Transport and Metabolism of Vitamin B6 in the Yeast Saccharomyces carlsbergensis 4228*

Active transport of pyridoxine, pyridoxal, and pyridoxamine occurs in resting cells of Saccharomyces cdsbergensis 4228 and can lead to intracellular concentrations of free vitamin much higher than those supplied externally. The initial K, for pyridoxine uptake is 3.6 x lo-’ M at 30” and pH 4.5, which are optimum for growth. Transport is inhibited by many unphosphorylated vitamin analogs, the most effective being 5’-deoxypyridoxine, 5’.deoxypyridoxal, toxopyrimidine, 4’.deoxypyridoxine, and 3-amino-3-deoxypyridoxine. Two distinct uptake systems that differ in structural specificity and ionic requirements are present. One, with optimum pH of 3.5, transports pyridoxal effectively, but not pyridoxamine; the other (optimum pH, 6.0) transports pyridoxamine effectively, but not pyridoxal. Both systems transport pyridoxine, while neither transports pyridoxal5’-phosphate. Other properties of these systems are similar, indicating that they share certain elements in common. An initial temperature optimum of 30” is observed for pyridoxine transport and, at this temperature, an “overshoot” in intracellular vitamin levels, with subsequent decrease to a constant level, occurs with time. It appears

Active transport of pyridoxine, pyridoxal, and pyridoxamine occurs in resting cells of Saccharomyces cdsbergensis 4228 and can lead to intracellular concentrations of free vitamin much higher than those supplied externally. The initial K, for pyridoxine uptake is 3.6 x lo-' M at 30" and pH 4.5, which are optimum for growth. Transport is inhibited by many unphosphorylated vitamin analogs, the most effective being 5'-deoxypyridoxine, 5'.deoxypyridoxal, toxopyrimidine, 4'.deoxypyridoxine, and 3amino-3-deoxypyridoxine.
Two distinct uptake systems that differ in structural specificity and ionic requirements are present. One, with optimum pH of 3.5, transports pyridoxal effectively, but not pyridoxamine; the other (optimum pH, 6.0) transports pyridoxamine effectively, but not pyridoxal. Both systems transport pyridoxine, while neither transports pyridoxal5'-phosphate. Other properties of these systems are similar, indicating that they share certain elements in common. An initial temperature optimum of 30" is observed for pyridoxine transport and, at this temperature, an "overshoot" in intracellular vitamin levels, with subsequent decrease to a constant level, occurs with time. It appears that intracellular vitamin, or a derivative, activates the exit mechanism for the vitamin. Exit rates also depend on the resuspension buffer and are increased in the presence of glucose and decreased by azide. Above 30" net uptake of pyridoxine drops initially, then rapidly increases to a second optimum at 50"; the uptake system is inactivated at about 55". The optimum at 50" apparently results from activation of inflow as exit is rapid and is accelerated by azide. No overshoot was detected at 50", so it appears that the exit system is not regulated by intracellular vitamin at this temperature. A phase transition in membrane lipids occurs at 30" and may be responsible for the change in properties of the inflow and exit mechanisms above this temperature.
Studies of vitamin B6 transport are complicated by the occurrence of this vitamin in several phosphorylated and nonphosphorylated forms. The process has been studied only to a small extent, mainly with pyridoxine as the vitamin source and in mammalian tissues (for a brief review, see Ref. 1). Concentration of the transported vitamin occurs in red blood cells, but the mechanism of uptake is not well understood (2-4).
Concentration also occurs in blood platelets (5) and Ehrlich tumor cells (6), but was ascribed to metabolism or intracellular binding of the vitamin.
Various microorganisms are auxotrophic for vitamin B6 and sometimes use only one or two of the individual forms of this vitamin (7,8). In some cases this nutritional specificity may result from inability of the organism to convert unphosphorylated forms of the vitamin to the active coenzymes, pyridoxal-P and pyridoxamine-P (9); in other instances specificity has been postulated to lie in the transport process (10,11). In no case have these various possibilities been thoroughly studied. which is, at least in part, due to contamination with pyridoxal or pyridoxamine (12). In the absence of thiamin, the organism grows well without externally added vitamin B6 (13).
We confirmed these relationships for the present culture, and have used it to study entry and exit mechanisms for vitamin B6 and the possible role of metabolism in uptake in this vitamin by nonproliferating cells.
Transport and Metabolism of Vitamin B6 modifications of procedures (16,17)  To determine the forms of the vitamin that accumulated, the filtered and washed cells were boiled with small amounts of water (approximately 0.2 mlimg of cells) for 10 min. Individual vitamin forms in the extract or the medium were then separated by successive column chromatography on acidic (Whatman Pll) and basic (Whatman DE52) cellulose (15). Measurement of Release of Vitamin B6 from Cells-Cells which had been allowed to accumulate labeled vitamin for various times ("loaded" cells) as described in the previous section were filtered, washed with water, resuspended in various buffer solutions at pH 4.5, and mcubated at 30" with shaking. At various times, aliquots were removed and filtered and the cell-free filtrate (0.9 ml) was added to counting vials to determine the amount of vitamm released.

Demonstration and General Characteristics of Pyridoxine Transport
["ClPyridoxine was rapidly accumulated by resting cells in Salts M + 1% Glc medium (Fig. 1A). Assuming that fresh cells contain 2.1 ml of free intracellular water/g of dry solids (21), peak intracellular concentrations of pyridoxine were from 50to 3000-fold higher than those supplied in the medium, depending on the initial substrate concentration. A marked "overshoot" in transported vitamin was seen with higher substrate concentrations.
Separate trials showed that uptake was proportional to cell concentration over the range tested (0.05 to 5.0 mg dry weight/ml) except when more than 50% of the vitamin in the medium had been transported.
Washed cells stored at O-4" in water or growth medium (lacking pyridoxine) retained their full uptake capacity for at least 5 hours; at 20" under these same conditions, or in Salts M in Salts M + 1% Glc was varied. Uptake was measured at 2.5 min (Curve 1), at the peak of net uptake (5 to 10 min, &rue 2), and at 60 min (steady state, &rue 3) after addition of the label.
Preincubation of cells in Salts M + 1% Glc for 30 to 66 min was necessary before maximal initial rates of uptake could be demonstrated (Fig. 1B). The steady state level, taken to be the level reached after 60 min uptake, was independent of preincubation time. Overshoot was reduced with shorter preincubation times and was virtually undetectable in the absence of preincubation.

Forms of Vitamin B6 Accumulated during Uptake Period
To determine to what extent transported ["Clpyridoxine was metabolized by the cells, boiled extracts were prepared after various uptake times and analyzed for individual forms of vitamin B6. At each time period analyzed, over 98% of the total cell label was extracted by boiling. The results (Table I) showed that maximal accumulation of vitamin occurred in the first 10 min, during which time pyridoxine accounted for more than 90% of the intracellular vitamin. By 2 hours most of the pyridoxine had been metabolized, mainly to pyridoxamine-P and pyridoxal-P, with nonphosphorylated vitamin (about equal amounts of pyridoxine, pyridoxal, and pyridoxamine) accounting for about 20% of the total cell label. The overshoot in total intracellular vitamin levels noted in Fig. lA therefore masked an even more pronounced overshoot in intracellular pyridoxine levels. Between 10 and 60 min, outflow of vitamin resulting from the overshoot phenomenon exceeds uptake, as shown by the increase in total vitamin in the medium (  (22)(23)(24) and yeast (25) and pyridoxine (10 rig/ml).
The uptake system(s) therefore appear to be constitutive in these cells.    3.5 to 4.0) is most effective for pyridoxal, whereas the high pH system (pH 5.5 to 6.5) is most effective for pyridoxamine. The effects of pH on the affinity of these and related compounds for the uptake systems are shown in Table  IV. Results at pH 4.5, which lies between the two optima for pyridoxine uptake but is the pH optimum for growth, are also included. Each of the compounds tested showed slightly lower affinity for the transport system in citrate-phosphate buffer at pH 4.5 (Table  IV) than in Salts M + 1% Glc solution at this same pH (cf. in the pH range studied (30). All analogs inhibited ["Clpyridoxine uptake competitively and had lower affinities at pH 7, as judged by K, values, than at pH 6. Pyridoxal-P, pyridoxine-P, and pyridoxamine-P had no affinity (K, > 100 FM) at any pH tested.
An overshoot in transported ["Clpyridoxine similar to that already noted at pH 4.5 (Fig. 1A) also was observed at pH 3.5 and 6.0.

Effect of Other Ions on Pyridoxine Uptake
Certain buffer effects led us to examine the effect of various ions on transport, even though no attempts were made to obtain ion-deficient media or cells. Since the rate of transport  Cells (1 mg/ml) were incubated at 30" for 30 min in 12.5 mM K,HPO,-12.5 mM K,-citrate-HCl buffer (+ 1% glucose), at the indicated pH values, before the addition of various levels of the compounds tested. Uptake was measured with 'C-or SH-labeled compounds after 1 and 2 min to determine K, and V,,,,. values.
K, values were determined by measuring uptake of ['Clpyridoxine (0.51 FM) in the presence of unlabeled analogs (5 or 50 GM). glucose (pH 4.5) was the same, the latter buffer was used as a base-line to evaluate these effects. At pH 3.5, 10 or 50 mM K+-activated transport 4-fold; 10 mM Na+ was without effect; while 50 mM Na+ stimulated Z-fold. At pH 4.5, 50 mM Li+, Na+, or K+ all increased the rate of transport 2-fold; additional NH,+ was without effect, while 50 mM triethanolamine inhibited by 50%. Uptake at pH 6 was stimulated maximally by 25 mM K+ (1.5-fold) but Na+ had no apparent effect over the range 10 to 50 mM. Di-and trivalent cations (Ca2+, Mg2+, Mn2+, and FeS+) and also phosphate, citrate, and Cl-were without apparent effect, while acetate (50 mM) inhibited uptake by 80%.
Effect of Temperature on Vitamin B6 Uptake Pyridoxine uptake showed an unexpected relationship to temperature (Fig. 3). Between 1 and 40", typical results for a transport system were seen: transport first increased from negligible values to an initial rate optimum near 35" and a steady state optimum of 30" (the optimum growth temperature), then decreased as the temperature was raised to 40". Above 40", however, a rapid rise in pyridoxine uptake occurred with an apparent optimum at 50", before inactivation occurred near 60". The Qr,, value between 20 and 30" and between 40 and 50" was 3.3 to 3.4, indicating an active process in each case. A phase transition in membrane lipids, occurring just above 30" (Fig. 4) may be related to the initial decrease in uptake rate. Some properties of the 50" system were examined briefly with results as follows: (a) the dual temperature optimum shown in Fig. 3 for uptake at pH 4.5 was also present at pH 3.5 and pH 6.0; (b) the affinities for pyridoxine, pyridoxamine, pyridoxal, and 5'-deoxypyridoxine at 50" were about one-half those shown in Table III (31)) and scans were performed at the specified temperatures on a Varian V4500 X-band EPR spectrometer equipped with a temperature control to maintain temperature to better than +0.5". Cells were grown on 5% glucose so very little mitochondrial membrane would have been present (32).
glucose varied from about 18-to loo-fold, depending upon the conditions; (d) preincubation of cells at 50" for 30 min without glucose virtually destroyed their uptake capacity; (e) only a very slight overshoot in transported pyridoxine levels was seen at 50" even with pyridoxine levels as high as 2.2 FM. This behavior contrasts markedly with the pronounced overshoot observed at 30" at only 0.8 pM substrate (Fig. lA).

Nature of Overshoot Phenomenon
The level of accumulated vitamin should represent a dynamic equilibrium determined by the inflow and exit rates at any given time. The overshoot in net uptake observed in Fig. 1 could be a result of a change in this equilibrium or, more trivially, could be caused by counterflow of external labeled vitamin with endogenous unlabeled vitamin. The latter possibility can be eliminated as little or no overshoot was observed when low levels of ["Clpyridoxine were supplied in the uptake medium (Fig. lA), when cells were not preincubated in the uptake medium (Fig. 1B) or when cells were grown on high levels of unlabeled pyridoxine HCl (100 rig/ml). In addition, if cells were equilibrated with high levels of pyridoxine (250 NM) or its analogs in the presence of azide (30 mM) and iodoacetate (1 mM), and resuspended in buffer containing [aH]pyridoxine (0.5 FM) plus metabolic inhibitors, no overshoot in intracellular labeled vitamin was observed, only an equilibration with that in the medium.
If the intracellular level of pyridoxine represents a dynamic equilibrium, small amounts of labeled vitamin should equilibrate with larger amounts of unlabeled intracellular vitamin. Data of Fig. 5 show this to be so. The initial rate of uptake of this equilibrating vitamin represents the actual inflow rate and, although rapid, appears to be unaffected by prior incubation with 0.2 or 2 PM unlabeled vitamin for periods up to 60 min, even when overshoot is in progress. When cells which had undergone overshoot and had achieved a steady state level of intracellular vitamin (e.g. those of Curve 3, Fig. 1B) were washed exhaustively and resuspended in uptake mixtures containing the same level of ["Clpyridoxine, only slight uptake sufficient to replace the small amount of vitamin lost by washing was observed; no re-overshoot occurred showing that accumulation of intracellular vitamin (or a product formed only in the presence of such high levels) was responsible for the activation of the exit mechanism.
To examine this conclusion further, the effects of permitting cells to accumulate ["Clpyridoxine from comparatively low external concentrations (0.04 or 0.2 FM) for various times on their subsequent behavior toward a high external concentration (1.6 1~) of the labeled vitamin were determined (Fig. 6). Incubation with 0.04 PM vitamin did not result in overshoot and did not affect the rate of inflow when 1.56 pM vitamin was added. In fact, addition of 1.56 ELM vitamin after 60 min  Initial exit rates were increased in the presence of glucose (Table  VI) and by addition of unlabeled analogs.
It is unlikely that this effect was due simply to inhibition of reuptake of released labeled vitamin as 5'.deoxypyridoxine, the most effective analog inhibitor of pyridoxine uptake, did not cause as large an increase in exit rate as did pyridoxine and pyridoxal (Table  VI). It is possible that excess external vitamin activated the exit mechanism but more probable that the vitamin was transported into the cell before this activation occurred. Exit rates were increased at 50" and were accelerated in the absence of glucose and in the presence of azide (Table VI).

Interconversion
and Efflur of Pyridoxine Metabolites-It was shown in Table  I  After 5 min, the cells were washed with water and an aliquot counted to determine total intracellular vitamin.
The remaining cells were resuspended (1 mg/ml) in the indicated media and release of label was followed with time. After 5 ruin, the loaded cells were washed with water and resuspended (1 mg/ml) in the indicated buffers containing unlabeled analogs (25 pM) or azide (30 ITIM PLP, pyridoxal-P; PNP, pyridoxine-P; PMP, pyridoxamine-P. ["Clpyridoxamine. One effect of these analogs would be to inhibit reuptake of released ["Clpyridoxine, but since 5'-deoxypyridoxine is the most effective analog inhibitor of uptake (cf.  (38)(39)(40).
The vitamin was taken up and retained against a concentration gradient and the transport process was dependent on energy, pH, temperature, and the ionic environment, and also displayed structural specificity and saturation kinetics. Uptake of pyridoxal, pyridoxamine, 5'.deoxypyridoxine, and 5'.deoxypyridoxal also displayed the properties of an active process in those characters measured. The K, of 3.6 x 10e7 M for uptake of pyridoxine at pH 4.5 is in the range reported for active transport systems acting on biotin (41) and thiamine (42)  Pyridoxine or a stable derivative of this vitamin appeared responsible for this activation in exit rate and the effect was time-dependent.
Since 5'-deoxypyridoxine and 5'.deoxypyridoxal both exhibited a similar overshoot in transport, 5'.phosphorylation of the vitamin apparently is not required either for this activation or for transport. p-Galactoside exit in E coli is accelerated in the absence of an energy source by a lowering of the exit K, (22,44), and high intracellular levels of pyridoxine may act similarly by lowering the exit K, for pyridoxine in S. carlsbergensis. The facts (a) that the cooperative effect on exit rate evoked by intracellular pyridoxine was manifested at about the same concentration that re-overshoot was inhibited, (b) that the apparent K, for inflow of pyridoxine decreases as the intracellular pyridoxine concentration increases (Table  II) (Fig. 1A). Pyridoxal-P does not effectively replace pyridoxal as a growth factor for yeast (10-12) or several bacteria (12), and because of this it is sometimes assumed that vitamin B6 phosphates do not in general cross cell membranes. However, pyridoxamine-P is an essential growth factor for some bacteria (8), and pyridoxal-P has been shown to cross the red blood cell membrane without hydrolysis (3). Similarly, many bacteria excrete vitamin B6 into their growth medium, mainly in the form of pyridoxal-P and sometimes pyridoxamine-P (46-48). Evidence has also been presented for the transport of pyridoxal-P or pyridoxine-P across placental membranes (16,49) and possibly the blood-brain barrier in mammals (16,50,51