Biotin-binding protein from chicken egg yolk. Assay and relationship to egg-white avidin.

1. Biotin in chicken egg yolk is non-covalently bound to a specific protein that comprises 0.03% of the total yolk protein (0.8 mg/yolk). This biotin-binding protein is not detectable by the normal avidin assay owing to the biotin being tightly bound. Exchange of [14C]biotin for bound biotin at 65 degrees C is the basis of an assay for this protein. 2. Biotin-binding protein from egg yolk is distinguishable from egg-white avidin on Sephadex G-100 gel filtration, although the sizes of the two proteins appear quite similar. 3. Biotin-binding protein is denatured at a lower temperature and freely exchanges biotin at lower temperatures than does avidin. 4. The biotin-binding protein in egg yolk is postulated to be responsible for the deposition of biotin in egg yolk. D-[carboxyl-14C]Biotin injected into laying hens rapidly appears in the egg bound to yolk biotin-binding protein and avidin. Over 60% of the radioactivity is eventually deposited in eggs. The kinetics of biotin deposition in the egg suggests a 25 day half-life for an intracellular biotinyl-coenzyme pool in the laying hen.     

Identification and molecular characterisation of a biotin-binding protein distinct from avidin of chicken egg white and comparison with yolk biotin-binding protein

By immunological and biochemical methods a biotin-binding protein, distinct from avidin, has been shown to be present in chicken egg white. This vitamin-binding protein (Mr 67,000) bound [14C]biotin, displayed thermally induced biotin exchange reaction and exhibited gross immunological cross-reactivity with the purified yolk biotin-binding protein. In vitro labelling of soluble proteins with radioactive amino acids in the oviduct tissue explants from estrogenised chicks revealed that approx. 2% of the total radioactive proteins was immunoprecipitated with anti-yolk biotin-binding protein antibodies. The protein could be purified to homogeneity by employing ion-exchange chromatography on DEAE-cellulose and biotin-AH Sepharose affinity chromatography. The purified protein specifically bound [14C]biotin, and exhibited complete immunological homology with the yolk biotin-binding protein but not with avidin. Its electrophoretic mobility (at pH 8.3), acidic nature, biotin-binding characteristics, immunological cross-reactivity and tryptic peptide maps were very similar to that of yolk biotin-binding protein, and not avidin.     

A radioimmunoassay for chicken avidin. Comparison with a [14C]biotin-binding method.

A double-antibody solid-phase radioimmunoassay for chicken avidin is reported. Avidin was labelled with 125I by the chloramine-T method. The bound and free avidin were separated with a second antibody bound to a solid matrix. In the logit-log scale the standard curve was linear from 1-2 to 100-200ng of avidin/ml. Cross-reaction of ovalbumin was less than 0.015%. Saturation of biotin-binding sites of avidin with an excess of biotin decreased radioimmunoassay values by about 15%. Recovery studies indicated that avidin can be assayed from all chicken tissues studied with radioimmunoassay, whereas the [14C]biotin/bentonite method gave poor recoveries for avidin in the liver and kidney. Radioimmunoassay and the [14C]biotin/bentonite method gave similar concentrations for oviduct avidin.     

Dietary dependent distribution of acetyl CoA carboxylase between cytoplasm and mitochondria of rat liver

Biotinyl proteins in cytoplasm and mitochondria of rat liver were examined by fluorography and the quantity of acetyl CoA carboxylase was determined after sodium dodecyl sulfate-denatured proteins were incubated with [14C] methyl avidin and separated by polyacrylamide gel electrophoresis. Results show that one-half of the total acetyl CoA carboxylase in liver of fed rats was associated with mitochondria in a relatively inactive form. Fasting shifted the distribution of the enzyme toward the mitochondrial fraction and refeeding previously fasted rats shifted the distribution towards cytoplasm. Thus, acetyl CoA carboxylase can be added to the list of ambiquitous enzymes whose subcellular distribution varies with physiological conditions.     

Use of avidin-sepharose to isolate and idenify biotin polypeptides from crude extracts

A method of isolating and identifying biotin polypeptides from crude cellular extracts is described. Protein samples are run on small avidin-Sepharose columns. After washing away nonspecifically bound protein, the biotin enzymes are eluted using an SDS-urea solution. The inactive polypeptides are then electrophoresed on SDS-polyacrylamide gels. The biotin polypeptides in the gels are identified by using fluorescent avidin or by analyzing for radioactive biotin-labeled polypeptides. A sensitive method for assaying biotin using avidin-Sepharose is also described.     

Site-directed alkylation of cysteine to test solvent accessibility of membrane proteins

This protocol describes a detailed method to study the static and dynamic features of membrane proteins, as well as solvent accessibility, by utilizing the lactose permease of Escherichia coli (LacY) as a model. The method relies on the use of functional single-Cys mutants, an affinity tag and a PhosphoImager. The membrane-permeant, radioactive thiol reagent N-[ethyl-1-14C]ethylmaleimide ([14C]NEM) is used to detect site-directed alkylation of engineered single-Cys mutants in situ. The solvent accessibility of the Cys residues is also determined by blockage of [14C]NEM labeling with membrane-impermeant thiol reagents such as methanethiosulfonate ethylsulfonate (MTSES). The labeled proteins are purified by mini-scale affinity chromatography and analyzed by gel electrophoresis. Gels are dried and exposed to a PhosphoImager screen for 1-5 d, and incorporation of radioactivity is visualized. Initial results can be obtained in 24 h.     

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli

This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with [14C]penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed that the serine residue in the middle of the sequence was covalently bonded to the [14C]penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented.     

Pyruvate carboxylase: isolation of the biotin-containing tryptic peptide and the determination of its primary sequency

The biotin-containing tryptic peptides of pyruvate carboxylase from sheep, chicken, and turkey liver mitochondria have been isolated and their primary structures determined. The amino acid sequences of the 19 residue peptides from chicken and turkey are identical and share a common sequence of 14 residues around biocytin with the 24-residue peptide isolated from sheep. The sequences obtained were: residue 1 → 11 Avian: Gly Ala Pro Leu Val Leu Ser Ala Met Biocytin Met Sheep: Gly Gln Pro Leu Val Leu Ser Ala Met Biocytin Met residues 12 → 19 or 24 Avian: Glu Thr Val Val Thr Ala Pro Arg Sheep: Glu Thr Val Val Thr Ser Pro Val Thr Glu Gly Val Arg A sensitive radiochemical assay for biotin was developed based on the tight binding of biotin by avidin. The ability of zinc sulfate to precipitate, without dissociating, the avidin-biotin complex provided a convenient procedure for separating free and bound biotin, and hence, for back-titrating a standard amount of avidin with [¹⁴C]biotin.     

Availability of avidin-bound biotin to the chicken embryo.

Avidin, an exceptionally stable protein in egg white, binds the vitamin biotin with very high affinity and can induce biotin deficiency when fed to animals. To determine if biotin bound to avidin is available to the chicken embryo, the fate of [3H]biotin complexed to avidin was monitored during embryonic development. The majority (greater than 85%) of the [3H]biotin was extraembryonic until the day before hatching, when embryos swallow egg white and withdraw the yolk sac into their abdomen. Thus, biotin in the egg white of chicken eggs contributes little to the biotin status of the chick prior to hatching. After hatching, much of the [3H]biotin was assimilated. About 30% of the total was found in the liver and kidneys by 4 days of age. The biotin in liver was associated with large proteins and not with avidin. In a separate experiment, biotin injected into the egg white of biotin-deficient eggs failed to increase embryonic development or hatchability. Both experiments suggest that biotin in egg yolk is the primary and virtually sole source of biotin for the chicken embryo.     

Studies on the flavins in rat liver mitochondrial outer membranes

The incorporation of radioactivity derived from [2-¹⁴C] riboflavin into the flavins of rat liver mitochondrial outer membranes was studied. These membranes were found to contain about 0.6 nmol of non-covalently bound flavins per mg protein; the majority is in the form of FAD (73%) and FMN (24%). The membranes also contain about 1.5 nmol per mg of covalently bound flavins.After labeling, radioactive flavins appeared in the non-covalently bound flavins for about 4 h. Most of this radioactivity was in FAD (77%). Neither the rate nor extent of this labelling was affected by cycloheximide (1 mg/kg) administered 30 min prior to the radioactive riboflavin. With the covalently bound flavins, radioactivity was incorporated into the coenzymes for at least 18 h, but the rate of incorporation was much slower. After cycloheximide, radioactive flavins continued to appear in covalently bound flavins for about 2 h, but then stopped. Labeling of both types of flavins after [¹⁴C] riboflavin was considerably slower than the incorporation of [³H] leucine into outer membrane proteins. These results suggest that with flavoproteins from the mitochondrial outer membranes, the incorporation of flavins occurs after synthesis of the various apoenyzmes is complete.     

Molecular weights of subunits of acetyl CoA carboxylase in rat liver cytoplasm

Monomeric [14C] methyl avidin was shown to bind to sodium dodecyl sulfate-denatured biotinyl proteins and remain bound through polyacrylamide gel electrophoresis which allowed their detection by fluorography. This method was used to show that purified rat liver acetyl CoA carboxylase contained two high molecular weight forms of the enzyme (MR = 241,000 and 252,000) while rapidly prepared, crude rat liver cytoplasm contained two larger molecular weight (MR = 257,000 and 270,000) forms. Thus, the enzyme had undergone substantial proteolysis during purification. The crude enzyme preparation also contained a smaller biotinyl protein (MR = 141,000) which is likely a proteolytic product of the larger forms of acetyl CoA carboxylase.     

Hepatic synthesis of carnitine from protein-bound trimethyl-lysine. Lysosomal digestion of methyl-lysine-labelled asialo-fetuin.

The biosynthesis of carnitine in the rat was studied by following the metabolism of two radioactive derivatives of asialo-fetuin. The first contained 14C-labelled methyl groups covalently bound to the 6-N-amino fraction of its lysine residues as 6-N-monomethyl- and dimethyl-lysine. By treating this protein with iodomethane, a second derivative was produced in which the radioactivity was preferentially incorporated as 6-N-[Me-14C]-trimethyl-lysine. These desialylated glycoproteins, like other asialo-proteins, were immediately cleared from the blood by rat liver. Within hepatocyte lysosomes, the 14C-labelled proteins were rapidly hydrolysed, producing free amino acids containing the various 6-N-[Me-14C]methylated lysine residues. The radioactive amino acids crossed the lysosomal membrane and were further metabolized in the cytosol. Carnitine was the major radioactive metabolite detected in extracts of the rat carcass and liver after intravenous injection of 6-N-[Me-14C]trimethyl-lysine-labelled asialo-fetuin. Within 3h, at least 34.6% of the trimethyl-lysine in the administered protein was converted into carnitine. Similarly, an isolated perfused rat liver converted 30% of the added peptide-bound trimethyl-lysine into carnitine within 90 min. On the other hand, in numerous attempts we failed to detect radioactive carnitine in both rat liver and carcass between 20 min and 22 h after injection of 6-N-[Me-14C]-monomethyl- and -dimethyl-lysine-labelled asialo-fetuin. These data provide evidence for a pathway of carnitine biosynthesis that involves trimethyl-lysine as a peptide-bound precursor as proposed by R.A. Cox & C.L. Hoppel [(1973) Biochem. J. 136, 1083-1090] and V. Tanphaichitr & H.P. Broquist [(1973) J. Biol. Chem. 248, 2176-2181]. The findings also show that rat liver can synthesize carnitine without the aid of other tissues, but cannot convert free partially methylated lysines into trimethyl-lysine.     

Determination of evolved 14CO2 in decarboxylase reactions with application to measurement of [14C]oxalic acid.

An assay is described for the determination of the radioactive purity of [14C]oxalic acid preparations and the quantity of [14C]oxalic acid in biological samples. In this method oxalate decarboxylase is used to convert oxalate to formate and CO2. The entire procedure is carried out in a scintillation vial. The 14CO2 released in the enzymic reaction is allowed to diffuse off in a fume hood following acidification. Scintillation fluid is added to reacted and unreacted vials and the radioactivity measured. The loss of radioactivity from the reacted versus the unreacted vials provides the quantity of evolved 14CO2. This value is equal to 50% of the [14C]-oxalate (dpm) present. The radioactive purity of four preparations of [U-14C]oxalic acid was 99.0% while a fifth batch had a purity of 88%. A single batch of [U-14C]oxalic acid had a radioactive purity of 99.0% following storage of an aqueous solution, at -20 degrees C for 7 years. Recovery of [14C]oxalic acid from rat fecal extracts was 101.3%. Eight replicate analyses of a [U-14C]oxalic acid preparation gave a coefficient of variation of 0.3%. Following subcutaneous infusion of [U-14C]oxalic acid to rats, 100.2 +/- 2.9%, mean +/- SD, of the 14C in fecal extracts was present as [14C]oxalic acid (n = 10). The procedure provides a rapid, sensitive, and specific method to determine [14C]oxalic acid. It avoids the time consuming and inconvenient procedure for trapping and counting the evolved 14CO2. The approach used to determine the evolved 14CO2 may find application in other radiochemical methods that require its measurement.     

Relationship between biotin-binding proteins from chicken plasma and egg yolk.

The plasma of laying hens contains a specific biotin-binding protein that appears to be identical with an egg-yolk biotin-binding protein. Both proteins are saturated with biotin and require elevated temperatures to effect the exchange of [14C]biotin for the protein-bound vitamin. The heat-exchange curve in each case is the same and differs sharply from that of avidin, the egg-white biotin-binding protein. On Sephadex G-100 gel filtration, plasma and yolk biotin-binding proteins were each eluted slightly ahead of avidin (mol.wt. 68,000), suggesting that they are of similar molecular weight. Plasma and yolk biotin-binding proteins required the same ionic strength to be eluted from a phosphocellulose ion-exchange column. Both the plasma and yolk biotin-binding proteins had a pI of 5; avidin has a pI of 10. Plasma biotin-binding protein cross-reacted with antiserum to yolk biotin-binding protein and showed a precipitin line of identity with purified yolk biotin-binding protein. It is suggested that biotin-binding plays an important role in mediating the transport of the vitamin from the bloodstream to the developing oocyte.     

5'-Fluorosulfonylbenzoyl derivatives of therapeutic nucleoside analogs

Several fluorosulfonylbenzoyl /FSB/ purine and pyrimidine nucleoside analogs of the clinically useful antimetabolites which belong to endo affinity labeling compounds were synthetized. Structures were confirmed by both 1H NMR and UV spectroscopy and by elemental analysis. Procedure for preparation of microamount of [3H] FSB-araC was developed. Bonding of radioactive FSB-araC to nucleotide utilizing enzymes in K562 myeloblasts soluble protein extract was compared with araCTP-degradating activities in this extract after DEAE-cellulose column chromatography. There was found that five major peaks of the radioactivity bound to the protein coincident with peaks of araCTP degradation. Bonding of [3H] FSB-araC in CCRF-CEM lymphoblasts exhibits only 3 major peaks of the bound radioactivity. This result correlates with the higher sensitivity of the CEM cells to araC.     

A chip-based method for studying the effects of exogenous proteins on neuronal axons

It has been reported that the activity of protein improved when it was adsorbed inside the pores of mesoporous silica (MPS). The current study investigated the activity of immobilized avidin to the biotin on MPS with various pore sizes (diameter=2.4-45.0 nm). The binding amount of immobilized avidin to biotin is 123 to 160 ng biotin/10 μg avidin on 2.7- to 5.4-nm pore MPS, but that on 12- to 45-nm pore MPS was markedly decreased (33-42 ng biotin/10 μg). Moreover, the binding amount was approximately 2- and 3-fold higher on the glycidoxypropyl (Gly)-functionalized 5.4- and 45-nm pore MPS in comparison with methyl (Me)-functionalized 5.4- and 45-nm pore MPS, respectively. Furthermore, avidin immobilized in native and Gly-grafted 45-nm pore MPS retained more than 70% and 50% binding activity to biotin, respectively, after incubating at 90°C for 3 h. In contrast, the activity was greatly reduced in the native and Gly-grafted 5.4-nm pore MPS under the same conditions (<36.9%). The immobilization also protected against effects of 0.01 M HCl and 50% MeOH; all of immobilized avidin proteins showed high activity (>50%) with biotin compared with that observed with free avidin (MeOH [<18.2%] and HCl [<32.7%]).     

Genetic engineering, expression, and activity of a chimeric monoclonal antibody-avidin fusion protein for receptor-mediated delivery of biotinylated drugs in humans

The genetic engineering, expression, and validation of a fusion protein of avidin (AV) and a chimeric monoclonal antibody (mAb) to the human insulin receptor (HIR) is described. The 15 kDa avidin monomer was fused to the carboxyl terminus of the heavy chain of the HIRMAb. The fusion protein heavy chain reacted with antibodies specific for human IgG and avidin, and had the same affinity for binding to the HIR extracellular domain as the original chimeric HIRMAb. The fusion protein qualitatively bound biotinylated ligands, but was secreted fully saturated with biotin by COS cells, owing to the high level of biotin in tissue culture medium. Chinese hamster ovary (CHO) cells were permanently transfected with a tandem vector expressing the fusion protein genes, and high expressing cell lines were isolated by methotrexate amplification and dilutional cloning. The product expressed by CHO cells had high binding to the HIR, and migrated as a homogeneous species in size exclusion HPLC and native polyacrylamide gel electrophoresis. The CHO cells were adapted to a 4 week culture in biotin depleted medium, and the HIRMAb-AV fusion protein expressed under these conditions had 1 unoccupied biotin binding site per molecule, based on a [3H]-biotin ultrafiltration assay. The HIRMAb-AV increased biotin uptake by human cells >15-fold, and mediated the endocytosis of fluorescein-biotin, as demonstrated by confocal microscopy. In summary, the HIRMAb-AV fusion protein is a new drug targeting system for humans that can be adapted to monobiotinylated drugs or nucleic acids.     

Acetyl-CoA pathway of autotrophic growth. Identification of the methyl-binding site of the CO dehydrogenase

CO dehydrogenase, a key enzyme of the acetyl-CoA pathway of autotrophic growth, has been methylated using 14CH3I or 14CH3-corrinoid enzyme plus ferredoxin. Acetyl-CoA was synthesized from the resulting 14CH3-CO dehydrogenase, CO, and CoASH, with about 50% yield of the available 14C and without addition of other enzymes except CO dehydrogenase disulfide reductase. Even the reductase could be replaced by dithioerythritol. Amino acid analysis of the 14CH3-CO dehydrogenase showed two radioactive peaks, one of which migrated as S-methylcysteine but very close to the methyl ester of glutamic acid. By oxidation with H2O2, the radioactive component of this peak was identified as S-methylcysteine sulfone. Amino acid analysis of the 14CH3-CO dehydrogenase after synthesis of acetyl-CoA demonstrated that there was a large decrease in radioactivity of the peak containing the S-methyl-cysteine. The compound present in the second peak has not been identified; there was no decrease in its radioactivity. By nonreducing gel electrophoresis of the 14CH3-CO dehydrogenase, followed by autoradiography, it was shown that the beta subunit is the methyl acceptor. These results demonstrate that a cysteine of the beta subunit is the methyl acceptor and that CO dehydrogenase per se catalyzes the synthesis of acetyl-CoA.     

The use of avidin-biotinylglycan as the model for in vitro glycoprotein processing

In an attempt to evaluate the effects of the protein matrix on the specificity of glycoprotein processing in Golgi membranes, we have developed a model neoglycoprotein consisting of biotinylated glycans bound noncovalently to avidin (Chen, V. J., and Wold, F. (1986) Biochemistry 25, 939-444) with which the protein effect on processing can be evaluated as the difference in substrate efficiency between a free biotinylated glycan and the same biotinylated glycan bound to avidin. The avidin (streptavidin)-glycan complex stability was found to be proper for the experimental design; the complex remains intact for extended periods of incubation at the concentrations used, but the glycan can be completely liberated and recovered by heating the complex at 95 degrees C for 10 min in the presence of a 10-fold molar excess of biotin. By measuring the relative rates of [14C]sugar incorporation into the free and bound substrates it was demonstrated that the protein indeed influences the processing reactions; under conditions where free glycans such as biotinyl-Asn-Glc-NAc2-Man5 and 6-(biotinamido)hexanoyl-Asn-Glc-NAc2-Man5 could be converted to the biantennary products R-Asn-GlcNAc2-Man3-GlcNAc2-Gal2-sialyl2 in the presence of UDP-GlcNAc, UDP-Gal and CMP-sialic acid and Golgi enzymes, the avidin-bound derivative without the extension arm gave only low levels of product and the streptavidin-bound one remained unaltered. The presence of the extension arm in the substrates significantly improved the yield of some products in the complex, apparently by reducing or eliminating the avidin inhibition of the early steps, but not of the late ones. There are consequently two types of effect of the protein matrix on processing efficiency. One is expressed only when the glycan is close to the protein surface and affecting primarily early steps (mannosidases and GlcNAc transferases). The other is apparently independent of the proximity of the glycan core and the protein, and affects primarily late steps, in particular the incorporation of the second sialic acid residue into a biantennary complex glycan.     

Preparation and characterization of a fully active biotinylated probe of cholecystokinin

In this study we describe the synthesis and purification of biotinylated cholecystokinin-8 (Bio-CCK-8) and characterize its use as a probe for the pancreatic cholecystokinin receptor. CCK-8 (0.1 umoles) was reacted with either radiolabeled d-[8,9(-3)H]biotin succinimide ester (0.5 umoles) or N-hydroxysuccinimidyl-biotin in dimethylformamide and triethylamine, and purified by anion exchange chromatography. Concentrations of Bio-CCK-8 and CCK-8 needed for half-maximal inhibition of [125]I-CCK-8 binding to pancreatic membranes were the same (1.0 and 1.3 nM). Bio-CCK-8 retained full biological activity as determined by stimulation of pancreatic protein secretion from rats, and the biotin group bound to CCK-8 retained its high sensitivity for avidin.