[71] Amino acid transport proteins

Glover, George I.

doi:10.1016/s0076-6879(77)46075-0

Cited by 6 publications

(3 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In order to identify functional proteins in intact cells or in crude extracts, radioactive molecules have been attached to specific proteins using both affinity and photoaffinity labeling techniques (Jakoby & Wilchek, 1977). Membrane proteins that have been identified by affinity and photoaffinity labeling protocols include ion channels (Ruoho & Kyte, 1977) and transporters for amino acids (Glover, 1977), biotin (Bayer & Wilchek, 1977), glucose (Carter-Su et al, 1982), and nucleosides (Young et al, 1983). Recently, a plasma membrane derived folate binding protein from murine L1210 cells has been identified by using a photoaffinity analogue of methotrexate Price et al, 1988).…”

mentioning

confidence: 99%

Affinity labeling of the folate-methotrexate transporter from Leishmania donovani

Beck¹,

Ullman²

1989

Biochemistry

View full text Add to dashboard Cite

An affinity labeling technique has been developed to identify the folate-methotrexate transporter of Leishmania donovani promastigotes using "activated" derivatives of the ligands. These "activated" derivatives were synthesized by incubating folate and methotrexate with a 10-fold excess of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) for 10 min at ambient temperature in dimethyl sulfoxide. Preincubation of intact cells with nonradioactive "activated" folate or methotrexate at a concentration of 40 microM inhibited the capacity of wild-type cells to transport submicromolar concentrations of unmodified ligand. When intact wild-type (DI700) Leishmania donovani or preparations of their membranes were incubated with a 0.4 microM concentration of either "activated" [3H]folate or "activated" [3H]methotrexate, the radiolabeled ligands were covalently incorporated into a polypeptide with a molecular weight of approximately 46,000, as demonstrated by SDS-polyacrylamide gel electrophoresis. No affinity labeling of a 46,000-dalton protein was observed when equimolar concentrations of "activated" radiolabeled ligands were incubated with intact cells or membranes prepared from a methotrexate-resistant mutant clone of Leishmania donovani, MTXA5, that is genetically defective in folate-methotrexate transport capability [Kaur, K., Coons, T., Emmett, K., & Ullman, B. (1988) J. Biol. Chem. 263, 7020-7028]. However, some labeling of a 46,000-dalton protein was observed when MTXA5 cells were incubated with higher concentrations of "activated" ligands. Time course studies indicated that maximal labeling of the 46,000-dalton protein occurred within 5-10 min of incubation of intact cells with "activated" ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

show abstract

mentioning

confidence: 99%

Affinity labeling of the folate-methotrexate transporter from Leishmania donovani

Beck¹,

Ullman²

1989

Biochemistry

View full text Add to dashboard Cite

show abstract

“…A major goal of many transport studies is the isolation of components of the transport system. Chemical modification of transport proteins has been carried out for a variety of transport systems by affinity labeling techniques (Glover, 1977) to tag carriers. Photoaffinity reagents have been used to probe various transport systems because the highly reactive photolysis products generated in situ can react with virtually any amino acid side chain (Bayley & Knowles, 1977;Chowdhry & Westheimer, 1979).…”

mentioning

confidence: 99%

Photoinactivation of peptide transport in Saccharomyces cerevisiae

Becker¹,

Dunsmore²,

Steinfeld³

et al. 1982

Biochemistry

View full text Add to dashboard Cite

Oligopeptides and dipeptides are transported into Saccharomyces cerevisiae by a carrier-mediated system. In the dark, leucyl-p-nitroanilide (Leu-p-NA) and leucyl-leucyl-4-azido-2-nitrophenylalanine [Leu-Leu-Phe-(4N3,2NO2)] are competitive inhibitors of peptide transport by S. cerevisiae cells. The photolysis of yeast cells in the presence of Leu-p-NA or Leu-Leu-Phe(4N3,2NO2) at 350 nm results in an irreversible inactivation of peptide transport. Protection against this inactivation is afforded by an excess of trimethionine, a transported peptide. Photolysis with Leu-p-NA or Leu-Leu-Phe(4N3,2NO2) does not affect amino acid or sugar transport, and cell viability is maintained throughout the irradiation procedure. A 5-min irradiation of S. cerevisiae with 2.4 microM Leu-p-NA or 15 microM Leu-Leu-Phe(4N3,2NO2) causes 50% inhibition of trimethionine uptake. p-Nitroaniline, a possible hydrolysis product generated from Leu-p-NA by cellular peptidase activity, has no effect on peptide transport. An exogenous energy source is not required for photoinactivation. The results suggest that a component(s) of the peptide transport system of S. cerevisiae is irreversibly modified by photolysis with Leu-p-NA or Leu-Leu-Phe-(4N3,2NO2) and provide the first example of the use of amino acid p-nitroanilides as photoaffinity labels.

show abstract

“…In this context, affinity-labeling techniques have led to the characterization of several transport systems (23)(24)(25), and photoaffinity labeling, in particular, has had relatively widespread use (26). These techniques-specifically, the use of aromatic azides-have been applied to the fl-galactoside transport system in this laboratory (27,28).…”

mentioning

confidence: 99%