Identification of the UDP-glucose-binding polypeptide of callose synthase from Beta vulgaris L. by photoaffinity labeling with 5-azido-UDP-glucose

The photoaffinity probe 5-azidouridine 5'-[beta-32P]diphosphate glucose (5N3[32P]UDP-Glc) was used to identify a 57-kDa polypeptide as a strong candidate for the UDP-Glc-binding polypeptide of UDP-glucose: (1,3)-beta-glucan (callose) synthase from red beet (Beta vulgaris L.) storage tissue. Unlabeled 5N3UDP-Glc was a competitive inhibitor of callose synthase with a Ki of 310 microM. Callose synthase was purified from plasma membranes by a two-step solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, followed by product entrapment, and photoincorporation of radioactivity from 5N3[32P]UDP-Glc was used to identify UDP-Glc-binding polypeptides that copurified with callose synthase activity. Photoinsertion into the 57-kDa band was closely correlated with all catalytic properties examined. Photolabeling of the 57-kDa polypeptide was enriched upon purification of callose synthase by product entrapment, was abolished with increasing levels of unlabeled UDP-Glc, was dependent upon the presence of divalent cations, and the pH dependence of photolabeling correlated with the pH activity profile of callose synthase. In addition, photolabeling of the 57-kDa band did not occur after phospholipase treatment, which destroys enzyme activity. The extent of labeling of this polypeptide thus correlates closely with the activity of callose synthase under a wide variety of conditions. These results imply that the polypeptide at 57 kDa represents the substrate-binding and cation-regulated component of the callose synthase complex of higher plants.     

Cloning of cellulose synthesis related genes from Acetobacter xylinum ATCC23769 and ATCC53582: comparison of cellulose synthetic ability between strains.

About 14.5 kb of DNA fragments from Acetobacter xylinum ATCC23769 and ATCC53582 were cloned, and their nucleotide sequences were determined. The sequenced DNA regions contained endo-beta-1,4-glucanase, cellulose complementing protein, cellulose synthase subunit AB, C, D and beta-glucosidase genes. The results from a homology search of deduced amino acid sequences between A. xylinum ATCC23769 and ATCC53582 showed that they were highly similar. However, the amount of cellulose production by ATCC53582 was 5 times larger than that of ATCC23769 during a 7-day incubation. In A. xylinum ATCC53582, synthesis of cellulose continued after glucose was consumed, suggesting that a metabolite of glucose, or a component of the medium other than glucose, may be a substrate of cellulose. On the other hand, cell growth of ATCC23769 was twice that of ATCC53582. Glucose is the energy source in A. xylinum as well as the substrate of cellulose synthesis, and the metabolic pathway of glucose in both strains may be different. These results suggest that the synthesis of cellulose and the growth of bacterial cells are contradictory.     

Synthesis and characterization of 5-azido-UDP-glucuronic acid. A new photoaffinity probe for UDP-glucuronic acid-utilizing proteins.

A new active site-directed photoaffinity analogue, [beta-32P]5-azido-UDP-glucuronic acid (UDP-GlcA), was enzymatically synthesized from [beta-32P]5-N3UDP-Glc using UDP-glucose dehydrogenase. The product was characterized by its mobility on ion exchange and two thin-layer chromatographic systems, by its UV absorbance at 288 nm, and the loss of this absorbance after UV irradiation of the compound. Photoincorporation of [beta-32P]5-N3UDP-GlcA into bovine liver UDP-Glc dehydrogenase (EC 1.1.1.22) was saturable with an apparent Kd of 12.5 microM, and was inhibited by the known active-site effectors UDP-GlcA, UDP-Glc, and UDP-xylose. When human liver microsomes with known UDP-glucuronosyltransferase (EC 2.4.1.17) activities were photolabeled with [beta-32P]5-N3UDP-GlcA, major photolabeled bands of 35-37 and 50-54 kDa were detected. When rat liver microsomes from phenobarbital-injected rats were photolabeled with [beta-32P]5-N3UDP-GlcA, there was a marked increase in photoincorporation of a 51-kDa protein as compared with control animals. Evidence is presented which suggests that the photolabeled 51-54-kDa proteins in the liver microsomes from both tissues are UDP-glucuronosyltransferase and that [beta-32P]5-N3UDP-GlcA represents a new alternative approach in the study of UDP-glucuronosyltransferase and other UDP-GlcA-utilizing enzymes.     

Photoaffinity labeling of glucosyltransferase of the dolichol cycle from rat mammary gland

UDP-Glc:dolichol phosphate glucosyltransferase from lactating rat mammary gland has been partially purified by a combination of (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography on DEAE-TSK, and affinity chromatography. The partially purified enzyme exhibited several protein bands when examined by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions; among these, a 35-kDa polypeptide was quite prominent and appeared to be enriched during purification. Photoaffinity labeling of the partially purified enzyme preparation with 5-azido-[beta-32P]UDP-Glc identified a 35-kDa polypeptide. Labeling of a solubilized enzyme preparation from crude and stripped microsomes also revealed a 35-kDa band on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Photoinsertion of the probe in this polypeptide is enhanced by the presence of dolichol phosphate and Mg2+. Competition studies with UDP-Glc, UDP-glucuronic acid, other sugar nucleotides, and Glc-1-phosphate provide evidence to validate the specificity of photoaffinity labeling. These studies indicate that this 35-kDa polypeptide is involved in the synthesis of dolichol-P-Glc in rat mammary tissue. The possibility that this polypeptide may represent glucosyltransferase has been discussed.     

The multiple nicotinamide nucleotide-binding subunits of bovine heart mitochondrial NADH:ubiquinone oxidoreductase (complex I).

Direct photoaffinity labeling of purified bovine heart NADH:ubiquinone oxidoreductase (complex I) with 32P-labeled NAD(H), NADP(H) and ADP has shown that five polypeptides become labeled, with molecular masses of 51, 42, 39, 30, and 18-20 kDa. The 51 and the 30-kDa polypeptides were labeled with either [32P]NAD(H), [32P]NADP(H) or [beta-32P]ADP. The 42-kDa polypeptide was labeled with [32P]NAD(H) and to a small extent with [beta-32P]ADP. It was not labeled with [32P]NADP(H). The 39-kDa polypeptide was labeled with [32P]NADPH and to a small extent with [beta-32P]ADP. Our previous studies had shown that this subunit also binds NADP, but not NAD(H) [Yamaguchi, M., Belogrudov, G.I. & Hatefi, Y. (1998) J. Biol. Chem. 273, 8094-8098]. The 18-20-kDa polypeptide was labeled only with [32P]NADPH. Among these polypeptides, the 51-kDa subunit is known to contain FMN and a [4Fe-4S] cluster, and is the NAD(P)H-binding subunit of the primary dehydrogenase domain of complex I. The possible roles of the other nucleotide-binding subunits of complex I have been discussed.     

Resolution of phosphoglucomatase and the 62-kDA acceptor for the glucosylphosphotransferase

The radioactive, photoactivatable labeling probe [beta-32P]5-azidouridine 5'-diphosphoglucose has recently been shown to label a 62-kDa protein in crude homogenates and in partially purified enzyme preparations without photoactivation. Here, we report that a portion of this radioactivity is due to labeling of phosphoglucomutase by contaminating levels of [32P]alpha Glc-1-P initially present at less than 1% of the total 32P. This conclusion is based in part on the ability of excess unlabeled alpha Glc-1-P and Glc-6-P, but not UDP-Glc, to block the labeling. In addition, the labeled protein in liver homogenates had a tryptic peptide pattern similar to that of authentic phosphoglucomutase. These findings, however, raised a second question. Assays for the UDP-Glc: glycoprotein glucosyl phosphotransferase (Glc phosphotransferase) have utilized [beta-32P]UDP-Glc and have resulted in the labeling of a small number of acceptors, including one of approximately 62 kDa. Despite the fact that these assays had routinely been performed in the presence of 1 mM alpha Glc-1-P, the coincidence in molecular weights led to these further studies. We conclude that the acceptor of approximately 62 kDa is distinct from phosphoglucomutase. This conclusion is based on differences in the time courses of incorporation, the specificity of blocking agents, the presence of covalently linked glucose, the products of acid hydrolysis and of beta-elimination, and isoelectric points.     

Synthesis and properties of 5-azido-UDP-glucose. Development of photoaffinity probes for nucleotide diphosphate sugar binding sites

A new active site directed photoaffinity probe, which is a model compound for studying nucleotide diphosphate sugar binding proteins, has been synthesized by coupling 5-azido-UTP and [32P]Glc-1-P using yeast UDP-glucose pyrophosphorylase to produce [beta-32P]5-azidouridine 5'-diphosphoglucose (5N3UDP-Glc). This probe has photochemical properties similar to that of 5-azidoUTP (Evans, R. K., and Haley, B. E. (1987) Biochemistry 26, 269-276). The efficacy of 5N3UDP-Glc as an active site directed probe was demonstrated using yeast UDP-Glc pyrophosphorylase. Saturation effects of photoinsertion were observed with an apparent Kd of 51 microM and the natural substrate, UDP-Glc, prevented photoinsertion of [beta-32P]5N3UDP-Glc with an apparent Kd of 87 microM. Prevention of photoinsertion was also seen with UTP and pyrophosphate with apparent Kd values less than 200 microM. UMP, UDP, ATP, and GTP were much less effective competitors. Selective photoinsertion was observed with several partially purified enzymes including UDP-Glc dehydrogenase, UDP-Gal-4-epimerase, Gal-1-P uridyltransferase, and phosphorylase a. The absence of nonselective photoinsertion into bulk proteins was demonstrated with crude homogenates of rabbit liver as well as with several UDP-Glc binding proteins. Of the six purified enzymes tested, only phosphoglucomutase has been shown to incorporate radiolabel from the photoprobe in the absence of UV irradiation. These results and a discussion of the utility of 5N3UDP-Glc for detecting UDP-Glc binding proteins and isolating active site peptides are presented.     

Application of 5-azido-UDP-glucose and 5-azido-UDP-glucuronic acid photoaffinity probes for the determination of the active site orientation of microsomal UDP-glucosyltransferases and UDP-glucuronosyltransferases.

A new approach to determining the active site orientation of microsomal glycosyltransferases is presented which utilizes the photoaffinity analogs [32P]5-Azido-UDP-glucose ([32P]5N3UDP-Glc) and [32P]5-Azido-UDP-glucuronic acid ([32P]5N3UDP-GlcA). It was previously shown that both photoprobes could be used to photolabel UDP-glucose:dolichol phosphate glucosyltransferase (Glc-P-Dol synthase), as well as the family of UDP-glucuronosyltransferases in rat liver microsomes. The effects of detergents, proteases, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on the photolabeling of these enzymes were examined in intact rat liver microsomes. Photolabeling of Glc-P-Dol synthase by either photoprobe was the same in intact or disrupted vesicles, was susceptible to trypsin digestion, and was inhibited by the nonpenetrating inhibitor DIDS. Photolabeling of the UDP-glucuronosyltransferases by [32P]5N3UDP-GlcA was stimulated 1.3-fold in disrupted vesicles as compared to intact vesicles, whereas photolabeling of these enzymes by [32P]5N3UDP-Glc showed a 14-fold increase when vesicles were disrupted. Photolabeled UDP-glucuronosyltransferases were only susceptible to trypsin digestion in disrupted vesicles, and this was further verified by Western blot analyses. The results indicate a cytoplasmic orientation for access of UDP-sugars to Glc-P-Dol synthase and a lumenal orientation of most UDP-glucuronosyltransferases.     

Cellulose biosynthesis and function in bacteria.

The current model of cellulose biogenesis in plants, as well as bacteria, holds that the membranous cellulose synthase complex polymerizes glucose moieties from UDP-Glc into beta-1,4-glucan chains which give rise to rigid crystalline fibrils upon extrusion at the outer surface of the cell. The distinct arrangement and degree of association of the polymerizing enzyme units presumably govern extracellular chain assembly in addition to the pattern and width of cellulose fibril deposition. Most evident for Acetobacter xylinum, polymerization and assembly appear to be tightly coupled. To date, only bacteria have been effectively studied at the biochemical and genetic levels. In A. xylinum, the cellulose synthase, composed of at least two structurally similar but functionally distinct subunits, is subject to a multicomponent regulatory system. Regulation is based on the novel nucleotide cyclic diguanylic acid, a positive allosteric effector, and the regulatory enzymes maintaining its intracellular turnover: diguanylate cyclase and Ca2(+)-sensitive bis-(3',5')-cyclic diguanylic acid (c-di-GMP) phosphodiesterase. Four genes have been isolated from A. xylinum which constitute the operon for cellulose synthesis. The second gene encodes the catalytic subunit of cellulose synthase; the functions of the other three gene products are still unknown. Exclusively an extracellular product, bacterial cellulose appears to fulfill diverse biological roles within the natural habitat, conferring mechanical, chemical, and physiological protection in A. xylinum and Sarcina ventriculi or facilitating cell adhesion during symbiotic or infectious interactions in Rhizobium and Agrobacterium species. A. xylinum is proving to be most amenable for industrial purposes, allowing the unique features of bacterial cellulose to be exploited for novel product applications.     

Isolation of tocopherol-binding proteins from the cytosol of smooth muscle A7r5 cells.

Cultured smooth muscle A7r5 cells were able to take up alpha-tocopherol (32 +/- 1.2 nmol/mg protein) the largest part of which (60%) was present in the cytosolic fraction. Using a tocopherol-based affinity chromatography and alpha-, beta-, gamma-, and delta-tocopherols as eluants, three polypeptides of molecular masses 81, 58 and 31 kDa were eluted. This preparation had alpha-[3H]tocopherol binding capability. The 58-kDa polypeptide could also be eluted by chromanol and the 81-kDa polypeptide could be eluted also by phytol. The 81-kDa polypeptide had the unique P-E-E-D-Q-X-Q-Y N-terminal sequence.     

The beta-glucan synthase from Lolium multiflorum. Detergent solubilization, purification using monoclonal antibodies, and photoaffinity labeling with a novel photoreactive pyrimidine analogue of uridine 5'-diphosphoglucose.

The membrane-bound beta-glucan synthase from Italian ryegrass (Lolium multiflorum L.) endosperm cells has been solubilized by both non-ionic and zwitterionic detergents. A complex relationship exists between the ratio of (1----3)-, (1----4)-, and (1----3, 1----4)-beta-glucan products of the solubilized enzyme, the cations present, and the concentration of the uridine 5'-diphosphoglucose substrate. Monoclonal antibodies directed against the beta-glucan synthase complex were generated by immunization of mice with an unfractionated microsomal reparation. Hybridoma cell lines were screened using a combination of indirect enzyme-linked immunosorbent assay followed by an enzyme-capture assay. The purified monoclonal antibodies were used with Pan-sorbin (stablized protein A-bearing staphylococcal cells) to immunoprecipitate an active beta-glucan synthase complex which had been solubilized from a microsomal preparation with 0.6% CHAPS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitated synthase complex revealed four major polypeptides of apparent molecular mass 30, 31, 54, and 58 kDa together with several minor components. The immunoprecipitated beta-glucan synthase complex was capable of synthesizing both (1----3)- and (1----4)-beta-glucans. A new photoreactive pyrimidine analogue of uridine 5'-diphosphoglucose, 5-[3-(p-azidosalicylamide]allyl-uridine 5'-diphosphoglucose was synthesized in a three-step reaction sequence involving mercuration of UDP-Glc, alkylation of 5-Hg-UDP-Glc, and acylation of 5-(3-amino)allyl-UDP-Glc and characterized by chemical and spectroscopic analysis. The analogue inhibits (Kiapp 16 microM) and, upon UV irradiation, irreversibly inactivates the beta-glucan synthase. The analogue was iodinated with Na125I to give a radiolabeled, photoreactive compound, and was used in photoaffinity labeling of UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, and several putative UDP-Glc-binding proteins from L. multiforum. The radiolabeled analogue specifically labeled the 31-kDa polypeptide in the immunoprecipitated synthase complex. The photolabeling of this polypeptide is strictly dependent on UV irradiation, is blocked by uridine 5'-diphosphoglucose and uridine 5'-diphosphate, and reaches saturation at analogue concentrations above 300 microM. These results indicate that the 31-kDa polypeptide in the beta-glucan synthase complex bears a uridine 5'-diphosphoglucose-binding site and is involved in the catalysis of beta-glucan synthesis.     

The beta-phosphoro[35S]thioate analogue of UDP-Glc is efficiently utilized by the glucose phosphotransferase and is relatively resistant to hydrolytic degradation

The beta-phosphoro[35S]thioate analogue of UDP-glucose ((beta-35S)UDP-Glc) is utilized with approximately the same efficiency as the parent compound by the UDP-glucose:glycoprotein glucose-1-phosphotransferase (glucosyltransferase), which catalyzes the transfer of alpha Glc-1-P from UDP-Glc to mannose-containing oligosaccharides on acceptor glycoproteins. The same endogenous acceptor glycoproteins are labeled by the glucosyltransferase using [beta-32P]UDP-Glc and (beta-35S)UDP-Glc. However, in liver homogenates, incorporation from [beta-32P]UDP-Glc ceases to increase after about 4 min of incubation, while incorporation from (beta-35S)UDP-Glc persists for at least 1 h. This difference is due to an approx. 10-fold slower hydrolytic rate for the phosphorothioate analogue than for the parent compound, a finding similar to previous work showing that a variety of nucleases and phosphodiesterases are less efficient in cleaving phosphorothioate DNA than the native polymer.     

Cloning and sequencing of the beta-glucosidase gene from Acetobacter xylinum ATCC 23769.

The beta-glucosidase gene (bglxA) was cloned from the genomic DNA of Acetobacter xylinum ATCC 23769 and its nucleotide sequence (2200 bp) was determined. This bglxA gene was present downstream of the cellulose synthase operon and coded for a polypeptide of molecular mass 79 kDa. The overexpression of the beta-glucosidase in A. xylinum caused a tenfold increase in activity compared to the wild-type strain. In addition, the action pattern of the enzyme was identified as G3ase activity. The deduced amino acid sequence of the bglxA gene showed 72.3%, 49.6%, and 45.1% identity with the beta-glucosidases from A. xylinum subsp. sucrofermentans, Cellvibrio gilvus, and Mycobacterium tuberculosis, respectively. Based on amino acid sequence similarities, the beta-glucosidase (BglxA) was assigned to family 3 of the glycosyl hydrolases.     

Localization of the tight ADP-binding site on the membrane-bound chloroplast coupling factor one

The photoaffinity analog 2-azido-ADP (2-azidoadenosine 5'-diphosphate) was used as a probe of the spinach chloroplast ATP synthase. The analog acted as a substrate for photophosphorylation. Several observations suggested that 2-azido-ADP and ADP bound to the same class of tight nucleotide binding sites: (a) 2-azido-ADP competitively inhibited ADP tight binding (Ki = 1.4 microM); (b) the concentration giving 50% maximum binding, K0.5 for analog tight binding (1 microM) was similar to that observed for ADP (2 microM); (c) nucleotide tight binding required prior membrane energization and was completely reversed by re-energization; (d) the tight binding of 2-azido-[beta-32P]ADP was completely prevented by ADP; (e) the analog inhibited the light-triggered ATPase activity at micromolar concentrations. Ultraviolet irradiation of washed thylakoid membranes containing tightly bound 2-azido-[beta-32P]ADP resulted in the covalent incorporation of the label into the membranes. Denaturing polyacrylamide gel electrophoresis of the labeled membranes demonstrated that the beta subunit of the coupling factor one complex was the only polypeptide in the thylakoid membranes which was labeled. These results identify the beta subunit of the coupling factor as the location of the tightly bound ADP on the thylakoid membranes.     

Affinity labeling the DNA polymerase alpha complex. Identification of subunits containing the DNA polymerase active site and an important regulatory nucleotide-binding site

Pyridoxal 5'-phosphate (PLP) inhibits DNA polymerase activity of the intact multifunctional DNA polymerase alpha complex by binding at either of two sites which can be distinguished on the basis of differential substrate protection. One site (PLP site 1) corresponds to an important nucleotide-binding site which is distinct from the DNA polymerase active site and which appears to correspond to the DNA primase active site while the second site (PLP site 2) corresponds to the dNTP binding domain of the DNA polymerase active site. A method for the enzymatic synthesis of high specific activity [32P]PLP is described and this labeled PLP was used to identify the binding sites described above. PLP inhibition of DNA polymerase alpha activity was shown to involve the binding of only a few (one to two) molecules of PLP/molecule of DNA polymerase alpha, and this label is primarily found on the 148- and 46-kDa subunits although the 63-, 58-, and 49-kDa subunits are labeled to a lesser extent. Labeling of the 46-kDa subunit by [32P]PLP is the only labeling on the enzyme which is blocked or even diminished in the presence of nucleotide alone, and, therefore, this 46-kDa subunit contains PLP site 1. Labeling of the 148-kDa subunit is enhanced in the presence of template-primer, suggesting that this subunit undergoes a conformational change upon binding template-primer. Furthermore, labeling of the 148-kDa subunit is the only labeling on the enzyme which can be specifically blocked only by the binding of both template-primer and the correct dNTP in a stable ternary complex. Therefore, the 148-kDa subunit contains PLP site 2, which corresponds to the dNTP binding domain of the DNA polymerase active site.     

Identification of 3-O-(4-benzoyl)benzoyladenosine 5'-triphosphate- and N,N'-dicyclohexylcarbodiimide-binding subunits of a higher plant H+-translocating tonoplast ATPase

The polypeptide composition of the NO-3-sensitive H+-ATPase of vacuolar membrane (tonoplast) vesicles isolated from red beet (Beta vulgaris L.) storage root was investigated by affinity labeling with [alpha-32P]3-O-(4-benzoyl)benzoyladenosine 5'-triphosphate [( alpha-32P]BzATP) and [14C]N,N'-dicyclohexylcarbodiimide [( 14C]DCCD). The photoactive affinity analog of ATP, BzATP, is a potent inhibitor of the tonoplast ATPase (apparent KI = 11 microM) and the photolysis of [alpha-32P]BzATP in the presence of native tonoplast yields one major 32P-labeled polypeptide of 57 kDa. Photoincorporation into the 57-kDa polypeptide shows saturation with respect to [alpha-32P]BzATP concentration and is blocked by ATP. [14C]DCCD, a hydrophobic carboxyl reagent and potent irreversible inhibitor of the tonoplast ATPase (k50 = 20 microM) labels a 16-kDa polypeptide in native tonoplast. The tonoplast ATPase is purified approximately 12-fold by Triton X-100 solubilization and Sepharose 4B chromatography. Partial purification results in the enrichment of two prominent polypeptides of 67 and 57 kDa. Solubilization, chromatography, and sodium dodecylsulfate-polyacrylamide gel electrophoresis of tonoplast labeled with [alpha-32P]BzATP or [14C]DCCD results in co-purification of the 57- and 16-kDa labeled polypeptides with ATPase activity. It is concluded that the tonoplast H+-ATPase is a multimer containing structurally distinct BzATP- and DCCD-binding subunits of 57 and 16 kDa, respectively. The data also suggest the association of a 67-kDA polypeptide with the ATPase.     

Direct photoaffinity labeling of proteins with adenosine 3'-[32P]phosphate 5'-phosphosulfate. Atractyloside inhibits labeling of a Mr = 34,000 protein in an adrenal medullary Golgi fraction

Direct photoaffinity labeling with radioactively labeled adenosine 3'-phosphate 5'-phosphosulfate (PAPS) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography was used to identify PAPS binding proteins in a Golgi membrane preparation of bovine adrenal medulla. [3'-32P]PAPS was synthesized from adenosine 5'-phosphosulfate (APS) and [gamma-32P]ATP using APS kinase prepared from yeast and was purified by reverse-phase ion pair high performance liquid chromatography. Upon irradiation with UV light, [3'-32P]PAPS, as well as [35S]PAPS under conditions which minimized sulfotransferase-catalyzed incorporation of 35SO4 from [35S]PAPS into proteins, bound selectively to a 34-kDa protein of the Golgi membrane preparation. PAPS binding to the 34-kDa protein was strongly inhibited by the presence of 50 microM atractyloside. The 34-kDa PAPS binding protein therefore appears to be similar to the mitochondrial ATP/ADP translocator with regard to both molecular weight and inhibition by atractyloside of adenine nucleotide binding. Photoaffinity labeling will be useful in the purification and functional identification of the 34-kDa protein.     

Localization of the high-affinity binding site for ATP on the membrane-bound chloroplast ATP synthase

The photoaffinity analog 2-azido-ADP has been used to investigate the high-affinity binding site(s) for ATP on the chloroplast thylakoid membrane. Photophosphorylation of 2-azido-ADP results in the rapid formation of 2-azido-ATP, which remains tightly bound to the membranes after extensive washing. The kinetic parameters of the tight binding of ATP and of 2-azido-ATP are similar (apparent Km = 1-2 microM; maximum extent = 0.2-0.4 nmol/mg of chlorophyll). Ultraviolet irradiation of washed thylakoid membranes containing tightly bound 2-azido-[gamma-32P]ATP induces covalent incorporation of the label exclusively into the beta subunit of the chloroplast coupling factor one. Previous results have shown that the tight binding site for ADP is also located on the beta subunit of the ATP synthase (Czarnecki, J. J., Abbott, M. S., and Selman, B. R. (1983) Eur. J. Biochem. 136, 19-24). To further characterize the tight binding sites for ADP and ATP, the membrane-bound coupling factor has been covalently modified with either tightly bound 2-azido-[gamma-32P]ATP or tightly bound 2-azido-[beta-32P]ADP. The photolabeled beta subunits have been isolated and subjected to partial proteolytic digestion and SDS-gel electrophoresis. The results of these experiments demonstrate that the tight binding sites for ADP and ATP are located on identical portions of beta subunit polypeptide.     

Identification of the uridine-binding domain of sucrose-phosphate synthase. Expression of a region of the protein that photoaffinity labels with 5-azidouridine diphosphate-glucose.

The uridine diphosphate-glucose (UDP-Glc) binding domain of sucrose-phosphate synthase (SPS) was identified by overexpressing part of the gene from spinach (Spinacia oleracea). Degenerate oligonucleotide primers corresponding to two tryptic peptides common to both the full-length 120-kD SPS subunit and an 82-kD form that photoaffinity labeled with 5-azidouridine diphosphate-glucose (5-N3UDP-Glc) were used in a polymerase chain reaction to isolate a partial cDNA clone. Comparison of the deduced amino acid sequence of spinach SPS with the sequences of potato sucrose synthase showed that the partial cDNA included one region that was highly conserved between the proteins. Expression of the partial cDNA clone of SPS in Escherichia coli produced a 26-kD fusion protein that photoaffinity labeled with 5-N3UDP-Glc. Photoaffinity labeling of the 26-kD fusion protein was specific, indicating that this portion of the SPS protein harbors the UDP-Glc-binding domain. Isolation of a modified peptide from the photolabeled protein provided tentative identification of amino acid residues that make up the uridine-binding domain of SPS.