Human pituitary growth hormone: immunochemical investigations of biologically active fragments

Guinea pig antisera to human growth hormone were tested for their ability to recognize the two biologically active fragments of the hormone produced by human plasmin digestion and a synthetic active fragment. A precipitin line was obtained with native human growth hormone, plasmin-treated human growth hormone, and its NH₂-terminal fragment (residues 1–134). In the microcomplement-fixation and radioimmunoassay experiments, the NH₂-terminal plasmin fragment (residues 1–134) showed a greater immunoreactivity than the COOH-terminal plasmin fragment (residues 141–191). This, in turn, was more active than the synthetic fragment (residues 95–136).     

Primary structure of equine pituitary prolactin

Equine prolactin was determined to be a single chain protein of 199 amino acid containing two tryptophan and six cysteine residues, as found in other mammalian prolactins. The primary sequence of equine prolactin was obtained by automated Edman analyses of S-carboxymethylated protein and proteolytic fragments of modified protein. Of the known prolactin sequences, equine prolactin shows closest homology with porcine (93%) and fin whale (87-91%) prolactins. Genetic mutations have produced changes in 17 of 199 residues of equine prolactin relative to its putative ancestral precursor. Since equine growth hormone has undergone alterations in 4 of 191 residues relative to this putative precursor protein, these results support the theory that prolactins are evolving at a faster rate than growth hormones. Consistent with the previously determined circular dichroic spectrum of equine prolactin, 60% of the protein is predicted to form alpha helices.     

Comparison of secreted and extracted forms of rat pituitary prolactin

Prolactin secreted by rat anterior pituitary explants into organ culture medium was purified by salt fractionation and gel filtration. A yield of 22 mg/g was obtained, which clearly represented de novo synthesis and secretion of the hormone. Comparative characterization studies were performed on the secreted prolactin and pituitary extracted rat prolactin obtained from the National Institute of Arthritis, Metabolism and Digestive Diseases. The biological and immunological activity estimates of both forms were comparable, although the specific activities of the secreted prolactin were somewhat lower than those of the pituitary prolactin. The secreted and extracted forms of prolactin appeared to be identical in primary structure as evidenced by similar amino acid compositions and identical NH₂-terminal sequences. Circular dichroism spectra suggested that there may be differences in tertiary structure, since the positive tryptophan band at 292 nm, which was observed with extracted hormone, was absent in the secreted prolactin.     

Aspartate transaminase from E. coli: amino acid sequences of the NH2-terminal 33 residues and chymotryptic pyridoxyl tetrapeptide.

The amino acid sequences of pyridoxal-binding tetrapeptide and the NH₂-terminal portion of aspartate transaminase from $\text{E.}$$\text{coli}$ B were analyzed and compared with those of the corresponding parts of the cytosolic and mitochondrial isozymes from pig heart. After borohydride reduction and chymotryptic digestion of the $\text{E.}$$\text{coli}$ enzyme, a pyridoxal-containing peptide was isolated, showing the sequence, Ser-Lys(Pxy)-Asn-Phe, identical with that of the cytosolic isozyme. The NH₂-terminal sequence was determined up to 33 residues with a liquid phase sequence analyzer. Nearly the same degree of homology was observed among the NH₂-terminal sequences of the three aspartate transaminases.     

Crotalus adamanteus phospholipase A2-α: Subunit structure,NH2-terminal sequence,and homology with other phospholipases

Automated Edman degradation of reduced and carboxymethylated phospholipase A₂-α from Crotalus adamanteus venom revealed a single amino acid sequence extending 30 residues into the protein from the amino terminus. The singularity of the sequence and the yields of the phenylthiohydantoin amino acids thus obtained indicate that the subunits comprising the phospholipase dimer are identical. Further chemical evidence in support of subunit identity was obtained by cleavage of phospholipase A₂-α with cyanogen bromide. Compositional analysis of the protein revealed one residue of methionine per monomer and the sequence determination placed this amino acid at position 10 in the sequence of 133 amino acids. Cyanogen bromide cleavage of the protein, followed by reduction and carboxymethylation afforded the expected 2 fragments: an NH₂-terminal decapeptide (CNBr-1) and a larger COOH-terminal fragment of 123 residues (CNBr-II). Automated Edman degradation of the latter has extended the sequence analysis to 54 residues in the NH₂-terminal segment of the monomer chain. Comparison of this sequence with those derived for phospholipases from other snake venoms, from bee venom, and from porcine pancreas has revealed striking homologies in this region of the molecules. As expected on the basis of their phylogenetic classification, the phospholipases from the pit vipers C. adamanteus and Agkistrodon halys blomhoffii are more similar to one another in sequence than to the enzyme from the more distantly related viper, Bitis gabonica. Furthermore, the very close similarities in sequence observed among all of these phospholipases in regions corresponding to residues 24 through 53 in the C. adamanteus enzyme suggest that this segment of the polypeptide plays an important role in phospholipase function and probably constitutes part of the active site.     

Targeting of NH2-terminal–processed Microsomal Protein to Mitochondria: A Novel Pathway for the Biogenesis of Hepatic Mitochondrial P450MT2

Cytochrome P4501A1 is a hepatic, microsomal membrane–bound enzyme that is highly induced by various xenobiotic agents. Two NH₂-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from β-naphthoflavone–induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH₂-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH₂-terminal 30–amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33–44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH₂-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.     

Elephant prolactin: isolation and characterization

Prolactin was isolated from anterior lobes of elephant pituitary glands. It consisted of 199 amino acids with three disulfide bridges and two tryptophan residues as found in prolactin from other species. The sequence of the NH2-terminal 28 amino acids was determined and shown homologous with the ovine hormone. In comparison with ovine prolactin, a marked difference was seen in the methionine content; the elephant hormone possessed only 18-34% lactogenic potency. The conformation of elephant prolactin was examined by zero order, second order and circular dichroism spectroscopy. The alpha helical content was estimated to be about 60%. In comparison with prolactins from other species, the second order spectra of elephant prolactin suggest that the local microenvironment for one or both tryptophan residues is somewhat different.     

Partial amino acid sequence of human thyroxine-binding globulin. Further evidence for a single polypeptide chain.

The NH₂-terminal amino acid of highly purified thyroxine-binding globulin has been identified by dansyl chloride, cyanate and Edman degradation methods. All three gave alanine as the only amino terminal residue. Carbamylation and Edman degradation of the denatured protein yielded 0.86 and 0.98 – 1.05 mole of alanine per mole of protein, respectively. These data further indicate that thyroxine-binding globulin is composed of a single polypeptide chain. Automated Edman degradation gave the partial sequence as: Ala-Ser-Pro-Glu-Gly-Lys-Val-Thr-Ala-Asp-Ser-Ser-Ser-Gln-(Pro)-X-Ala-(Ser)-Leu-Tyr- A computer search revealed no homology of the NH₂-terminal segment of thyroxine-binding globulin with human prealbumin. The NH₂-terminal portion of prealbumin contains part of the thyroxine binding site.     

Effect of cyclic AMP on RNA and protein synthesis in Candida albicans.

Sequence analysis by the automated Edman degradation shows that dopamine $\text{β}$-hydroxylase (dopamine $\text{β}$-monooxygenase; EC 1.14.17.1) from bovine adrenal medulla contains equal amounts of NH₂-terminal alanine and serine residues. The sequence data are in agreement with the proposal that this enzyme consists of two types of polypeptide chains which are identical in the NH₂-terminal ends, except that one of the chains lack the NH₂-terminal tripeptide Ser-Ala-Thr.     

Human somatotropin: semisynthesis of the hormone by noncovalent interaction of the NH2-terminal fragment with synthetic analogs of the COOH-terminal fragment

Complementation of the natural NH₂-terminal fragment (consisting of 134 amino acid residues) with synthetic analogs of COOH-terminal fragments of 52 or 47 amino acids of reduced-carbamidomethylated human somatotropin gave recombinants with full growth-promoting activity as evidenced by the tibia test. Radioimmunoassay data show that the semisynthetic recombinant hormones possess nearly full immunoreactivity as compared to the native molecule.     

Amino Acid Sequence Analysis of Reverse Transcriptase Subunits from Avian Myeloblastosis Virus

The NH₂-terminal amino acid sequences of the α and β chains of avian myeloblastosis αβ DNA polymerase were determined by using microsequence analysis in the subnanomole range and were found to be identical up to 17 residues. The common sequence was as follows: Thr-Val-Ala-Leu-His-Leu-Ala-Ile-Pro-Leu-Lys-Trp-Lys-Pro-Asn-His-Thr-. This result provides convincing chemical evidence that the α chain is derived from the NH₂-terminal region of the β chain by proteolytic cleavage, whereas the amino acid composition for these α and β subunits and p32 DNA endonuclease suggests that the latter is derived from the carboxyl-terminal region of the β chain.     

Enzymatic Probe for Accessibility of NH<Subscript>2</Subscript>-Terminal Residues in Immunoglobulins

ARGINYL-tRNA-protein transferase is a soluble enzyme from mammalian tissues which catalyses the transfer of arginine from arginyl-tRNA into peptide linkage specifically with NH₂-terminal aspartic or glutamic acid residues of protein acceptors^1,2. Molar equivalents of arginine are transferred to appropriate NH₂-terminal residues². The reaction differs in many respects from the transfer of amino-acids associated with protein synthesis de novo and is thought to be a mechanism for regulating the activity of acceptor proteins³. Many immunoglobulins possess aspartic or glutamic acid in the NH₂-terminal position and it was interesting to examine whether arginylation of such residues might result in an alteration of activity of these proteins, for there is considerable evidence that the antibody-combining site is contained in the NH₂-terminal region of both light^4–7 and heavy^8,9 chains.     

Antihypertensive Effect of Boysenberry Seed Polyphenols on Spontaneously Hypertensive Rats and Identification of Orally Absorbable Proanthocyanidins with Vasorelaxant Activity

An E. coli expression clone coding for human proinsulin, which was fused to NH₂-terminal β-galactosidase, was engineered for the separation from host proteins by introducing peptide devices, and for the sequential removal of the fused polypeptide by cyanogen bromide in front of the NH₂ terminal residue (methionine) of the human proinsulin gene. Short synthetic genes encoding oligopeptide residues including (Glu)_n, (His)_n, (Trp)_n, and (Ser)_n (n = 10 or 11), which have certain characteristic physical properties such as metal-affinity, polarity, hydrophobicity, and hydrophilicity, respectively, were inserted at the junction region of the gene fusion. Interestingly, it was found that among the oligopeptides, the oligohistidine residue as an affinity-tag has greatly facilitated the procedures for FPI purification, particularly in the manner of selective metal-affinity precipitation. The chelating peptide covering the NH₂-terminal beta-galactosidase portion could then be removed simply after purification to generate a protein with the natural amino acid sequence of proinsulin by cyanogen bromide.     

Amino acid sequence of the first 65 residues of IgA myeloma protein

Amino acid sequence of the first 65 residues of human IgA myeloma protein was determined. MH₂-terminal fragment was obtained by specific scission at Asn-Gly bond with NH₂OH from completely reduced and alkylated protein. Peptides were obtained by tryptic and chymotryptic digestions of the fragment following column chromatography in a volatile buffer system. After purification by high voltage electrophoresis, peptides were sequenced by manual Edman-dansyl technique. The fragment consists of 65 amino acid residues and has the sequence characteristic of V_HIII subgroup of heavy chains.     

Roles of the NH2-terminal Domains of Cardiac Ryanodine Receptor in Ca2+ Release Activation and Termination

The NH₂-terminal region (residues 1–543) of the cardiac ryanodine receptor (RyR2) harbors a large number of mutations associated with cardiac arrhythmias and cardiomyopathies. Functional studies have revealed that the NH₂-terminal region is involved in the activation and termination of Ca²⁺ release. The three-dimensional structure of the NH₂-terminal region has recently been solved. It is composed of three domains (A, B, and C). However, the roles of these individual domains in Ca²⁺ release activation and termination are largely unknown. To understand the functional significance of each of these NH₂-terminal domains, we systematically deleted these domains and assessed their impact on caffeine- or Ca²⁺-induced Ca²⁺ release and store overload-induced Ca²⁺ release (SOICR) in HEK293 cells. We found that all deletion mutants were capable of forming caffeine- and ryanodine-sensitive functional channels, indicating that the NH₂-terminal region is not essential for channel gating. Ca²⁺ release measurements revealed that deleting domain A markedly reduced the threshold for SOICR termination but had no effect on caffeine or Ca²⁺ activation or the threshold for SOICR activation, whereas deleting domain B substantially enhanced caffeine and Ca²⁺ activation and lowered the threshold for SOICR activation and termination. Conversely, deleting domain C suppressed caffeine activation, abolished Ca²⁺ activation and SOICR, and diminished protein expression. These results suggest that domain A is involved in channel termination, domain B is involved in channel suppression, and domain C is critical for channel activation and expression. Our data shed new insights into the structure-function relationship of the NH₂-terminal domains of RyR2 and the action of NH₂-terminal disease mutations.     

Abolition of anion-activation of mitochondrial F1-ATPase by the partial ADP-induced hysteretic inhibition

An M_r 21 000 polypeptide, designated APPG, has been purified by reverse-phase, high-performance liquid chromatography (RP-HPLC), from acid extracts of porcine anterior pituitary glands. This acidic protein possesses an isoelectric point of 4.9. Amino acid analysis shows that it is not a glycoprotein and estimates it to contain about 173 amino acids. NH₂-terminal sequence analysis allowed the determination of the first 50 residues unambiguously. A computer data bank search using a mutation data matrix and comparison with 269 012 protein segments indicated that this is a novel polypeptide sequence. However, this search revealed suggestive sequence homologies to a number of peptides of known sequence, including duck proinsulin (30%), Rous sarcoma virus transforming protein TVFV60 (24%) and pig secretin (26%).     

Structural evidence for a liver-specific glyceraldehyde-3-phosphate dehydrogenase.

The amino acid sequences near the amino termini of glyceraldehyde-3-phosphate dehydrogenase from bovine and porcine liver have been determined. Using classical peptide isolation techniques as well as automated Edman degradation, the NH₂-terminal 30 residues of the bovine liver enzyme were determined to be Val-Lys-Val-Gly-Val-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg-Leu-Val-Thr-Arg-Ala-Ala-Phe-Asn-Ser-Gly-Lys-Val-Asp-Ile-Val-Phe-Ile. Twenty-two residues from the NH₂-terminus of the porcine liver enzyme, determined using the automated Edman degradation, were identical to the corresponding sequence from bovine liver enzyme. Both liver enzymes have Asn at position 6. The corresponding residue 6 in the muscle and yeast glyceraldehyde-3-phosphate dehydrogenases is Asp. This evidence suggests that the Asn-6 residue is specific for the liver tissues. The exchange of Asn for Asp may significantly alter the allosteric properties of muscle and liver enzymes especially the activity of the liver enzymes in gluconeogenesis.     

Structural studies of Bacillus subtilis glutamine synthetase. Further purification, sulfhydryl groups, and the NH2-terminal amino acid sequence.

A new procedure for the isolation of Bacillus subtilis glutamine synthetase in a high state of purity is described. Automated Edman degradation of the reduced and carboxy-methylated protein revealed a single NH₂-terminal amino acid sequence: H₂N-Ala-Lys- Tyr-Thr-Arg⁵-Glu-Asp-Ile-Gln-Lys¹⁰-Leu-Val-Ser-Glu-Ser¹⁵-CM-Cys-Val-Thr- Tyr-Ile²⁰-Ser-Leu-Gly-Phe-Ser²⁵-Asn-Ser-Leu-Gly- -. The recovery of phenylthiohydantoin(PTH)-amino acids and the single sequence obtained are consistent with the view that the dodecameric enzyme of molecular weight 600,000 is composed of identical subunits. Earlier observations of multiple sequences (80% PTH-Ala and 20% PTH-Gly as NH₂ terminal residues) appear to have been due to impurities removed by the final purification step described herein, which involves column chromatography on hydroxyapatite. Evidence for the existence of one disulfide bond and two free cysteine residues per subunit of dodecameric glutamine synthetase was obtained by alkylation of the denatured enzyme in the presence and absence of reducing agents. This distribution of the four cysteine residues in the enzyme monomer was confirmed by titration of the enzyme denatured in sodium dodecyl sulfate with 5,5′-dithiobis(2-nitrobenzoic acid).