Binding and Transport of Thiamine by Lactobacillus casei

Transcription

1 JOURNAL OF BACTRIOLOGY, Mar. 1978, P /78/ $2./ Copyright 1978 Amerian Soiety for Mirobiology Vol. 133, No. 3 Printed in U.S.A. Binding and Transport of Thiamine by Latobaillus asei GARY B. HNDRSON* AND DWARD M. ZVLY Department of Biohemistry, Sripps Clini and Researh Foundation, La Jolla, California 9237 Reeived for publiation 6 Otober 1977 The relationship between thiamine transport and a membrane-assoiated thiamine-binding ativity has been investigated in Latobaillus asei. Thiamine transport proeeds via a system whose general properties are typial of ative uptake proesses; entry of the vitamin into the ells requires energy, is temperature dependent, exhibits saturation kinetis, and is inhibited by substrate analogs. A onsiderable onentration gradient of unhanged thiamine an be ahieved by the system, although the vitamin is slowly metabolized to thiamine pyrophosphate. Consistent with these results, L. asei also ontains a high-affinity, thiamine-binding omponent whih ould be measured by inubation of intat ells with labeled substrate at 4 C (onditions under whih transport is negligible). Binding was insensitive to iodoaetate, ourred at a level (.5 nmol per 11 ells) nearly 2-fold higher than ould be aounted for by failitated diffusion, and was found to reside in a omponent of the ell membrane. Partiipation of this binder in thiamine transport is supported by the observations that the proesses of binding and transport showed similarities in their (i) regulation by the onentration of thiamine in the growth medium, (ii) binding affinities for thiamine, and (iii) suseptibility to inhibition by thiamine analogs. Cells of Latobaillus asei ontain a highly effiient transport system for the aumulation of the vitamin foli aid and various folate ompounds (1, 4, 8, 19, 2). In onjuntion with this transport apaity, the ells also have the ability to bind folate. The omponent responsible for this binding ould be deteted as uptake of the vitamin at 4C and was shown to reside in the ell membrane (5, 6). When membrane preparations were treated with the nonioni detergent Triton X-1 in the presene of labeled folate, the bound folate was released from the partiulate fration and was reovered stoihiometrially in a water-soluble form (5, 6). The extrated binding omponent was then purified to homogeneity (6, 7) and shown to be an unusually hydrophobi protein (Mr 25,) that ould bind large amounts of Triton X-1 (8.5 g/g of protein). The partiipation of this purified binding protein in the folate transport system of intat ells has been established by several riteria (5-8). In a preliminary ommuniation (G. B. Henderson and. M. Zevely, Fed. Pro. 35:1357, 1976), we demonstrated that L. asei ells have the ability to transport several other B-vitamins, inluding thiamine. When examined in more detail, the thiamine transport system of L. asei was found to be similar in many respets to the thiamine uptake systems in both L. fermentii 119 (14-17) and sherihia oli (9, 1, 18). L. asei also ontains onsiderable amounts of a thiamine-binding ativity whih an be deteted by the same proedure (5) employed to measure folate binding by these ells. The general properties of this thiamine binder and its relationship to thiamine transport are desribed in the present ommuniation. MATRIALS AND MTHODS Radiolabeled thiamine. [thiazole-2- '4C]thiamine hydrohloride (14 mci/mmol) and [3S]thiamine hydrohloride (189 mci/mmol) were purhased from Amersham/Searle. Radioativity was measured with the use of a dioxane-based sintillation fluid (4). Growth of ells. L. asei subsp. rhamnosis (ATCC 7469) was grown from a 1% inoulum for 24 h at 3 C in the basi medium desribed by Flynn et al. (2) exept that the presribed amount of thiamine was omitted and the folate onentration was inreased to 5,uM. Vitamin-free asein hydrolysate (Nutritional Biohemials) served as the soure of aid-hydrolyzed asein. The yield of ells grown under these onditions was 5 to 7 g/liter. Thiamine binding. The binding of thiamine by L. asei was measured by the same type of proedure as desribed previously for folate (5). Assay mixtures onsisted of.8 ml of a washed ell suspension (19 ells per ml in.5 M potassium phosphate, ph 6.8),.1 ml of [14C]thiamine (unless otherwise stated), and the indiated additions in a final volume of 1. ml. After inubation for 5 min at 4C, the ell suspensions were diluted to 8 ml with ie-old phosphate buffer (see above) and entrifuged (12, x g) for 5 min at 4 C. The ell pellets were suspended in.2 ml of water and transferred to ounting vials with two 3-ml portions of sintillation fluid. Control values were ob-

2 VOL. 133, 1978 BINDING AND TRANSPORT OF THIAMIN 1191 tained from ell samples to whih unlabeled thiamine (1,M) was added 1 min before the labeled vitamin. Results after subtration of the ontrol are expressed in nanomoles of thiamine bound per 1' ells (a. 2.2 mg of ells, dry weight). Thiamine transport. The transport of thiamine (at 37 C) was determined by the same proedure that was used to measure thiamine binding, exept that the ells were preinubated with 5 mm gluose for 5 min at 37C before the addition of labeled thiamine. Thiamine transport (at 37C) was orreted for the amount of vitamin bound by the ells (at 4) under the same onditions. Conentration gradients were based on a ell volume of 2.4 x 1-12 ml; the latter was alulated as desribed by Winkler and Wilson (21). Preparation of subeliular frations. L. asei ells (1 g) were suspended in 2 ml of.1 M potassium phosphate (ph 7.5) and frozen in a dry ie-aetone bath. After 5 min, the ells were thawed, and [14C]- thiamine was added to a final onentration of 5,uM. The mixture was then subjeted to soni osillation (Biosonik III) for two 1-min intervals at 4 C, entrifuged for 2 min at 32, x g, and separated into a supemnatant and partiulate fration. After the latter fration was suspended in 2 ml of buffer, bound thiamine was measured by the same proedure employed for analysis of whole ells (see above). Binding ativity in the supemnatant fration was determined by a Sephadex G-25 olumn hromatography proedure desribed previously (5). Protein in intat ells (after soni treatment for 2 min at 23C) and in the supernatant and partiulate frations was determined by the method of Lowry et al. (11), using bovine serum albumin as the standard. Thiamine metabolism. L. asei ells (3 x 11) were suspended in 15 ml of.5 M potassium phosphate (ph 6.8) and inubated as desribed in the legend to Fig. 3. The ells were diluted to 4 ml with ie-old.5 M sodium aetate (ph 4.), entrifuged for 5 min at 4C (12, x g), and resuspended in 2. ml of.2 M sodium aetate (ph 4.). Samples were then heated for 1 min at 8 C and entrifuged as above to remove partiulate material. After lyophilization, the supernatant fration was dissolved in 25 PI of water and hromatographed on ellulose sheets (astman) in the solvent system desribed by Kawasaki et al. (9). Setions (1-m) of the dried plates were ut out, and their radioativity was determined by liquid sintillation ounting. RSULTS General harateristis of thiamine uptake by L. asei. The time ourse for the uptake of thiamine at 37C and in the presene of gluose is shown in Fig. 1. Under these onditions, the initial rate of uptake was rapid (a..6 nmol/min per 11 ells) and was linear for approximately 1 min. A seond phase haraterized by a muh slower rate of thiamine uptake (a..1 nmol/min per 11 ells) was observed after 1 min. In various experiments, a total of 1.7 to 2. nmol of thiamine was taken up during a 3- min inubation. When the temperature was lowered to 4C and gluose was omitted, uptake of labeled thiamine was again rapid but reahed a plateau within 5 min (Fig. 1). Total uptake of thiamine at 4C was approximately.5 nmol per 11 ells. Thus, by ontrolling the temperature, the uptake of thiamine (like that of folate [5]) an be separated experimentally into two omponents: (i) the apparent binding of thiamine by ells (uptake at 4) and (ii) the transport of thiamine into ells (uptake at 37C minus binding at 4). Relationship between thiamine uptake at 4C by intat ells and thiamine-binding ativity in subeliular frations. Intat ells were disrupted in the presene of [14C]thiamine by soni osillation (see Materials and Methods) and were separated into a partiulate (membrane) and a soluble (ytoplasmi) fration by entrifugation. Although thiamine transport was almost ompletely lost by this treatment, thiamine-binding ativity ould be reovered in subellular frations (Table 1). (The purifiation and properties of the membrane protein ontaining this thiamine-binding ativity will be presented elsewhere.) Bound thiamine was on- 2. =.5 37 ~._ Time, min FIG. 1. Time dependene for the uptake of thiamine (1. MM) by L. asei. Uptake at 4 C in the absene ofgluose (binding) and at 37 C in the presene of 5 mm gluose (binding plus transport) were determined as desribed in the text. TABL 1. Distribution of thiamine-binding ativity in subellular frations of L. asei Thia- Sp at Pro- (nmol/ mie Cellular prepn tein mound mg of (mg) (nmol) protein) Intat ells Cytoplasmi fration Membrane fration

3 1192 HNDRSON AND ZVLY tained predominantly in the insoluble, membrane fration and in an amount representing 73% of the binding apaity of intat ells; only 1% of the total ativity appeared in the ytoplasmi fration. The speifi ativity for thiamine bound by the partiulate fration (.37 nmol/mg of protein) was higher than the orresponding values for ells (.3) or the soluble fration (.9). In an experiment not shown in Table 1, thiamine-binding ativity (.43 nmol/mg of protein) was also reovered in good yield (6%) in a membrane fration prepared by treatment of intat ells with lysozyme (6). nergy requirements. Transport (but not binding) of thiamine was highly dependent upon a soure of energy (Table 2). When ells were depleted of their energy reserves by prior inubation in phosphate buffer, the 37C transport proess was barely detetable unless gluose (5 mm) was added to the assay mixtures. In addition, treatment of the ells with 5 mm iodoaetate to blok glyolysis abolished the transport of thiamine, but did not affet its binding. Countertransport of thiamine ompounds. fflux of ['4C]thiamine ompounds from L. asei was also determined (Fig. 2). Cells were preloaded by exposure to ['4C]thiamine for 2 min at 37C in the presene of gluose. After removal of free thiamine by entrifugation, the samples were inubated at 37C in the presene and absene of unlabeled thamine (1,uM), and the release of the 14C label was monitored. Plateau values were observed in both ases within 3 min; at this time, ells to whih no additional substrate was added had lost 45% of their labeled material, but, in ells inubated TABL 2. ffet ofgluose and iodoaetate on the binding and transport of thiamine by energydepleted ells ofl. aseia Thiamine (nmol/1' ells) Addition (5 mm) Bound rted None Gluoseb lodoaetate plus gluosec.55.1 a Cells were exhausted of their energy reserves by inubation in.5 M potassium phosphate buffer (ph 6.8) for 6 min at 23C. Binding and transport were determined after inubation of the ells with 1.,uM labeled substrate for 5 min at 4 and 37C, respetively. b Samples were preinubated with 5 mm gluose for 5 min at 37 C prior to the determination of binding or transport. Samples were inubated sequentially with 5 mm iodoaetate (5 min, 37C) and then with 5 mm gluose (5 min, 37C) prior to the determination of binding and transport. - ~~~~~NO ADDITION u) 4 - J. BACTRIOL. 1O 2 3 Time, min FIG. 2. ffet of thiamine on the release of 4Cthiamine ompounds from L. asei. Cells (11 ) were suspended in 2ml of.5mpotassium phosphate (ph 6.8) ontaining 5 mm gluose and 1. S&M ll4cjthia. mine. Aftr inubation for 2 mn at 37 C, the ells were reovered by entrifugation for 5 min at 4 C (12, x g) and resuspended at 4C in 2 ml of phosphate buffer. CeU sampls (.9 ml) and.1 ml of either buffer alone or buffer ontaining I mm unlabekd thiamine were ombined and inubated again at 37C. At the indiated times, sampes were removed and entrifuged as above, and radioativity assoiated with the eupellets was determined. with unlabeled vitamin, radioativity was released in somewhat higher amounts (57% after 3 min) and at a more rapid initial rate. Addition of thiamine pyrophosphate (1,uM) to samples already ontaining unlabeled thiamine (1 IM) did not inrease the rate or extent of release of the labeled material. Analysis of bound and transported material. When analyzed by thin-layer hromatography (Fig. 3A), the labeled material bound to ells at 4C hromatographed with an Rf value (.62) idential to that of the thiamine standard. xtrats from ells into whih labeled vitamin was atively transported in the presene of gluose also ontained primarily unhanged thiamine, although small amounts of thiamine pyrophosphate (Rf =.14) ould be deteted (Fig. 3A). In ontrast, labeled material assoiated with ells that had been preloaded with [14C]_ thiamine and then exposed to unlabeled vitamin for 3 min at 37C (see Fig. 2) onsisted predominantly of thiamine pyrophosphate (Fig. 3B). Analysis of the material released into the medium during the 3-min efflux period showed that it was mostly unhanged thiamine, although a signifiant amount (a. 25%) of an unidentified metabolite (Rf =.95) was also present. When preloaded ells were inubated

4 VOL. 133, 1978 BINDING AND TRANSPORT OF THIAMIN 1193 C 6 TPpB 4 TP << ~~~~Thbiamine , Distane from Origin, m FIG. 3. Metabolism of ["Cithiamine by L. asei. (A) Thin-layer hromatography of the thiamine ompounds assoiated with ells that had been inubated with either 1. t&m ['4C]thiamine for 5 min at 4C () or 1. p.m ['4C]thiamine and 5 nm gluose for 1 min at 37 C (). (B) Thin-layer hromatography of the thiamine ompounds assoiated with ells whih, subsequent to their aumulation of /'4C]thiamine, were inubated (3 min at 37QC) in the presene of unlabeled thiamine (1 pm). Preloading of the ells with labeled vitamin (see legend to Fig. 2) was ahieved by inubation with 1. p.m ['4CJthiamine and 5 mm gluose for 2 min at 37 C. Arrows show the positions to whih standards of thiamine, thiamine phosphate (TP), and thiamine pyrophosphate (TPP) migrate under these onditions (see referene 1). for 3 min in the absene of unlabeled thiamine (see Fig. 2), only the unknown ompound appeared in the external medium. Regulation by thiamine in the growth medium. The ability of L. asei to bind and transport labeled thiamine was dependent upon the onentration of vitamin added to the growth medium (Fig. 4). (Trae amounts of thiamine may be present in the vitamin-free asein hydrolysate used in the medium.) Both binding and transport were maximal in ells propagated in the presene of very small amounts of added thiamine (less than l' M). At higher onentrations (1'- to 1-6 M), both ativities were repressed oordinately. From the data in Fig. 4, the onentrations for a 5% loss in thiamine binding and transport were alulated to be 17 and 37 nm, respetively. The loss of binding and transport ativity was not due to saturation of these proesses by added thiamine. Cells that had been exposed to unlabeled vitamin (1,uM) and gluose (5 mm) for 3 min at 37 C and then washed with 1 volumes of vitamin-free buffer retained 6% of their apaity for the uptake (transport plus binding) of [14C]thiamine. Cells grown in the presene of 1,uM thiamine lost greater than 9% of their ability to take up thiamine (Fig. 4) under these same onditions. Kinetis of thiamine binding and transport. In L. asei ells grown in the absene of thiamine, the binding (5 min, 4 C) and transport (2 min, 37 C) of ['4C]thiamine remained onstant when tested over a range (5 to 5 nm) of substrate onentrations. [3S]thiamine was also bound and transported maximally at the lowest substrate onentration (1 nm) permitted by the speifi ativity of this isotope. It was ononluded aordingly that the dissoiation onstant (KD) for binding and the Mihaelis onstant for transport (Km) are both less than 1 nm Ṫhe extremely tight assoiation of thiamine with ells was also evident from the results shown in Table 3. When ells that had been first exposed (at 4 C) to.1,um ['4C]thiamine were treated with unlabeled thiamine at 1 times I 2 Q.- ID s. _ lo-le 1-s7~ lo-6s Thiamine, M FIG. 4. ffet of thiamine added to the growth medium upon the binding () and transport () of thiamine. Assay mixtures ontained 1. pm/4c]thiamine and were inubated for 5 min at 4 C or 2 min at 37 C for the measurement of binding and oftransport ativity, respetively. TABL 3. ffet of unlabeled thiamine on the binding (at 4 C) of '4C]thiamine by L. aseia [14C]thiamine Addition A Addition B bound (.1 jim) (1,uM) (nmol/11 ells) ['4C]thiamine None.52 ['4C]thiamine Thiamine.45 Thiamine [I4C]thiamine.17 a L. asei ells (.8 ml) were ombined with addition A (.1 ml of a 1.,M solution), inubated for 5 min at 4 C, and then exposed to addition B (.1 ml of a 1 tim solution). After inubation for an additional 5 min at 4 C, [14C]thiamine bound by the ells was determined as desribed in the text. - a 2 a a] 1 a

5 1194 HNDRSON AND ZVLY this level, the unlabeled material was unable to displae the previously bound vitamin. Similarly, when ells were exposed in the onverse experiment to a low onentration (.1,uM) of unlabeled thiamine, the addition of a large exess (1,uM) of ['4C]thiamine did not readily replae the previously bound substrate. In ontrast, when these ompetition experiments were performed at 37 C in ells treated with 1 mm iodoaetate to blok transport, a rapid exhange ourred between bound and free thiamine regardless of the order of addition. Inhibition of ['4C]thiamine binding and transport by thiamine ompounds. The ompetition of unlabeled thiamine, thiamine phosphate, and thiamine pyrophosphate with ["4C]thiamine for both binding and transport is illustrated in Table 4. To ensure ompetitive onditions (ompare Table 3), the [14C]thiamine and the unlabeled thiamine ompounds were mixed before their addition to the ells. The results show that eah of these ompounds was a good inhibitor of the binding and transport of labeled thiamine and that the sequential addition of phosphate groups onto the vitamin gave rise to analogs with a progressively lower ability to ompete with the parent ompound. Moreover, as the onentrations of thiamine and its phosphorylated derivatives were varied, the binding and transport of thiamine were eah inhibited to the same degree. TABL 4. transport by thiamine ompoundsa Inhibition of ['4C]thiamine binding and Inhibi- Inhibi- Thiamine om- Conn tion of pound (UM) binding tion of trans- (%) (%) Thiamine Thiamine phos phate Thiamine pyro phosphate a Assay samples were prepared at 4 C by ombining.1 ml of 1. fim ['4C]thiamine and.1 ml of the indiated unlabeled thiamine ompound, followed by the addition of.8 ml of ells. The binding and transport of ['4C]thiamine were then determined after inubation for 5 min at 4 C or 2 min at 37 C, respetively. Control levels (no addition) were.5 nmol bound per 11 ells and.49 nmol transported per 1' ells. J. BACTRIOL. DISCUSSION Thiamine uptake at 37 C by non-proliferating ells of L. asei possesses several harateristis ommon to arrier-mediated ative transport systems. The uptake proess is dependent upon a soure of energy (Table 2), is sensitive to temperature (Fig. 1), displays speifiity (Table 4), and follows saturation kinetis. Rapid exhange between intraellular and extraellular thiamine ould also be demonstrated (ompare Fig. 2 and 3), indiating that the vitamin was not tightly bound by intraellular omponents and was free to reross the membrane. Intraellular thiamine was, however, slowly onverted to thiamine pyrophosphate, whih did not exit from the ells. A onsiderable onentration gradient ould also be ahieved by the system. When the uptake of thiamine after 1 min at 37 C was orreted for both the small amount of vitamin onverted to its phosphorylated forms and the amount of vitamin bound by the ells, the ratio of the internal and external thiamine onentrations was approximately 4. In both. oli (9) and L. fermentii (14-16), free thiamine does not aumulate but is onverted rapidly to thiamine pyrophosphate upon entry into the ell. However, mutant studies have shown that, at least in. oli, energy an be used to ahieve onentration gradients for free thiamine (1). The energy required for thiamine transport was readily generated by the metabolism of gluose (Table 2). Similarly, transport ould be abolished when iodoaetate was used to blok glyolysis. Sine the ells aumulate free thiamine, the energy requirement appears to be oupled diretly to the transport system and not to the subsequent metabolism of this vitamin. In addition to the ative transport proess, a onsiderable portion (a. 38%) of the total uptake of thiamine by L. asei is referable to the binding of the vitamin by the ells. The binding omponent responsible for this uptake is deteted at low temperature (or at 37 C in the absene of energy prodution), has a high affinity for thiamine (KD < 1 nm), resides in the ell membrane, and is present in relatively large amounts (.5 nmol per 11 ells). This latter value exeeds by 2-fold the amount of thiamine whih ould be expeted to be taken up by ells via failitated (or passive) diffusion. That the energy-independent uptake of thiamine at 4 C is mediated by a speies with a very high affinity for the vitamin was further indiated by experiments (Table 3) whih showed that at 4C high onentrations of unlabeled thiamine did not readily displae ['4C]thiamine one the latter was taken up by ells. The insensitivity of the binding ativity to iodoaetate suggests that re-

6 VOL. 133, 1978 ative sulfhydryl groups are not present at the thiamine-binding site. The latter result is not harateristi of various amino aid-binding proteins in the ell membrane of. oli whih, upon treatment with p-hloromeruribenzoate, lose the apaity to bind their respetive substrates (3).. oli also ontains a periplasmi binding protein whih appears to mediate the uptake of thiamine (9, 12, 18). The latter binder also has a high affinity for thiamine (KD 29 nm) but is present in onsiderably lower amounts per ell (18) than the L. asei thiamine-binding omponent. L. fermentii transports thiamine with a Km of 48 nm (15) and presumably ontains a binding omponent with a similar KD value. L. asei onentrates another vitamin, foli aid, by a system whih an also be separated into a transport and binding phase (4, 5, 7). The membrane omponent responsible for folate binding is present in amounts (.35 nmol per 11 ells) slightly lower than the thiamine binder and is insensitive to iodoaetate and other sulfhydryl reagents. Studies on regulation (5), ellular loation (5, 6), the strutural properties of the purified protein (6), and kinetis in wildtype and mutant ells of L. asei (7) have provided substantial evidene that the isolated folate binder is a omponent of the folate transport system. Analysis of the thiamine-binding omponent of L. asei has led to a similar onlusion. videne supporting this viewpoint is as follows. (i) The binding of thiamine by ells is mediated by a omponent of the ell membrane. (ii) Repression of both binding and transport of thiamine by the level of the vitamin in the growth medium ours in a parallel fashion (Fig. 4). (iii) The KD for thiamine binding and the KM for thiamine transport are of the same magnitude (<1 nm). (iv) When various onentrations of thiamine, thiamine phosphate, and thiamine pyrophosphate were tested as inhibitors of [I4C]thiamine binding and transport, the relative order of affinities (thiamine > thiamine phosphate > thiamine pyrophosphate) and the apparent values for the affinity onstants were the same for eah proess (Table 4). Although another means to explore this relationship would be to isolate ells defetive in thiamine transport, seletion of suh mutants would be diffiult, sine a growth requirement for thiamine has not been established in L. asei (13). ACKNOWLDGMNTS This investigation was supported by grants to F. M. Huennekens from the National Caner Institute (Publi Health Servie grant CA-6522) and the Amerian Caner Soiety (CH-31). G.B.H. is a reipient of a Publi Health Servie postdotoral fellowship (CA-616) from the National Caner Institute. BINDING AND TRANSPORT OF THIAMIN 1195 We are indebted to F. M. Huennekens and Karin Vitols for advie in the preparation of the manusript and to Maureen Gordon for expert tehnial assistane. LITRATUR CITD 1. Cooper, B. A Studies of [:H]foli aid uptake by Latobaillus asei. Biohim. Biophys. Ata 28: Flynn, L M., V. B. Williams, B. D. O'Dell, and A. G. Hogan Medium for assay of vitamins with lati aid bateria. Anal. Chem. 23: Gordon, A., F. J. Lombardi, and H. R. Kabak Solubilization and partial purifiation of amino aidspeifi omponents of the D-latate dehydrogenaseoupled amino aid transport system. Pro. Natl. Aad. Si. U.S.A. 69: Henderson, G. B., and F. M. Huennekens Transport of folate ompounds into Latobaillus asei. Arh. Biohem. Biophys. 164: Henderson, G. B.,. M. Zevely, and F. M. Huennekens Folate transport in Latobaillus asei: solubilization and general properties of the binding protein. Biohem. Biophys. Res. Commun. 68: Henderson, G. B.,. M. Zevely, and F. M. Huennekens Purifiation and properties of a membraneassoiated, folate-binding protein from Latobaillus asei. J. Biol. Chem. 252: Henderson, G. B.,. M. Zevely, R. J. Kadner, and F. M. Huennekens The folate and thiamine transport proteins of Latobaillus asei. J. Supramol. Strut. 6: Huennekens, F. M., and G. B. Henderson Transport of folate ompounds into mammalian and baterial ells, p In W. Pfleiderer (ed.), Chemistry and biology of pteridines. Walter de Gruyter, Berlin. 9. Kawasaki, T., I. Miyata,. Kimiko, and Y. Nose Thiamine uptake by sherihia oli. Arh. Biohem. Biophys. 131: Kawasaki, T., and K. Yamada The uptake system of free thiamine in mutants of sherihia oli. Biohem. Biophys. Res. Commun. 47: Lowry, O. H., N. J. Rosebrough, A. L Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Matsuura, A., A. Iwashima, and Y. Nose Purifiation of thiamine-binding protein from sherihia oli by affinity hromatography. Biohem. Biophys. Res. Commun. 51: Morishita, T., T. Fukada, M. Shirota, and T. Yura Geneti basis of nutritional requirements in Latobaillus asei. J. Bateriol. 12: Neujahr, H. Y Transport of B-vitamins in miroorganisms. I. On the permeability of Latobaillus fermentii to 'S-thiamine. Ata Chem. Sand. 17: Neujahr, H. Y Transport of B-vitamins in miroorganisms. II. Fators affeting the uptake of labelled thiamine by non-proliferating ells of Latobaillus fermentii. Ata Chem. Sand. 2: Neujahr, H. Y Transport of B-vitamins in miroorganisms. III. Chromatographi studies on the radioativity extrated from non-proliferating ells of Latobaillus fermentii after exposure to labelled thiamine. Ata Chem. Sand. 2: Neujahr, H. Y Transport of B-vitamins in miroorganisms. IV. The non-speifiity of the effet of exogenous ATP on the uptake of labelled thiamine by non-proliferating ells of L. fermentii. A reappraisal. Ata Chem. Sand. 2: Nishimune, T., and R. Hayashi Thiamine-binding protein and thiamine uptake by sherihia oli.

7 1196 HNDRSON AND ZVLY J. BACTRIOL. Biohim. Biophys. Ata 244: utilization of methyl tetrahydrofolates by Latobaillus 19. Shane, B., and. L. R. Stokstad Transport and asei. J. Biol. Chem. 251: metabolism of folates by bateria. J. Biol. Chem. 21. Winkler, H. H., and T. H. Wilson The role of 25: energy oupling in the transport of fl-galatosides by 2. Shane, B., and. L. R. Stokstad Transport and sherihia oli. J. Biol. Chem. 241: