Calmodulin tagging provides a general method of using lanthanide induced magnetic field orientation to observe residual dipolar couplings in proteins in solution.

A general method is presented for magnetic field alignment of proteins in solution. By tagging a target protein with calmodulin saturated with paramagnetic lanthanide ions it is possible to measure substantial residual dipolar couplings (RDC) whilst minimising the effects of pseudocontact shifts on the target protein. A construct was made consisting of a calmodulin-binding peptide (M13 from sk-MLCK) attached to a target protein, dihydrofolate reductase in this case. The engineered protein binds tightly to calmodulin saturated with terbium, a paramagnetic lanthanide ion. By using only a short linker region between the M13 and the target protein, some of the magnetic field alignment induced in the CaM(Tb3+)4 is effectively transmitted to the target protein (DHFR). 1H-15N HSQC IPAP experiments on the tagged complex containing 15N-labelled DHFR-M13 protein and unlabelled CaM(Tb3+)4 allow one to measure RDC contributions in the aligned complex. RDC values in the range +4.0 to -7.4 Hz were measured at 600 MHz. Comparisons of 1H-15N HSQC spectra of 15N-DHFR-M13 alone and its complexes with CaM(Ca2+)4 and CaM(Tb3+)4 indicated that (i) the structure of the target protein is not affected by the complex formation and (ii) the spectra of the target protein are not seriously perturbed by pseudocontact shifts. The use of a relatively large tagging group (CaM) allows us to use a lanthanide ion with a very high magnetic susceptibility anisotropy (such as Tb3+) to give large alignments while maintaining relatively long distances from the target protein nuclei (and hence giving only small pseudocontact shift contributions).

Synthesis and high-performance liquid chromatographic purification of tritiated thyrotrophin-releasing hormone-like peptides.

A simple method is presented for the synthesis and RP-HPLC purification of tritium-labelled thyrotrophin-releasing hormone (TRH)-like tripeptides. These peptides differ from TRH (pGlu-His-Pro-amide) in that they possess a neutral or acidic residue in place of the histidine of TRH. The method involves the preparation of the appropriate dipeptide by a solid-phase peptide synthesis procedure using 9-fluorenylmethoxycarbonyl (Fmoc) protection. Very small amounts of tritiated glutamine are then converted into tritiated pyroglutamic acid, and coupling to the dipeptide is effected using a mixed anhydride derived from Fmoc-phenylalanine and the tritiated pyroglutamic acid. The required labelled product is then separated from unlabelled material by reversed-phase HPLC, as the hydrophobicity of the phenylalanine-containing product ensures that it is strongly retained. The availability of a series of tritium-labelled markers prepared by this method has permitted the unequivocal identification of certain naturally occurring TRH-like peptides.

Peptide amidation.

Many hormones, neurotransmitters and growth factors are peptides that carry an amide group at their carboxyl terminus which is essential for their biological activity. The amide is formed by hydroxylation of an additional glycine residue present in the biosynthetic precursor and the hydroxyglycine derivative dissociates to form the peptide amide and glyoxylic acid. Recent discoveries have shown that two enzymes are involved that act sequentially.

4-Phenyl-3-butenoic acid, an in vivo inhibitor of peptidylglycine hydroxylase (peptide amidating enzyme).

The ability of a series of non-peptide carboxylic acids to act as substrates or inhibitors of the peptide-amidating enzyme (peptidyl-glycine hydroxylase) was assessed by determining their ability to reduce the rate of enzymic conversion of D-tyrosyl-valyl-glycine or D-tyrosyl-phenylalanyl-glycine to the corresponding dipeptide amide. The inclusion of a phenyl substituent in a position distal to the carboxyl group promoted the inhibitory action. The inhibition was found to be irreversible when an olefinic double bond, alpha or beta to the carboxyl group, was present in the molecule; the inhibition appeared to be associated with a covalent interaction between the amidating enzyme and the inhibitor. With 4-phenyl-3-butenoic acid the inhibitory properties were manifest only in the presence of cofactors of the enzyme. When 4-phenyl-3-[2-14C]butenoic acid was used, the radioactivity was shown to be incorporated into protein that co-chromatographed with active enzyme. Incubation of rat thyroid carcinoma CA77 cells in the presence of 4-phenyl-3-butenoic acid led to a decrease in the levels of intracellular amidating activity and of thyrotropin-releasing hormone, an amidated peptide produced by these cells. The inhibitory effects reached a maximum at approximately 15 h after which the enzyme levels returned to the control values even though the concentration of 4-phenyl-3-butenoic acid in the cells remained unchanged. The results indicate that a mechanism exists in these cells for regulation of amidating activity.

Peptide amidation: evidence for multiple molecular forms of the amidating enzyme.

Amidating enzyme extracted from porcine pituitary was separated into glycosylated and non-glycosylated forms by fractionation on a column of Concanavalin-A Sepharose. The molecular weights of the species present were assessed by HPLC gel exclusion chromatography, which demonstrated that both the glycosylated and the non-glycosylated forms of the enzyme comprise multiple components. The apparent molecular weights of the non-glycosylated forms ranged from approximately 35 kDa to 100 kDa; the glycosylated enzyme contained species with molecular weights ranging from 65 kDa to 135 kDa. Similar proportions of glycosylated to non-glycosylated enzyme (approximately 1:4) were found in the anterior and posterior regions of the pituitary; higher proportions (approximately 1:1) were observed in the thyroid, adrenals and pancreas. The glycosylated forms of the amidating enzyme were shown to exhibit the same mandatory requirement for copper as the non-glycosylated forms, and no differences were seen in respect of their stimulation by dopamine or their pH optima. Both forms catalysed the hydroxylation of glyoxylic acid phenylhydrazone, indicating a common mechanism of action. By these criteria, glycosylation does not affect the activity of the amidating enzyme.

Substrate recognition by UDP-N-acetyl-alpha-D-galactosamine: polypeptide n-acetyl-alpha-D-galactosaminyltransferase. Effects of chain length and disulphide bonding of synthetic peptide substrates.

A synthetic peptide AcTPPP, based on a threonine-containing sequence present in bovine myelin basic protein, is a potent acceptor of glycosyl transfer from UDP-N-acetylgalactosamine catalyzed by extracts of baby hamster kidney (BHK) cells or rabbit lymph node tissue. In contrast, the disulphide-linked peptide (AcTCPPP)2, based on a glycosylated sequence present in the hinge region of rabbit immunoglobulin G, is not an acceptor and inhibits glycosylation of AcTPPP. Extension of the cystine-containing peptide at the N-terminus produced weak acceptors but strong acceptors resulted when the cystine residue was reduced to form monomeric peptides. The acceptor specificity of the N-acetylgalactosaminyl-transferase activity of BHK cells is very similar to that of rabbit lymph node tissue. The results indicated that tissues actively secreting immunoglobulin do not contain a transferase activity adapted specifically for glycosylation of sequences containing cystine residues, and suggested that addition of an N-acetylgalactosamine to a threonine residue in the hinge region of rabbit immunoglobulin takes place during biosynthesis prior to the formation of the inter-chain disulphide bridge of fully assembled immunoglobulins.

Processing reactions in the later stages of hormone activation.

Three tiers of processing have been investigated in the reactions that transform prohormones into their mature end products. Evidence is presented that the proteolytic reactions that convert lipotropin into shortened forms of beta-endorphin take place in individually distinct stages. After these cleavages have occurred, the removal of basic residues by carboxypeptidase H and amidation of the products are effected by independent reactions which do not synergise. Experiments are also described which show that the amidating enzyme can accept certain imino acids as substrates and utilises a mechanism that involves hydroxylation; it is implicit that peptide amidation proceeds by a similar mechanism. These results point to a general concept that pro-hormone processing involves consecutive reactions which take place in a predetermined order.

Biosynthesis of peptide neurotransmitters: studies on the formation of peptide amides.

A high proportion of peptide transmitters and peptide hormones terminate their peptide chain in a C-terminal amide group which is essential for their biological activity. The specificity of an enzyme that catalyses the formation of the amide was investigated with the aid of synthetic peptide substrates. With peptides containing l-amino acids the enzyme exhibited an essential requirement for glycine in the C-terminal position; amidation did not take place with peptides that had leucine, alanine, glutamic acid, lysine or N-methylglycine at the C-terminus and a peptide extended by the attachment of lysine to the C-terminal glycine did not act as a substrate. Amidation did occur with a peptide containing C-terminal D-alanine but no reaction was detected with peptides having C-terminal, D-serine or D-leucine. In tripeptides with a neutral amino acid in the penultimate position, amidation, took place readily but the reaction was slower when this position was occupied by an acidic or a basic residue. A series of overlapping peptides with C-terminal glycine, based on partial sequences of calcitonin, underwent amidation at similar rates, indicating that the amidating enzyme recognizes only a limited sequence at the C-terminus of its substrates. The results provide evidence that the amidating enzyme has a highly compact substrate binding site.

Enzyme-catalysed peptide amidation. Isolation of a stable intermediate formed by reaction of the amidating enzyme with an imino acid.

A series of hydrazones and semicarbazones of glyoxylic acid were shown to have a potent inhibitory effect on the enzyme-catalysed conversion of D-Tyr-Val-Gly to D-Tyr-Val-NH2. Among the derivatives tested, the inhibitory activity was increased by the presence of hydrophobic substituents and decreased by polar substituents. The inhibition produced by glyoxylic acid phenylhydrazone was shown to be competitive. No inhibition was obtained with pyruvic acid phenylhydrazone, which possesses a methyl group in place of the alpha-H of glyoxylic acid phenylhydrazone. The inhibitory potencies of these non-peptide substances are in accord with the specificity exhibited by the amidating enzyme in its reaction with peptide substrates. The inhibition produced by the glyoxylic acid derivatives was shown to be due to their ability to act as substrates for the peptide-amidating enzyme. The product formed from [14C]glyoxylic acid phenylhydrazone was identified as oxalic acid phenylhydrazide by co-chromatography in three chromatographic systems. The results demonstrate that the enzyme-catalysed oxidation of glyoxylic acid phenylhydrazone takes place by a mechanism involving hydroxylation. It is implicit that peptide amidation catalysed by the same enzyme proceeds by a similar mechanism.

Biosynthesis of the C-terminal amide in peptide hormones.

Recent developments in the study of peptide amidation are reviewed. The main areas covered are assay procedures, purification of amidating enzymes, co-factors and regulation, mechanism and specificity of the amidating reaction, and multiple forms of the amidating enzyme and glycosylation. Discussion is presented on aspects that are poorly understood and new areas open to investigation are indicated.

The amidating enzyme in pituitary will accept a peptide with C-terminal D-alanine as substrate.

A series of tripeptides which terminated in d-alanine, d-serine, d-leucine or l-alanine was synthesized and the peptides tested for their ability to act as substrates for an amidating enzyme present in porcine pituitary. The peptides were allowed to compete with a radiolabelled substrate 125I d-Tyr Phe Gly in the presence of a rate limiting concentration of amidating enzyme and the degree of conversion to 125I d-Tyr Phe amide was determined by ion exchange chromatography. An accelerated procedure was developed for investigating the rates of reaction. The results showed that d-Tyr Phe d-Ala has a significant affinity for the amidating enzyme; no affinity could be demonstrated with d-Tyr Phe 1-Ala, d-Tyr Phe d-Ser or d-Tyr Phe d-Leu. Direct evidence that d-Tyr Phe d-Ala can undergo amidation was obtained by incubating the 125I labelled tripeptide with the pituitary enzyme. Amidation took place readily with d-Tyr Phe d-Ala but not with the other tripeptides; thus, while the enzyme is unable to catalyse the conversion of a peptide terminating in 1-alanine, it can accept a peptide terminating in d-alanine. The results indicate that the amidating enzyme has a highly compact substrate binding site.

Substrate specificity of an amidating enzyme in porcine pituitary.

A series of tripeptides was synthesised and tested as substrates for an amidating enzyme present in porcine pituitary. The rates of conversion of the tripeptides to the corresponding dipeptide amides were determined by ion exchange chromatography of the radio-iodinated peptides. The experiments showed that the amidating enzyme has a mandatory requirement for glycine in position 3 of the tripeptide substrates; peptides containing lysine, glutamic acid, leucine or alanine in the C-terminal position did not undergo reaction. In studies of the substrate requirements at position 2 of the tripeptides, facile reaction took place with neutral amino acids in this position but much slower reactions occurred with basic or acidic residues. With the neutral substrates the enzyme exhibited an optimum pH value of 6.8; with histidine in position 2 the optimum reaction occurred at a higher pH, consistent with a preference shown by the enzyme for an uncharged amino acid in the penultimate position of the peptide substrate.

Prohormones of beta-melanotropin (beta-melanocyte-stimulating hormone, beta-MSH) and corticotropin (adrenocorticotropic hormone, ACTH): structure and activation.

It is proposed that all peptide hormones and releasing factors are biosynthesized in the form of precursor molecules which are biologically inactive. Enzymic activation may take place by hydrolytic cleavage to release a terminal COOH group or by transmidation to form a COOH-terminal amide. Studies with pituitary prohormones and hormones are providing data that support this hypothesis. Evidence has been obtained that the 91 residue beta-lipotropin (beta-LPH) is the prohormone of beta-melanotropin (beta-MSH). The specificity of the pituitary enzymes involved in release of the hormone was demonstrated by the isolation of five constituent fragments of LPH, which were obtained in homogeneous form from the pituitary gland of the pig. The enzymes have specificities similar to trypsin and carboxypeptidase B; carboxypeptidase A and aminopeptidase activities do not appear to be involved. Mild digestion of beta-LPH by trypsin in vitro has confirmed the susceptibility of the peptide bond on the carboxy side of the paired basic residues at positions 59 and 60, adjacent to the COOH-terminus of beta-MSH, and tryptic digestion of a model peptide demonstrated the same specificity. The paired basic residues at positions 39 and 40 adjacent to the NH2-terminus of beta-MSH were more resistant to tryptic attack, both in LPH and in a model peptide. In the gland it is apparent that LPH is cleaved on the carboxy side of the paired lysyl residues at positions 39 and 40, whereas in the synthetic peptide cleavage takes place in between these residues. The activating enzyme may differ from trypsin; alternatively, explanation may be found in the conformation of the prohormone. Prediction of secondary indicates that both pairs of basic residues lie adjacent to beta-bends on the surface of the molecule and occupy sites accessible to enzymic attack. It seems likely that alpha-MSH and corticotropin (ACTH) share a common pro hormone. The release of ACTH could involve cleavage of a -Gly-Ser- bond in the prohormone to expose the NH2-terminus of the hormone. With alpha-MSH, a concerted acetylation and cleavage may take place to form the N-acetylserine residue; the COOH-terminus may be released as an amide by direct transamidation of a -Val-Gly- bond in the prohormone. Release of either hormone would be accompanied by the release of contiguous fragments of the prohormone. We have isolated two novel polypeptides from pig pituitary in substantial quantity and have determined the primary structures. They may represent fragments of a prohormone to alpha-MSH or ACTH.