Synthesis of the peptide-transport carrier of barley scutellum during the early stages of germination

Development of peptide-transport activity in the scutella of isolated barley (Hordeum vulgare l. cv. Maris Otter, Winter) embryos is shown to increase rapidly after about 15 h of imbibition, with the bulk of the transport activity being expressed by 24 h. This development is prevented by treatment of 15 h embryos with cycloheximide. Protein synthesis is found to increase in a closely related manner and also to be abolished by cycloheximide. Measurement of the incorporation of bound [³⁵S]methionine by 15 to 21-h embryos indicates that de-novo protein synthesis during this period is greater in the scutellum than in the embryonic axis. Previous studies have shown that the peptide-transport system possesses essential dithiol groups, probably located at the substrate-binding site (Walker-Smith and Payne 1983 b, 1984b). Treatment of 15-h embryos with the non-penetrant thiol reagent p-chloromercuribenzene sulphonic acid did not affect development of peptide-transport activity during the following 6 h, whereas with 3-d embryos identical treatment inhibited uptake almost completely during a subsequent 6-h period. Radioautography revealed that amongst the proteins synthesised during this early phase of germination and labelled in vitro with [³⁵S]methionine some are found within the epithelial plasmalemmae of the scutellum, which is the location of the peptide-transport carrier identified previously by externally labelling with a radioactive thiol reagent. The results provide evidence that protein(s) of the peptide-transport system are synthesised and inserted into the scutellum during early germination, allowing the system to play a major role in the nitrogen nutrition of the embryo.Abbreviations Gly Glycine - Phe phenylalanine     

Isolation and identification of proteins from the peptide-transport carrier in the scutellum of germinating barley (Hordeum vulgare L.) embryos

Peptide-transport proteins, intrinsic to the epithelial plasmalemmae of the scutella of germinating barley (Hordeum vulgare L.) embryos, have been selectively labelled with p-chloro-[²⁰³Hg]mercuribenzenesulphonate using both a substrate-screening technique and a procedure developed to label exclusively vicinal dithiol groups, which were shown previously (Walker-Smith and Payne, 1983, FEBS Lett. 160, 25–30) to be essential components of the peptide-transport system. After radioactive labelling, proteins from the scutellar membranes have been solubilised with lithium diiodosalicylate plus sodium dodecyl sulphate and separated by using polyacrylamide gel electrophoresis. Fluorography and silver staining of these gels has for the first time allowed identification of two presumptive components of the peptide-transport system. These components only become detectable in an extract of the scutellar epithelia after 15 h imbibition, concomitant with a dramatic increase in peptide-transport activity, and they remain present at least 3 d after the onset of germination. [³⁵] Methionine was shown to be incorporated into these proteins between 15–20 h after imbibition, but its incorporation during a similar 5 h period into scutella isolated after 3 d was undetectable, implying a slow turnover of these proteins during the later stages of germination.Abbreviations Ala₂, Ala₃ dialanine, trialanine - CHAPS 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulphonate - p-CMBS p-chloromercuribenzenesulphonic acid - NEM N-ethylmaleimide - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Characteristics of the protein carrier of the peptide-transport system in the scutellum of germinating barley embryos

Through the use of the protein reagents N-ethylmaleimide, p-chloromercuribenzenesulphonic acid and phenylarsine oxide, it is shown that in the scutellum of the germinating barley embryo, the transport of peptides, but not the transport of amino acids or glucose is specifically thiol-dependent. Furthermore, these essential thiol groups are shown to exist as redox-sensitive, vicinal-dithiols that lie at the substrate-binding sites of the peptide-transport proteins. The binding of N-ethylmaleimide to these dithiols is shown to be very fast, matching the kinetics of inhibition of peptide transport by this reagent. A technique for the specific labelling of the dithiols with N-ethyl[2,3-¹⁴C]maleimide is described, which allows the carrier proteins to be visualized at the scutellar epithelium using radioautography and permits calculation of the approximate amount of peptide-transport protein present per scutellum. In related studies, the importance of arginyl and histidyl residues to both amino-acid and peptide transport is shown, although other residues, e.g. carboxyl ligands do not seem to be critically involved.Abbreviations Ala alanine - Gly glycine - Leu Leucine - NEM N-ethylmaleimide - PAO phenylarsine oxide - PCMBS p-chloromercuribenzenesulphonic acid - Phe phenylalanine     

Characteristics of the active transport of peptides and amino acids by germinating barley embryos

Use of two different assays involving either radioactively labelled substrates or a fluorescent-labelling procedure, gave good agreement for the rates of transport of peptides and amino acids into the scutellum of germinating grains of barley (Hordeum vulgare cv. Maris Otter, Winter). However, evidence was obtained for the enzymic decarboxylation of transpored substrate, which can cause underestimates of transport rates when using radioactively labelled substrates. The peptide Gly-Phe, was shown to be rapidly hydrolysed after uptake, and autoradiography of transported Gly-[U-¹⁴C]Phe indicated a rapid distribution of tracer, i.e. [U-¹⁴C] phenylalanine into the epithelium and sub-epithelial layers of the scutellum. The developmental patterns of transport activity indicate that peptide transport is more important nutritionally during the early stages of germination (1–3 d) whereas amino acids become relatively more important later (4–6 d). A range of amino acids is shown to be actively transported and several compete for uptake. At physiological concentrations, e.g. 2mM, transport of peptides and amino acids is inhibited about 80% by protonophore uncouplers, but at higher concentrations (10–100 mM) passive uptake predominates.Abbreviations Gly glycine - Leu leucine - Phe phenylalanine - Pro proline     

Amino Acid and Peptide uptake in the scutella of germinating grains of barley, wheat, rice, and maize

Scutella separated from germinating grains of barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), rice (Oryza sativa L.), and maize (Zea mays L.) took up the four amino acids and the three peptides tested from incubation media. The uptake of amino acids by wheat scutella was similar to that of barley scutella and was via at least four uptake systems: two nonspecific amino acid uptake systems, one system specific for proline, and another system specific for basic amino acids. The scutellum of rice apparently has two nonspecific systems and a system specific for the basic amino acids, but the proline-specific system is lacking. The scutellum of maize seems to have the same systems as the scutellum of rice, but one (or both) of the nonspecific systems differs from that of the other species studied in taking up arginine only slowly. No great differences were observed in the uptake of peptides in the four species studied. The rates of uptake of different amino acids and peptides were of the same order of magnitude in the four cereals. The fact that carboxypeptidase activities in the endosperms of wheat and barley are 20-to 100-fold higher than those in rice and maize, does thus not seem to be reflected in the uptake properties of the scutella.     

Manipulation,by nutrient limitation,of the biosynthetic activity of immobilized cells of Capsicum frutescens Mill. cv. annuum

The relationship between the synthesis and accumulation of protein and capsaicin was investigated in cultured cells of Capsicum frutescens Mill. cv. annuum immobilized in reticulate polyurethane. Cells were cultured in media containing reduced concentrations of essential nutrients, in an attempt to manipulate the rates of protein synthesis. Cells cultured in the absence of orthophosphate for 7 d demonstrated no reduction in the incorporation of l-[U-¹⁴C]phenylalanine into soluble protein or an increase in incorporation into capsaicin, compared with controls supplied with orthophosphate. By day 15 of culture, however, a differential incorporation of label was observed. Over a 21-d culture period the intracellular phosphate did not completely disappear. Cells cultured in the absence of nitrate and phosphate combined, however, exhibited some reduction in incorporation of [¹⁴C]phenylalanine into protein and an increased incorporation into capsaicin after 7 d of culture, but the differences were greater at day 15, when increases in the total capsaicin content of the cultures were apparent. There was observed a relationship between the intracellular nitrate concentration, the culture growth index, and the incorporation of [¹⁴C]phenylalanine into soluble protein — each of these factors was inversely related to the incorporation of label into capsaicin and the total capsaicin content of the cultures.Abbreviations HPLC high-performance liquid chromatography - Phe phenylalanine     

Peptide transport by germinating barley embryos: Uptake of physiological di- and oligopeptides

The uptake of a variety of physiological di- and oligopeptides by germinating barley (Hordeum vulgare L.) embryos is described. Peptides as large as pentaalanine can be absorbed. Evidence is presented suggesting the peptides are absorbed intact and subsequently undergo rapid intracellular hydrolysis. Uptake shows stereospecificity. The transport of peptides is generally faster than the transport of amino acids, making it likely that the former could play an important role in the mobilization of the protein storage reserves during germination. The peptide transport system in barley is compared with similar systems from other groups of organisms.Abbreviations Gly-sar glycylsarcosine - Gly-sar-sar glycylsarcosylsarcosine - Gly-sar-sar-sar Glycylsarcosylsarcosylsarcosine     

Apparent transinhibition of peptide uptake in the scutellum of barley grain

The uptake of glycylsarcosine (Gly-Sar) into scutella separated from germinating grains of barley ( Hordeum vulgare L. cv. Himalaya) is inhibited by other peptides; in most cases the inhibition is not purely competitive but of a mixed type (simultaneous increase in the apparent K_m and decrease in V_max) (Sopanen, T. 1979. FEBS Lett. 108: 447–450). The aim of the present experiments was to elucidate the mechanism of the mixed inhibition by studying how peptides already taken up into the cells affect the uptake of Gly-Sar.
When scutella were preincubated in the presence of various peptides, 11 of the 13 peptides tested inhibited the subsequent uptake of Gly-Sar by 10 to 45%. The inhibition, studied in detail with leucylleucine and prolylproline, was due to a decrease in V_max. The two peptides having no effect were glycylglycine and D-alanyl-L-alanine which are the only peptides known to date acting as purely competitive inhibitors when present together with the substrate Gly-Sar.
Preincubation with leucine, proline and alanine was not inhibitory, although preincubation with the corresponding dipeptides was. This result, together with the demonstration of intact leucylleucine in the scutella after preincubation with leucylleucine, indicates that the inhibition was caused by the intact peptides.
The results support the notion that in the mixed type inhibition the increase in the apparent K_m is due to competition for the carrier at the outside of the membrane, while the decrease in V_max is due to peptides taken up and binding to the carrier at the inside of the membrane.     

Peptide transport by germinating barley embryos: Evidence for a single common carrier for di- and oligopeptides

Competition for uptake of a range of amino acids and peptides by germinating barley (Hordeum vulgare L.) embryos was studied. Peptides and amino acids show no competition and are apparently absorbed by independent transport systems. However, peptides of widely different structures do compete and it seems that only a single peptide transport system is present in barley embryos, capable of handling both di- and oligopeptides. The ability of physiological peptides to totally inhibit the uptake of glycylsarcosine indicates they share a common uptake system which previously has been shown to have the properties of an active transport process. The characteristics of the barley peptide transport system are compared with those found in other organisms.     

Affinity labeling of the active site and the reactive sulfhydryl associated with activation of rat liver phenylalanine hydroxylase

A pterin analogue, 5-[(3-azido-6-nitrobenzylidene)amino]-2,6-diamino-4-pyrimidinone (ANBADP), was synthesized as a probe of the pterin binding site of phenylalanine hydroxylase. The photoaffinity label has been found to be a competitive inhibitor of the enzyme with respect to 6,7-dimethyltetrahydropterin, having a Ki of 8.8 +/- 1.1 microM. The irreversible labeling of phenylalanine hydroxylase by the photoaffinity label upon irradiation is both concentration and time dependent. Phenylalanine hydroxylase is covalently labeled with a stoichiometry of 0.87 +/- 0.08 mol of label/enzyme subunit. 5-Deaza-6-methyltetrahydropterin protects against inactivation and both 5-deaza-6-methyltetrahydropterin and 6-methyltetrahydropterin protect against covalent labeling, indicating that labeling occurs at the pterin binding site. Three tryptic peptides were isolated from [3H]ANBADP-photolabeled enzyme and sequenced. All peptides indicated the sequence Thr-Leu-Lys-Ala-Leu-Tyr-Lys (residues 192-198). The residues labeled with [3H]ANBADP were Lys198 and Lys194, with the majority of the radioactivity being associated with Lys198. The reactive sulfhydryl of phenylalanine hydroxylase associated with activation of the enzyme was also identified by labeling with the chromophoric label 5-(iodoacetamido)fluorescein [Parniak, M. A., & Kaufman, S. (1981) J. Biol. Chem. 256, 6876]. Labeling of the enzyme resulted in 1 mol of fluorescein bound per phenylalanine hydroxylase subunit and a concomitant activation of phenylalanine hydroxylase to 82% of the activity found with phenylalanine-activated enzyme. Tryptic and chymotryptic peptides were isolated from fluorescein-labeled enzyme and sequenced. The modified residue was identified as Cys236.     

Citrate transport into barley mesophyll vacuoles — comparison with malate-uptake activity

Citrate uptake into barley (Hordeum vulgare L.) mesophyll vacuoles was found to be saturable with a K _m of about 200 M. Uptake appears to occur via the citrate^3- form, as indicated by concentration-dependent uptake studies at different pHs. Free citrate and not the Mg-citrate complex was taken up by the vacuoles, even though slow transport of the Mg complex could not be excluded. Citrate transport into vacuoles was competitively inhibited by malate (K _i=0.68 mM). Various organic acids and protein-modifying agents affected the uptake of malate and citrate to a similar extent. These results indicate that both organic acids cross the tonoplast by means of the same carrier. Accumulation of citrate was ATP-dependent and could be inhibited by ionophores. Bovine serum albumin strongly stimulated citrate uptake, but other proteins tested did not show a similar stimulatory effect.Abbreviation BSA bovine serum albumin We wish to thank Esther Vogt for her help with the experiments and Professor N. Amrhein (ETH, Zürich, Switzerland) and Dr. Michael Kertesz (ETH, Zürich) for helpful discussions. This work was supported by the Swiss National Foundation grant No. 31-25196.88.     

Transport of phenylalanine into vacuoles isolated from barley mesophyll protoplasts

The energy-dependent transport of phenylalanine into isolated vacuoles of barley (Hordeum vulgare L.) mesophyll protoplasts has been studied by silicone-layer floatation filtering. The uptake of this aromatic amino acid into the vacuolar compartment is markedly increased by MgATP, showing saturation kinetics; the K _m values were 0.5 mM for MgATP and 1.2 mM for phenylalanine. V _max for phenylalanine transport was estimated to 140 nmol phenylalanine·(mg·Chl)^-1·h^-1. The transport shows a distinct pH optimum at 7.3 and is markedly inhibited by 40 mM nitrate. Azide (1 mM) and vanadate (400 M) had no or little effect on rates of transport while p-fluorophenylalanine seemed to be an effective inhibitor, indicating a possible competition at an amino-acid carrier. Ionophores such as valinomycin, nigericin or gramicidin were strong inhibitors of phenylalanine transport, indicating that this process is coupled to both the transmembrane pH gradient (pH) and the transmembrane potential ().Abbreviations and symbols BSA bovine serum albumin - Chl chlorophyll - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - pH transmembrane pH gradient - transmembrane potential     

Reconstitution of the tonoplast amino-acid carrier into liposomes

The tonoplast amino-acid transporter of barley (Hordeum vulgare L.) mesophyll cells was functionally reconstituted by incorporating solubilized tonoplast membranes, vacuoplast membranes or tonoplast-enriched microsomal vesicles into phosphatidylcholine liposomes. (i) Time-, concentration- and ATP-dependence of amino-acid uptake were similar to results with isolated vacuoles. Although the orientation of incorporation could not be controlled, the results indicate that the transporter functions as a uniport system which allows regulated equilibration by diffusion between the cytosolic and vacuolar amino-acid pools. (ii) The ATP-modulated amino-acid carrier was also successfully reconstituted from barley epidermal protoplasts and Valerianella or Tulipa vacuoplasts, indicating its general occurrence. (iii) Fractionation of solubilized tonoplasts by size-exclusion chromatography followed by reconstitution of the fractions for glutamine transport gave two activity peaks: the first eluted in the region of high-molecular-mass vesicles and the second at a size of 300 kDa for the Triton-protein micelle.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacryl-amide gel electrophoresis This work was part of our research efforts within the Sonderforschungsbereich 176 of the University. We gratefully acknowledge experimental support by Marion Betz and valuable discussions with Professors U. Heber and U.-I. Flügge and Dr. Armin Gross (University of Würzburg) and Dr. E. Martinoia (ETH, Zürich, Switzerland).     

Identification of peptide‐binding sites within BSA using rapid,laser‐induced covalent cross‐linking combined with high‐performance mass spectrometry

We are developing a rapid, time‐resolved method using laser‐activated cross‐linking to capture protein‐peptide interactions as a means to interrogate the interaction of serum proteins as delivery systems for peptides and other molecules. A model system was established to investigate the interactions between bovine serum albumin (BSA) and 2 peptides, the tridecapeptide budding‐yeast mating pheromone (α‐factor) and the decapeptide human gonadotropin‐releasing hormone (GnRH). Cross‐linking of α‐factor, using a biotinylated, photoactivatable p‐benzoyl‐L‐phenylalanine (Bpa)–modified analog, was energy‐dependent and achieved within seconds of laser irradiation. Protein blotting with an avidin probe was used to detect biotinylated species in the BSA‐peptide complex. The cross‐linked complex was trypsinized and then interrogated with nano‐LC–MS/MS to identify the peptide cross‐links. Cross‐linking was greatly facilitated by Bpa in the peptide, but some cross‐linking occurred at higher laser powers and high concentrations of a non‐Bpa–modified α‐factor. This was supported by experiments using GnRH, a peptide with sequence homology to α‐factor, which was likewise found to be cross‐linked to BSA by laser irradiation. Analysis of peptides in the mass spectra showed that the binding site for both α‐factor and GnRH was in the BSA pocket defined previously as the site for fatty acid binding. This model system validates the use of laser‐activation to facilitate cross‐linking of Bpa‐containing molecules to proteins. The rapid cross‐linking procedure and high performance of MS/MS to identify cross‐links provides a method to interrogate protein‐peptide interactions in a living cell in a time‐resolved manner.     

Sucrose transport into vacuoles isolated from barley mesophyll protoplasts

Sucrose transport has been investigated in vacuoles isolated from barley mesophyll protoplasts. Rates of sucrose transfer across the tonoplast were even higher in vitro than in vivo indicating that the sucrose transport system had not suffered damage during isolation of the vacuoles. Sucrose transport is carrier-mediated as shown by substrate saturation of transport and sensitivity to a metabolic inhibitor and to competitive substrates. A number of sugars, in particular maltose and raffinose, decreased uptake of sucrose. Sorbitol was slowly taken up but had no effect on sucrose transport. The SH-reagent p-chloromercuribenzene sulfonate inhibited sucrose uptake completely. The apparent K_m of the carrier for sucrose uptake was 21 mM. Transport was neither influenced by ATP and pyrophosphate, with or without Mg²⁺ present, nor by protonophores and valinomycin (with K⁺ present). Apparently uptake was not energy dependent. Efflux experiments with preloaded vacuoles indicated that sucrose unloading from the isolated vavuoles is mediated by the same carrier which catalyses uptake. The vacuole of mesophyll cells appears to represent an intermediary storage compartment. Uptake of photosynthetic products into the vacuole during the light apparently minimizes osmotic swelling of the small cytosolic compartment of vacuolated leaf cells when photosynthetic productivity exceeds the capacity of the phloem for translocation of sugars.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazincethane-sulfonic acid - pCMBS p-chloromercuribenzene sulfonate Dedicated to Professor Dr. W. Simonis on the occasion of his 75th birthday     

Mobilization of proline in the starchy endosperm of germinating barley grain

In germinating grains of barley, Hordeum vulgare L. cv. Himalaya, free proline accumulated in the starchy endosperm during the period of rapid mobilization of reserve proteins. When starchy endosperms were separated from germinating grains and homogenized in a dilute buffer of pH 5 (the pH of the starchy endosperm), the liberation of proline continued in these suspensions. The process was completely inhibited by diisopropylfluorophosphate, indicating that it was totally dependent on serine carboxy-peptidases. The carboxypeptidases present in the starchy endosperms of germinating grains were fractionated by chromatography on DEAE-cellulose. Four peaks were obtained, all with different activity spectra on the seven carbobenzoxydipeptides (Z-dipeptides) tested. Two of the peaks corresponded to previously known barley carboxypeptidases; these as well as a third peak hydrolyzed substrates of the types Z-X-Y and Z-X-Pro (X and Y denote any amino acid residue except proline). The fourth peak corresponded to a proline carboxypeptidase specific for substrates of the Z-Pro-X type. Apparently, in the hydrolysis of longer proline-containing peptides there must be sequential cooperation between the two carboxypeptidase types. The carboxypeptidases in extracts of starchy endosperms also liberated proline from the peptides Ala-Ala-Ala-Pro and Ala-Ala-Pro while Ala-Pro and Pro-Ala were not attacked. The dipeptides, however, were rapidly hydrolyzed around pH 7 by extracts prepared from the scutella of germinating grains. It is concluded that one part of the proline residues of the reserve proteins is liberated in situ in the starchy endosperm through the combined action of acid proteinases and carboxypeptidases, while another part is taken up in the form of small peptides by the scutellum, where proline is liberated by amino- and/or dipeptidases in some neutral compartment.Abbreviations DFP diisopropylfluorophosphate - DTT dithiothreitol - TNBS 2,4,6-trinitrobenzenesulphonic acid - Z N-carbobenzoxy - TLC thin layer chromatography A preliminary account of these results was given at the Meeting of the Federation of European Plant Physiological Societies in Edinburgh in July 1978. Abstract No. 181     

Identification of embryonal antigens of maize: Globulins as primary reserve proteins of the embryo

Normal and -irradiated caryopses of Zea mays L. were germinated, and the degradation of embryonal antigens (EA) was followed in the endosperms, scutella and embryonic axes of the seedlings, using double immunodiffusion, immunoelectrophoresis and quantitative immunoprecipitation. The predominant transient EA were presumed to be storage proteins related to the reserve globulins of dicotyledonous seeds. Therefore globulins were isolated from maize scutella, purified by (NH₄)₂SO₄ fractionation and isoelectric precipitation, and the molecular weights of the polypeptide units were estimated by discontinuous sodium-dodecyl-sulphate slab electrophoresis. The globulins were found to be identical with the predominant EA and amounted to about 40% of the protein nitrogen in the embryos of mature, non-germinated caryopses. The presumed reserve function of the globulins and the characteristic time course of their degradation in embryonic axes and scutella of maize seedlings are discussed in relation to the two-step pattern of mobilization of nitrogen reserves in germinating cereal caryopses.Abbreviations AG, AR and AS antisera to globulins and to root and scutellum proteins, respectively - CA common antigens - DAP days after pollination - EA embryonal antigens - ME 2-mercaptoethanol - SDS-PAGE discontinuous sodium-dodecylsulphate slab polyacrylamide-gel electrophoresis - TCA trichloroacetic acid     

The influence of intracellular pools of phenylalanine derivatives upon the synthesis of capsaicin by immobilized cell cultures of the chilli pepper,Capsicum frutescens

In an attempt to determine the potential factors controlling the biosynthesis of the secondary metabolite capsaicin by immobilized cell cultures of the chilli pepper, Capsicum frutescens Mill, labelling techniques using the radioactive precursor [¹⁴C]phenylalanine have been employed. Following preincubation treatments with either capsaicin (the end-product of the pathway) or sinapic acid, [¹⁴C]phenylalanine was applied and the movement of the label through the pathway and its eventual fate was followed. Results have shown that capsaicin, through a feedback-inhibition mechanism, negatively influences its own synthesis. Furthermore, capsaicin synthesis in these cells is not controlled via the activity of the enzymes phenylalanine ammonia-lyase and cinnamate 4-hydroxylase which may determine the rate of entry of metabolites into the phenylpropanoid pathway. The importance of other sinks for phenylalanine derivatives, which may compete for capsaicin precursors, has also been investigated. Surprisingly, protein proved to be only a relatively minor sink for phenylalanine with the great majority of the label rapidly ending up in covalently bound phenolics in the cell wall. Attempts to prevent this by applying sinapic acid were only partially successful. The importance of these results in relation to the possible control mechanisms which operate to control secondary metabolite synthesis in vitro is discussed.     

Labeling of the active site of cytoplasmic aspartate aminotransferase by -chloro-L-alanine

Syncatalytic inactivation of pig heart cytoplasmic aspartate aminotransferase by β-chloro-[U-¹⁴C]L-alanine resulted in the incorporation of radioactivity corresponding to one mole of the label per mole of the monomeric unit of the enzyme. A borohydride-reduced and then carboxymethylated preparation of the labeled enzyme was digested by trypsin. A radioactive peptide was isolated and found to contain a covalently linked pyridoxyl derivative which absorbed at 325 nm. The amino acid sequence of this peptide was Tyr-Phe-Val-Ser-Glu-Gly-Phe -Glu-Leu-Phe-Cys-Ala-Gln-Ser-Phe-Ser-Lys-Asn-Phe-Gly-Leu-Tyr-Asn-Glu-Arg. In the peptide the phosphopyridoxyl group seems to be covalently bound via alanyl moiety derived from β-chloro-L-alanine, the β-carbon atom of which is covalently linked to the ?-nitrogen atom of the lysyl residue(Lys). From a comparison with the amino acid composition of the phosphopyridoxyl peptide isolated from the tryptic digest of a borohydride-reduced holoenzyme, it was concluded that the modified lysul residue was identical to that involved in binding pyridoxal phosphate to the apoenzyme.