation with CO₂ and Mg²⁺ like other higher plant carboxylases, had an activation equilibrium constant (K_cK_Mg) of 1.90 × 10⁵ to 2.41 × 10⁵ micromolar² (pH 8.2 and 25°C). Kinetic parameters of carboxylation and oxygenation catalyzed by the completely activated enzyme were examined at 25°C and the respective optimal pHs. The K_m(CO₂), K_m(RuBP), and V_max values for carboxylation were 8 micromolar, 31 micromolar, and 1.79 units milligram⁻¹, respectively. The K_m(O₂), K_m(RuBP), and V_max values for oxygenation were 370 micromolar, 29 micromolar, and 0.60 units milligram⁻¹, respectively.

Comparison of rice leaf RuBP carboxylase with other C₃ plant carboxylases showed that it had a relatively high affinity for CO₂ but the lowest catalytic turnover number (V_max) among the species examined.

     

Transport of glucose and galactose in kidney-cortex cells

1. The aerobic transport of d-glucose and d-galactose in rabbit kidney tissue at 25° was studied. 2. In slices forming glucose from added substrates an accumulation of glucose against its concentration gradient was found. The apparent ratio of intracellular ([S]_i) and extracellular ([S]_o) glucose concentrations was increased by 0·4mm-phlorrhizin and 0·3mm-ouabain. 3. Slices and isolated renal tubules actively accumulated glucose from the saline; the apparent [S]_i/[S]_o fell below 1·0 only at [S]_o higher than 0·5mm. 4. The rate of glucose oxidation by slices was characterized by the following parameters: K_m 1·16mm; V_max. 4·5μmoles/g. wet wt./hr. 5. The active accumulation of glucose from the saline was decreased by 0·1mm-2,4-dinitrophenol, 0·4mm-phlorrhizin and by the absence of external Na⁺. 6. The kinetic parameters of galactose entry into the cells were: K_m 1·5mm; V_max 10μmoles/g. wet wt./hr. 7. The efflux kinetics from slices indicated two intracellular compartments for d-galactose. The galactose efflux was greatly diminished at 0°, was inhibited by 0·4mm-phlorrhizin, but was insensitive to ouabain. 8. The following mechanism of glucose and galactose transport in renal tubular cells is suggested: (a) at the tubular membrane, these sugars are actively transported into the cells by a metabolically- and Na⁺-dependent phlorrhizin-sensitive mechanism; (b) at the basal cell membrane, these sugars are transported in accordance with their concentration gradient by a phlorrhizin-sensitive Na⁺-independent facilitated diffusion. The steady-state intracellular sugar concentration is determined by the kinetic parameters of active entry, passive outflow and intracellular utilization.     

Functional Importance of Arginine 64 in Chlamydomonas reinhardtii Phosphoribulokinase

Phosphoribulokinase (EC 2.7.1.19) was investigated in wild-type Chlamydomonas reinhardtii and in mutant strains deficient in this enzyme activity. Immunoblot analysis revealed substantial amounts of phosphoribulokinase in mutant 12-2B but none in mutant F-60. The pH optimum of the wild-type enzyme was 8.0 and that of the 12-2B enzyme was 6.5. The mutant kinase possessed a K_m value for ribulose 5-phosphate of about 45 millimolar, nearly three orders of magnitude greater than the wild-type value of 56 micromolar. K_m values for ATP in the range of 36 to 72 micromolar were observed with both wild-type and mutant enzymes. The V_max of the wild-type enzyme was about 450 micromoles per minute per milligram of protein, and values for the mutant enzyme were 140 micromoles per minute per milligram at pH 6.5 and 36 micromoles per minute per milligram at pH 7.8. Thermal stabilities of the wild-type and mutant kinases were similar. Sequence analysis of the 12-2B phosphoribulokinase gene revealed a C to T transition that caused an arginine to cysteine change at position 64 of the enzyme. This arginine residue is conserved in phosphoribulokinases from vascular plants, algae, and photosynthetic bacteria and appears to function in binding ribulose 5-phosphate.     

The raz1 mutant of Arabidopsis thaliana lacks the activity of a high-affinity amino acid transporter

The raz1 mutant of Arabidopsis thaliana (L.) Heynh. has been selected as resistant to the toxic proline analogue, azetidine-2-carboxylic acid (2AZ). Seedlings of the mutant tolerated fivefold higher concentrations of 2AZ (ED₅₀ = 0.25 mM) than the wild-type seedlings (ED⁵⁰ = 0.05 mM). The mutant gene was found to be semi-dominant and the corresponding RAZ1 locus was mapped on chromosome 5 at 69.6±1.8 cM. The resistance to 2AZ could be fully and exclusively accounted for by the lower uptake rate of the proline analogue in the mutant. The influx of L-proline in roots of wild-type seedlings could be dissected into two components: (i) a component with a high affinity and a low capacity for l-proline (K _m≈20 gmM, V _max≈60 nmol·(g FW)^-1·h^-1) and also a high affinity for L-2AZ (K _i≈40 μM) and (ii) a low-affinity, high-capacity component (K _m≈5 mM: V _max = 1300 nmol·(g FW)^-1·h^-1). Clearly, the raz1 mutation affects the activity of a high-affinity transporter, because the high-affinity uptake of proline in the mutant was at least fivefold lower than in the wild-type, whereas the low-affinity uptake was unchanged.     

Kinetics of phosphorus absorption by mycorrhizal and nonmycorrhizal tomato roots

Kinetics of P absorption were investigated in mycorrhizal (Glomus fasciculatus) and nonmycorrhizal tomato (Lycopersicon esculentum) roots to determine why increased ion absorption by mycorrhizae occurs. Initial rates of absorption of ³²P were measured at 1 to 100 micromolar KH₂PO₄ (pH 4.6). Absorption rates of mycorrhizae were about twice those of control roots. Augustinsson-Hofstee analysis yielded two linear phases; V_max and K_m were calculated for each phase. In the low phase (1 to 20 micromolar), V_max values for the mycorrhizal and nonmycorrhizal roots were each 0.10 micromoles P per gram fresh weight per hour while K_m values were 1.6 and 3.9 micromolar KH₂PO₄, respectively. For the high phase (30 to 100 micromolar), V_max values for mycorrhizal and nonmycorrhizal roots were 0.32 and 0.25 micromoles P per gram fresh weight per hour and K_m values were 35 and 42 micromolar, respectively. These results indicate that at the lower phase concentrations, similar to those expected in most soil solutions, a major factor contributing to the increased uptake was an apparent greater affinity of the absorbing sites for H₂PO₄⁻ (lower K_m).     

Carrier-Mediated Uptake of Abscisic Acid by Suspension-Cultured Amaranthus tricolor Cells

Abscisic acid (ABA) uptake by Amaranthus tricolor cell suspensions was found to include both a nonsaturable component and a saturable part with K_m of 3.74 ± 0.43 micromolar and an apparent V_max of 1.5 ± 0.12 nanomoles per gram per minute. These kinetic parameters as well as the uptake by intact cells at 0°C or by frozen and thawed cells, are consistent with operation of a saturable carrier. This carrier-mediated ABA uptake was partially energized by ΔpH: it increased as the external pH was lowered to pH 4.0; it decreased after the lowering of the ΔpH by the proton ionophore carbonylcyanide-m-chlorophenylhydrazone or after the altering of metabolically maintained pH gradient by metabolic inhibitors (KCN, oligomycin). The carrier is specific for ABA among the plant growth regulators tested, is unaffected by (RS)-trans-ABA and was inhibited by (S)-ABA, (R)-ABA, and also by the ABA analog LAB 173711.     

Thymidine transport by cultured Novikoff hepatoma cells and uptake by simple diffusion and relationship to incorporation into deoxyribonucleic acid

The initial rate of thymidine-³H incorporation into the acid-soluble pool by cultured Novikoff rat hepatoma cells was investigated as a function of the thymidine concentration in the medium. Below, but not above 2 µM, thymidine incorporation followed normal Michaelis-Menten kinetics at 22°, 27°, 32°, and 37°C with an apparent K_m of 0.5 µM, and the V_max values increased with an average Q₁₀ of 1.8 with an increase in temperature. The intracellular acid-soluble ³H was associated solely with thymine nucleotides (mainly deoxythymidine triphosphate [dTTP]). Between 2 and 200 µM, on the other hand, the initial rate of thymidine incorporation increased linearly with an increase in thymidine concentration in the medium and was about the same at all four temperatures. Pretreatment of the cells with 40 or 100 µM p-chloromercuribenzoate for 15 min or heat-shock (49.5°C, 5 min) markedly reduced the saturable component of uptake without affecting the unsaturable component or the phosphorylation of thymidine. The effect of p-chloromercuribenzoate was readily reversed by incubating the cells in the presence of dithiothreitol. Persantin and uridine competitively inhibited thymidine incorporation into the acid-soluble pool without inhibiting thymidine phosphorylation. At concentrations below 2 µM, thymidine incorporation into DNA also followed normal Michaelis-Menten kinetics and was inhibited in an apparently competitive manner by Persantin and uridine. The apparent K_m and K_i values were about the same as those for thymidine incorporation into the nucleotide pool. The over-all results indicate that uptake is the rate-limiting step in the incorporation of thymidine into the nucleotide pool as well as into DNA. The cells possess an excess of thymidine kinase, and thymidine is phosphorylated as rapidly as it enters the cells and is thereby trapped. At low concentrations, thymidine is taken up mainly by a transport reaction, whereas at concentrations above 2 µM simple diffusion becomes the principal mode of uptake. Evidence is presented that indicates that uridine and thymidine are transported by different systems. Upon inhibition of DNA synthesis, net thymidine incorporation into the acid-soluble pool ceased rapidly. Results from pulse-chase experiments indicate that a rapid turnover of dTTP to thymidine may be involved in limiting the level of thymine nucleotides in the cell.     

Regulation of sulfate transport in filamentous fungi

Inorganic sulfate enters the mycelia of Aspergillus nidulans, Penicillium chrysogenum, and Penicillium notatum by a temperature-, energy-, pH-, ionic strength-, and concentration-dependent transport system (“permease”). Transport is unidirectional. In the presence of excess external sulfate, ATP sulfurylase-negative mutants will accumulate inorganic sulfate intracellularly to a level of about 0.04 m. The intracellular sulfate can be retained against a concentration gradient. Retention is not energy-dependent, nor is there any exchange between intracellular (accumulated) and extracellular sulfate. The sulfate permease is under metabolic control. Sulfur starvation of high methionine-grown mycelia results in about a 1000-fold increase in the specific sulfate transport activity at low external sulfate concentrations. l-Methionine is a metabolic repressor of the sulfate permease, while intracellular sulfate and possibly l-cysteine (or a derivative of l-cysteine) are feedback inhibitors. Sulfate transport follows hyperbolic saturation kinetics with a Michaelis constant (Km) value of 6 × 10⁻⁵ to 10⁻⁴m and a V_max (for maximally sulfurstarved mycelia) of about 5 micromoles per gram per minute. Refeeding sulfur-starved mycelia with sulfate or cysteine results in about a 10-fold decrease in the V_max value with no marked change in the Km. Azide and dinitrophenol also reduce the V_max.     

Basic Properties of Rotary Dynamics of the Molecular Motor Enterococcus hirae V1-ATPase

V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V₁ moiety of Enterococcus hirae V-ATPase (EhV₁) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV₁ using single-molecule analysis employing a load-free probe. EhV₁ rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (V_max) of 107 revolutions/s and a Michaelis constant (K_m) of 154 μm at 26 °C. At all ATP concentrations tested, EhV₁ showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V₁-ATPase (TtV₁). At 10 μm ATP (⪡K_m), the distribution of the durations of the ATP-waiting pause fit well with a single-exponential decay function. The second-order binding rate constant for ATP was 2.3 × 10⁶ m⁻¹ s⁻¹. At 40 mm ATP (⪢K_m), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV₁. Our results identify the common properties of rotary catalysis of V₁-ATPases that are distinct from those of F₁-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.     

Evidence that the uptake of tri-iodo-l-thyronine by human erythrocytes is carrier-mediated but not energy-dependent

We investigated 3,3′,5-tri-iodo-l-thyronine transport by human erythrocytes and by `ghosts'' prepared from these cells. Uptake of tri-iodothyronine by erythrocytes at 37°C was time-dependent with a maximum reached after 60min. Tracer analysis after incubation for 1min revealed only one saturable binding site, with K<sub>m</sub> 128±19nm (mean±s.e.m.; n=7) and V<sub>max.</sub> 4.6±0.7pmol of tri-iodothyronine/min per 6×10<sup>7</sup> cells. After 10min incubation K<sub>m</sub> 100±16nm (n=10) was found with V<sub>max.</sub> 7.7±1.2pmol of tri-iodothyronine/10min per 6×10<sup>7</sup> cells. At 0°C the uptake system is still active, with K<sub>m</sub> 132±26nm and V<sub>max.</sub> 1.8±0.3pmol of tri-iodothyronine/10min per 6×10<sup>7</sup> cells. The V<sub>max.</sub> with intact cells is 5-fold greater than the V<sub>max.</sub> with membranes derived from the same amount of cells when uptake studies are performed in media with similar free tri-iodothyronine concentrations. This indicates that at least 80% of tri-iodothyronine taken up by the intact erythrocytes enters the cell. This saturable uptake system can be inhibited by X-ray-contrast agents in a dose-dependent fashion. (±)-Propranolol, but not atenolol, has the same effect, indicating that the membrane-stabilizing properties of (±)-propranolol are involved. Furthermore, there is no inhibition by ouabain or vanadate, which indicates that tri-iodothyronine uptake is not dependent on the activity of Na<sup>+</sup>+K<sup>+</sup>-dependent adenosine triphosphatase. We have prepared erythrocyte `ghosts'', resealed after 2.5min with 0mm-, 2mm- or 4mm-ATP inside. Inclusion of ATP and integrity of the membrane of the erythrocyte `ghosts'' were verified on the basis of an ATP-concentration-dependent functioning of the Ca<sup>2+</sup> pump. No difference was found in the uptake of tri-iodothyronine by erythrocyte `ghosts'' with or without ATP included, indicating that uptake of tri-iodothyronine is not ATP-dependent. The following conclusions are drawn. (1) Tri-iodothyronine enters human erythrocytes. (2) There is only one saturable uptake system present for tri-iodothyronine, which is neither energy (i.e. ATP)-dependent nor influenced by the absence of an Na⁺ gradient across the plasma membrane. This mode of uptake of tri-iodothyronine by human erythrocytes is in sharp contrast with that of rat hepatocytes, which uptake system is energy-dependent and ouabain-sensitive [Krenning, Docter, Bernard, Visser & Hennemann (1978) FEBS Lett. 91, 113–116; Krenning, Docter, Bernard, Visser & Hennemann (1980) FEBS Lett. 119, 279–282]. (3) X-ray-contrast agents inhibit tri-iodothyronine uptake by erythrocytes in a similar fashion to that by which they inhibit the uptake of tri-iodothyronine by rat hepatocytes [Krenning, Docter, Bernard, Visser & Hennemann (1982) FEBS Lett. 140, 229–233].     

Rubidium transport in Neurospora crassa

Rb⁺ transport in low-K⁺ cells of Neurospora crassa is biphasic, transport at millimolar Rb⁺ being added to a transport process which saturates in the micromolar range. Both processes exhibit Michaelis-Menten kinetics, but in the micromolar phase the kinetic parameters depend on the K⁺ content of the cell (the lower the K⁺ content the lower the K_m and the higher the V_max). Normal-K⁺ cells, suspended in a buffer with millimolar K⁺, do not present Rb⁺ transport in the micromolar range. Millimolar transport in these cells presents kinetics which depend on the K⁺ in buffer (the higher the K⁺ the higher the K_m), although the K⁺ content of the cells is constant. Na⁺ inhibits competitively Rb⁺ transport in low-K⁺ and normal-K⁺ cells, but, even when the differences between the Rb⁺K_m values are more than three orders of magnitude, the apparent dissociation constant for Na⁺ is the same, and millimolar, in both cases.     

Identification,Purification, and Characterization of a Novel Amino Acid Racemase,Isoleucine 2-Epimerase,from Lactobacillus Species

Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no γ-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The K_m and V_max values of the enzyme for l-isoleucine were 5.00 mM and 153 μmol·min⁻¹·mg⁻¹, respectively, and those for d-allo-isoleucine were 13.2 mM and 286 μmol·min⁻¹·mg⁻¹, respectively. Hydroxylamine and other inhibitors of pyridoxal 5′-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5′-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.     

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k_cat/K_m = 1000 ± 100 m⁻¹ s⁻¹ versus 700 ± 100 m⁻¹ s⁻¹). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k_cat/K_m = 80 ± 40 m⁻¹ s⁻¹). In the presence of 3-HSA the K_mapp for O₂ was 100 ± 10 μm. The crystal structure of HsaA to 2.5-Å resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme''s substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val³⁶⁷–Val³⁹⁴) could adopt two conformations differing by a rigid body rotation of 25° around Arg³⁶⁶. This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme''s substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids.     

Functional Characterization of Key Enzymes involved in l-Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter⁻¹ of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low K_m value for ammonium (10 mM) and a high K_m value for l-glutamate (250 mM), and the V_max value is 7-fold higher for l-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar K_m values (1 to 1.4 mM) and V_max values (4 U/mg) for both l-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating l-glutamate production in this organism.     

Potassium transport in Chlamydomonas reinhardtii: isolation and characterization of transport-deficient mutant strains

Putative potassium-transport-deficient mutant strains of Chlamydomonas reinhardtii Dang. were induced by ultra-violet mutagenesis and were identified by their dependence on abnormally high concentrations of potassium for growth. Potassium transport studies employing ⁸⁶Rb as a tracer were carried out with wild-type cells and with three independently isolated KDP (potassium-dependent phenotype) clones. Wildtype cells exhibit two transport activities. Transport activity A was expressed when cells were grown in medium supplemented with 10 mM KCl. The transporter with type-A activity does not discriminate between either Rb⁺ or K⁺ as a substrate and has a K_m for Rb⁺ equal to 1 mM and a V_max equal to 31 nmol Rb⁺ h^-1 10^-6 cells. Transport activity B was expressed when cells were starved of potassium for 24 h. The transporter with type-B activity prefers K⁺ to Rb⁺ as a substrate; it has a K_m for Rb⁺ equal to 2.5 mM and a V_max equal to 210 nmol Rb⁺ h^-1 10^-6 cells. All three mutant clones exhibit transport activity comparable to type-A when grown in 10 mM KCl. When starved of potassium for 24 h, two KDP clones demonstrate no transport activity and the third clone continues to exhibit only type-A activity.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DES diethylstilbesterol - KDP potassium-dependent phenotype     

Metal Ion Interactions with Phosphoenolpyruvate Carboxylase from Crassula argentea and Zea mays

Metal ion interactions with phosphoenolpyruvate carboxylase from the CAM plant Crassula argentea and the C₄ plant Zea mays were kinetically analyzed. Fe²⁺ and Cd²⁺ were found to be active metal cofactors along with the previously known active metals Mg²⁺, Mn²⁺, and Co²⁺. In studies with the Crassula enzyme, Mg²⁺ yielded the highest V_max value but also generated the highest values of K_m(metal) and K_m(pep). For these five active metals lower K_m(metal) values tended to be associated with lower K_m(pep) values. PEP saturation curves showed more kinetic cooperativity than the corresponding metal saturation curves. The activating metal ions all have ionic radii in the range of 0.86 to 1.09 Å. Ca²⁺, Sr²⁺, Ba²⁺, and Ni²⁺ inhibited competitively with respect to Mg²⁺, whereas Be²⁺, Cu²⁺, Zn²⁺, and Pd²⁺ showed mixed-type inhibition. V_max trends with the five active metals were similar for the C. argentea and Z. mays enzymes except that Cd²⁺ was less effective with the maize enzyme. K_m(metal) values were 10- to 60-fold higher in the enzyme from Z. mays.     

Ionic interconversion of pacemaker and nonpacemaker cultured chick heart cells

Trypsin-dispersed cells from hearts (ventricles) of 7 to 8 day chick embryos were cultured 3 to 21 days. The cells became attached to the culture dish and assembled into monolayer communities. By means of a bridge circuit, one microelectrode was used for simultaneously passing current and recording membrane potentials (V_m). The input resistance, calculated by the measured ΔV_m for a known step of current, averaged 10 MΩ. Electrotonic depolarization of nonpacemaker cells had no effect on frequency of firing. Within 2 min after addition of Ba⁺⁺ (5 to 10 mM) to the Tyrode bath, the cells became partially depolarized and quiescent nonpacemaker cells developed oscillations in V_m which led to action potentials. With time, the depolarization became nearly complete and the input resistance increased 2 to 10 times. During such sustained depolarizations, action potentials were no longer produced and often tiny oscillations were observed; however, large action potentials developed during hyperpolarizing pulses. Thus, the automaticity of the depolarized cell became apparent during artificial repolarization. Sr⁺⁺ (5 to 10 mM) initially produced hyperpolarization and induced automaticity in quiescent nonpacemaker cells. Elevated [K⁺]_o (20 to 30 mM) suppressed automaticity of pacemaker cells and decreased R_m concomitantly. Thus, Ba⁺⁺ probably converts nonpacemaker cells into pacemaker cells independently of its depolarizing action. Ba⁺⁺ may induce automaticity and depolarization by decreasing g _K, and elevated [K⁺]_o may depress automaticity by increasing g _K. The data support the hypothesis that the level of g _K determines whether a cell shall function as a pacemaker.     

Biosynthesis of Arabinogalactan-Protein in Lolium multiflorum (Ryegrass) Endosperm Cells : III. Subcellular Distribution of Prolyl Hydroxylase

The peptidyl prolyl hydroxylase responsible for the formation of hydroxyproline during arabinogalactan-protein biosynthesis in Lolium multiflorum (ryegrass) endosperm cells is a membrane-associated enzyme which will catalyze the hydroxylation of poly(l-proline) in the presence of oxygen, α-ketoglutarate, ferrous ion, and ascorbate. The K_m for poly(l-proline) (8000 molecular weight) is 40 micromolar. The enzyme will also hydroxylate the protocollagen analog (Pro-Pro-Gly)₅·4H₂O.