Thomas Fairwell

The amino acid sequence of human APOA-I, an apolipoprotein isolated from high density lipoproteins.

Abstract The complete amino acid sequence of human A-I has been determined by manual and automated Edman degradation of intact and peptide fragments of A-I. A-I is a single chain protein of 243 residues with the following amino acid composition: Asp16, Asn5, Thr10, Ser15, Glu27, Gln19, Pro10, Gly10, Ala19, Val13, Met3, Leu37, Tyr7, Phe6, Trp4, Lys21, His5, and Arg16. The amino acid sequence contains no linear segments of hydrophobic or hydrophilic residues. A detailed correlation of the amino acid sequence, conformation, and self association of A-I will add further insight into the molecular mechanisms involved in protein-protein and protein-lipid interactions.

Peptides of postulated inhibin activity: Lack of in vitro inhibin activity of a 94-residue peptide isolated from human seminal plasma, and of a synthetic replicate of its C-terminal 28-residue segment

A 94‐residue polypeptide isolated from human seminal plasma and its chemically synthesized C‐terminal 28‐residue segment were studied in an in vitro inhibin bioassay utilizing rat pituitary cell cultures. Both peptides have previously been claimed to have inhibin activities, and the effects on the secretion and cellular content of gonadotrophins (FSH and LH) were now assessed in the in vitro assay. No inhibition was found. After 72 h of culture, both the cellular content and the spontaneous as well as the LHRH‐stimulated release of bioactive or immunoactive FSH and LH remained unaffected. Similarly, no effects were found on the storage and/or release of prolactin, growth hormone, or thyrotropin. We conclude that both the native 94‐residue peptide and the synthetic replicate of its C‐terminal 28‐residue segment, do not influence the pituitary FSH secretion when assessed in this in vitro system.

Recent studies on the chemistry of human, bovine and porcine parathyroid hormones

Abstract The amino acid sequence of the NH 2 -terminal 34 residues of human parathyroid hormone (PTH) has been determined and duplicated synthetically to produce a peptide that is biologically active. In the amino acid sequences of the bovine and porcine hormones, the glutamic acid function at position 22 has been revised to glutamine. Among these initial 34 residues, human PTH differs from bovine PTH by 5 residues and from porcine PTH by 4 residues. Native human PTH and the synthetic human PTH (1–34) peptide are not rigid structures, and significant changes in conformation were observed during pH titration. In addition, at physiologic pH, native human PTH appeared to differ in structure from human PTH (1–34) in the region of the tryptophan residue (residue 23). The fluorescence spectrum of human PTH revealed a maximum at 344 nm, but the spectrum of human PTH (1–34) had a peak at 343 nm; the spectrum of human PTH (1–34) was normalized to 346 nm in 6 M guanidine hydrochloride, but there was no shift with the intact hormone. Fluorescence titration of human PTH in the alkaline region revealed no loss of tryptophanyl fluorescence in aqueous solution or in 6 M guanidine hydrochloride. The synthetic human PTH (1–34) peptide, however, showed an approximately 25 per cent loss of indole fluorescence during alkaline titration which could be normalized with denaturing reagents. These studies suggest that synthetic fragments of the native hormone may not have the same tertiary conformation as the same sequence in the intact hormone. These findings may be of major significance with regard to the biologic activity and immunologic cross reactivity of synthetic fragments and the native hormone.

Human plasma proapoa-I: Isolation and amino-terminal sequence

Human apoA-I is synthesized as preproapoA-I, a 267 amino acid precursor apolipoprotein. PreproapoA-I initially undergoes intracellular co-translational proteolytic cleavage into proapoA-I. ProapoA-I is secreted from the cell and was isolated from thoracic duct lymph in the apoA-I1 isoform position. The amino-terminal sequence of proapoA-I isolated from human lymph revealed the presence of 6 additional amino acids, Arg-His-Phe-Trp-Gln-Gln, on the amino-terminal end of apoA-I consistent with the proapoA-I sequence determined by nucleic acid sequence analysis of cloned apoA-I. Our results indicate that proapoA-I is present in human plasma, and undergoes post-translational proteolytic cleavage to mature plasma apoA-I.

Human proapoA-ITangier: isolation of proapoA-ITangier and amino acid sequence of the propeptide.

The metabolic defect in Tangier disease is an increased catabolism of apoA-ITangier. The plasma concentration of proapoA-ITangier (apoA-I1 isoform) is increased in patients with Tangier disease. ProapoA-ITangier has been purified to homogeneity, and the amino acid sequence of the propeptide determined by automated Edman degradation. The propeptide sequence was Arg-His-Phe-Trp-Gln-Gln which is identical to the propeptide sequence of normal proapoA-I. These studies indicate that the increase in plasma proapoA-ITangier is not due to a structural defect in the propeptide sequence of proapoA-ITangier and a defect in conversion of proapoA-ITangier to mature apoA-ITangier. The increased catabolism of apoA-ITangier is due to a primary structural defect in mature apoA-ITangier.

Differences in α-amino acetylation of isozymes of yeast alcohol dehydrogenase

Native proteins and peptides often have acetylblocked oi-amino groups. This N-terminal modification was first discovered in a viral coat protein [I ] and a hormonal peptide [2] but is now known to be very common, affecting many classes of naturally occurring polypeptide chains [3]. The proteins are usually recovered m completely blocked form [3], and the modification is enzymatically performed on the nascent polypeptide chain with acetyl-CuA [4]. The blockage can be prevented during translation in cellfree systems by artificial removal of acetyl-CoA [5]. Once attached, however. the Niu-acetyl group is comparatively inert [6]. The functional significance of acetylation is unknown, but it has been suggested that it may protect against premature protein catabolism [3]. Yeast alcohol dehydrogenase normally has an acyl-blocked N-terminus [7]. Acetylatlon of this protein was therefore likely but not directly shown until the present report. During studies of the two major lsozymes, it was discovered that the N-terminus of the protein subumts may also be recovered in unblocked form under certain physiological conditions [8]. Although isozyme separations wer-e incomplete, the presence of unblocked molecules appeared to be associated with growth conditions and isozyme patterns. Since both blocked and unblocked (acetylated and unacetylated) molecules have activity, this finding provided an opportunity to investigate the possible roles of acetylation in protein metabolism and enzyme function. We show here first that both isozyme I and 11 of yeast alcohol dehydrogenase are indeed normally

The chemical ionization mass spectrometric analysis of phenylthiohydantoin and 2-anilino-5-thiazolinone amino acids obtained from the Edman degradation of proteins and peptides

Abstract The development of accelerated automated methods for the sequence analysis of peptides and proteins has resulted in the need for an unambiguous and rapid method for identification of phenylthiohydantoin amino acids obtained from the Edman degradation. Chemical ionization mass spectrometry has been shown to be an extremely rapid, sensitive, and reliable method for the identification of all amino acid derivatives. Phenylthiohydantoin amino acids are identified by their protonated molecular ion or fragment ions which are characteristic of each amino acid derivative. This method can be used for identification of thiohydantoin as well as the thiazolinone derivatives of amino acids. Currently the chemical ionization method represents the fastest, easiest, and most versatile single method for identification of these amino acid derivatives. This method should facilitate the development of a totally automated system for the sequence analysis of peptides and proteins.

Acetyl-blocked N-terminal structures of sorbitol and aldehyde dehydrogenases

Two new dehydrogenase structures, the 354‐residue polypeptide chain of sorbitol dehydrogenase (from sheep liver) and the 500‐residue polypeptide chain of cytoplasmic aldehyde dehydrogenase (from human liver), have blocked N‐termini. The N‐terminal peptides were purified by reverse‐phase high‐performance liquid chromatography and submitted to mass spectrometry after derivatization. They were also analyzed by dipeptidyl carboxypeptidase digestion, utilizing gas chromatography‐mass spectometry for dipeptide identifications. Results are consistent and establish that sorbitol dehydrogenase has N‐terminal acetylalanine and aldehyde dehydrogenase N‐terminal acetylserine in amino acid sequences that are compatible with estimates from chemical analyses. The two N‐terminal residues found are typical of acetylated proteins in general, extend the group of known acetylated dehydrogenases, and show that these intracellular proteins are frequently N‐terminally acetylated.

Acetylated N-terminal structures of class III alcohol dehydrogenases Differences among the three enzyme classes

The protein chains of mammalian alcohol dehydrogenases typically lack free α‐amino groups. The blocked N‐terminal regions of the class III type of the rat (ADH‐2), human (χχ) and horse enzymes were isolated by digestions with proteases, and characterized by mass‐spectrometry supplemented with chemical analysis of the peptides and their redigestion fragments. Results were confirmed by synthesis of the corresponding peptides, followed by chromatographic comparisons of the native and synthetic products. The N‐terminal regions of the three class III alcohol dehydrogenase subunits are homologous but differ from the class I and II enzymes in both the exact start position and the amino acid sequence, which suggests that different N‐terminal structures are typical for each of the three classes.

Automated Edman degradations: studies with a large sequencer cup and high-speed drive.

The major limitations with the current methodology employed in the automated Edman degradation of polypeptides and proteins is the progressive increase in overlap of penultimate amino acid residues during degradation and the appearance of new amino-terminal amino acid residues resulting from nonspecific cleavage of peptide bonds during acid cleavage. These limitations have been significantly reduced with automated degradations performed on a modified Beckman 890B sequencer equipped with a large reaction cup (75% increase in surface area), high speed drive (1800/3600 rpm), and cold trap attached to the high vacuum pump. Automated Edman degradations performed with the modified Beckman sequencer on apomyoglobin and bovine parathyroid hormone were of higher repetitive yield, and the individual steps in the sequence contained significantly less overlap and nonspecific background of amino acids due to acidolysis. Degradations with the modified sequencer on apomyoglobin were routinely performed for 65–70 cycles, and 2 to 20 mg of protein was efficiently sequenced with the large reaction cup. With the new modified sequencer, a single degradation can be extended further down the protein chain, and large as well as small proteins can be degraded. This improved system will facilitate the structural analysis of polypeptides and proteins and will reduce the necessity for extensive cleavage of proteins for overlapping peptide fragments.