A novel isotope labeling protocol for bacterially expressed proteins

Summary A novel protocol for isotopically labeling bacterially expressed proteins is presented. This method circumvents problems related to poor cell growth, commonly associated with the use of minimal labeled media, and problems with protein induction encountered, less commonly, when using enriched labeled media. The method involves initially growing the bacterial cells to high optical density in a commercially available enriched labeled medium. Following a suitable growth period, the cells are transferred to a different (minimal) labeled medium, appropriate for induction. The method is demonstrated using the protein melanoma growth stimulating activity (MGSA).     

Affinity labeling of adenine nucleotide-related enzymes with reactive adenine nucleotide analogs. II. Affinity labeling of phosphoglycerate kinase with a reactive AMP analog.

Affinity labeling of yeast and B. stearothermophilus phosphoglycerate kinases with a reactive AMP analog, N6-(p-bromoacetaminobenzyl)-AMP was examined. Complete loss of enzyme activity was observed when 1 mol of the reagent had reacted per mol of either enzyme. Results on the effect of pH and substrate addition on the inactivation, titration of SH groups before and after modification, and kinetic studies with AMP analogs suggest that the modification occurs at one amino group at or near the substrate binding site. General affinity labeling of kinases is discussed based on the results obtained.     

The adenine nucleotide binding site on yeast hexokinase PII. Affinity labeling of Lys-111 by pyridoxal 5'-diphospho-5'-adenosine

The adenine nucleotide analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), is shown to be a potent and specific inhibitor of yeast hexokinase PII. Evidence that the analog binds specifically at the ATP binding site includes the demonstration that glucose binding enhances PLP-AMP binding and that PLP-AMP and ATP bind competitively with an apparent Ki(PLP-AMP) = 23 microM. In addition, from the relationship between the degree of inhibition and extent of modification, it is estimated that the incorporation of 1 mol of PLP-AMP/mol of subunit is required for complete inhibition. Borohydride reduction of the Schiff's base complex formed between hexokinase and [3H]PLP-AMP gives a stable product. The reduced derivative was digested with trypsin and a single radioactive peptide was isolated by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis identified Lys-111 as the modified residue. Taking into account the known structures of the binary complexes (Shoham, M., and Steitz, T. A. (1980) J. Mol. Biol. 140, 1-14), the results suggest that Lys-111, located in the smaller of the two lobes of hexokinase, moves into the active site upon formation of the ternary complex.     

The radioactive labeling of proteins with an iodinated amidination reagent.

An iodinated derivative of the imidoester methyl p-hydroxybenzimidate HCl (MPHBIM) has been synthesized for the selective labeling of proteins to high specific activity with radioactive iodine. In the first step, MPHBIM is reacted with radioactive iodide in the presence of chloramine T, and the iodinated derivative is precipitated from acidified solution to achieve partial purification. In the second step, the iodinated imidoester is redissolved at slightly alkaline pH and reacted with protein amino groups, to which it couples by amidine linkage. The coupling reaction proceeds in the presence of sulfhydryl reagents used to protect proteins. The main advantage of this two-step labeling procedure is that it avoids direct contact of the protein with potentially deleterious materials such as chloramine T or contaminants of the radioactive iodide.     

A new solid-state reagent to iodinate proteins: I. Conditions for the efficient labeling of antiserum

A new solid-state reagent for iodinating polypeptides and proteins was used to radiolabel antiporcine insulin antiserum. Reaction conditions of pH, temperature, buffer, reactant concentration, and the type of reaction vessel were optimized to achieve almost quantitative incorporation of radioiodide (99%), good recovery of protein (92%), and a moderate specific activity (9.9 μCi/μg of antiserum). A second set of conditions was found to produce antiserum of high specific activity (161 μCi/μg) which retained its immunological ability to recognize insulin. The nature of the actual iodinating species and the effects on the reaction of sodium azide, detergents, and chaotropic reagents such as urea and high salt were investigated.     

Synthesis and characterization of phenothiazine labeled oligodeoxynucleotides: novel 2'-deoxyadenosine and thymidine probes for labeling DNA.

A facile procedure for the incorporation of phenothiazine at the terminus of oligodeoxynucleotides is reported. Phenothiazine is covalently linked to the 5'-position of 2'-deoxyadenosine and thymidine. Next, the corresponding phosphoramidites are prepared, and then the labeled nucleosides are incorporated in DNA using an automated DNA solid-phase synthesizer. Phenothiazine labeled oligodeoxynucleotides form stable B-form duplexes with similar melting temperatures, CD spectra, and DSC traces compared to unlabeled DNA duplexes. The favorable photophysical properties of phenothiazine are also retained after covalent attachment to the oligodeoxynucleotide.     

A novel method for selective isotope labeling of bacterially expressed proteins

Summary A novel method for isotope labeling in selected amino acids is presented for use with the T7 RNA polymerase system. The protocol is illustrated with the DNA-binding domain from the E2 protein of bovine papillomavirus, BPV-1. On addition of rifampicin, protein expression occurs exclusively from the gene controlled by the T7 promoter. Since the bacteria are now dedicated to the production of E2 protein, labeling with specific amino acids is efficiently performed. For example, 10 mg/l of ¹⁵N-labeled phenylalanine is shown to be sufficient for incorporation of the label, without scrambling, and without the use of an auxotrophic strain.     

Affinity labeling of adenine nucleotide-related enzymes with reactive adenine nucleotide analogs. I. Affinity labeling of glyceraldehyde 3-phosphate dehydrogenase and myokinase with a reactive AMP analog.

Rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GPD) and myokinase (MK) were rapidly inactivated by a reactive AMP analog, N6-(p-bromoacetaminobenzyl)-AMP, under mild conditions. Complete inactivation was observed when 4 and 0.3 mol of the reagent with respect to enzyme were reacted with GPD and MK, respectively. The inactivation of both enzymes were favored at higher pH and the enzymes were protected by addition of adenine nucleotide substrate. Modified GPD or MK had no affinity for AMP-Sepharose, in contrast to the native enzymes. From these results, the inactivation of GPD and MK by the reactive AMP analog can be regarded as an affinity labeling. The posibility that the present AMP analog may be used as a general affinity labeling reagent for various adenine nucleotide-related enzymes is discussed based on the results obtained.     

Thyroid hormone effect on rat heart mitochondrial proteins and affinity labeling with N-bromoacetyl-3,3',5-triiodo-L-thyronine. Lack of direct effect on the adenine nucleotide translocase

N-bromoacetyl-3,3',5-tri[3'-125I]iodo-L-thyronine was used to label intact heart mitochondria from eu, hypo- and hyperthyroid rats in order to identify proteins involved in T3-regulated mitochondrial processes. The results show strong labeling, competed for by T3 and other analogues, of two proteins with a molecular mass of 48,000 and 49,200 Da. No labeling is seen of the adenine nucleotide translocase, a likely target, neither at 0 degree C, at room temperature, nor after preincubation with the substrates or specific inhibitors. No difference in labeling intensity or distribution is seen in mitochondria from eu-, hypo- or hyperthyroid rats, and the abundance of the adenine nucleotide translocase is unchanged, but five other proteins show differential abundance.     

Adenosine 5'-diphosphate dialdehyde: an affinity labeling reagent for phenol-sulfotransferase

Adenosine 5′-diphosphate (5′-ADP) was oxidized with periodic acid to 2′-O-[(R)-formyl(adenin-9-yl)methyl]-3′-diphosphate-3′-deoxy-(S)-glyceraldehyde (ADP-dialdehyde). ADP-dialdehyde, but not 2′, 3′-acyclic ADP, inhibited phenol-sulfotransferase (PST). The inhibition of PST by ADP dialdehyde was irreversible. A kinetic analysis of the enzyme inactivation suggests the formation of a dissociable enzyme-inhibitor complex prior to the inactivation step. PST could be completely protected from inactivation by the inclusion of 3′-phosphoadenosine-5′-phosphosulfate in the preincubation mixture. These results are consistent with ADP-dialdehyde being an affinity labeling reagent for PST.     

Functional linkage of adenine nucleotide binding sites in mammalian muscle 6-phosphofructokinase

6-Phosphofructokinases (Pfk) are homo- and heterooligomeric, allosteric enzymes that catalyze one of the rate-limiting steps of the glycolysis: the phosphorylation of fructose 6-phosphate at position 1. Pfk activity is modulated by a number of regulators including adenine nucleotides. Recent crystal structures from eukaryotic Pfk revealed several adenine nucleotide binding sites. Herein, we determined the functional relevance of two adenine nucleotide binding sites through site-directed mutagenesis and enzyme kinetic studies. Subsequent characterization of Pfk mutants allowed the identification of the activating (AMP, ADP) and inhibitory (ATP, ADP) allosteric binding sites. Mutation of one binding site reciprocally influenced the allosteric regulation through nucleotides interacting with the other binding site. Such reciprocal linkage between the activating and inhibitory binding sites is in agreement with current models of allosteric enzyme regulation. Because the allosteric nucleotide binding sites in eukaryotic Pfk did not evolve from prokaryotic ancestors, reciprocal linkage of functionally opposed allosteric binding sites must have developed independently in prokaryotic and eukaryotic Pfk (convergent evolution).     

p-Diazobenzoyl biocytin--a new biotinylating reagent for the labeling of tyrosines and histidines in proteins

A new biotin-containing reagent, p-diazobenzoyl biocytin (DBB), has been developed for labeling tyrosines and histidines in proteins. The reagent has used to label these residues in both model proteins and erythrocyte membrane proteins on dot blots and blot transfers. In some cases, sub-nanogram levels of individual proteins could be detected. The utility of DBB as a versatile alternative to biotin-containing N-hydroxysuccinimide esters for the general labeling of proteins is discussed.     

Site-specific modification of a single-chain antibody using a novel glyoxylyl-based labeling reagent.

A novel, highly specific protein modification approach is described. By using conventional molecular cloning techniques, a protein can be constructed and expressed such that the N-terminal residue is replaced by cysteine. Its 1,2-aminothiol structure reacts very specifically with a glyoxylyl group at pH 7 or below, forming a relatively stable thiazolidine bridge. Therefore, a glyoxylyl-based labeling agent (e.g., radioactive tags, fluorescent probes, biotin) can be used to specifically modify a protein at its N-terminus. To highlight this novel approach, a recombinant anti-insulin single chain antibody (scFv) was specifically biotinylated at its N-terminus even in the presence of other proteins in the total cell lysate. The glyoxylyl-biotinylated scFv retained binding activity similar to unmodified scFv.     

A three-step procedure for the isolation of peptides derived from adenine nucleotide binding sites of enzymes labeled with p-fluorosulfonyl [14C]benzoyl-5'-adenosine.

A simple three-step procedure is described for the purification of a labeled peptide from a tryptic digest of the β-subunit of the F₁-ATPase after the enzyme had been inactivated with p-fluorosulfonyl-[¹⁴C]benzoyl-5′-adenosine. The procedure involves: (1) anion-exchange chromatography of a tryptic digest of the labeled β-subunit on diethylaminoethyl-Sephadex; (2) treatment of the peptides in the radioactive peak from the first step with 0.1m NaOH under conditions in which the ester bond in the label is hydrolyzed; and (3) anion-exchange chromatography of the treated peptides under conditions identical to those of the first step after removal of the NaOH by gel filtration. Cleavage of the ester bond in the second step releases adenosine and specifically introduces an additional negative charge onto the labeled peptide. Thus, it is resolved from the peptides that contaminate it in the third step.