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.     

Human pituitary growth hormone. 44. Effects of plasmin-modified hormone and its fragments on ornithine decarboxylase activity and lipolysis.

The rat hepatic ornithine decarboxylase stimulating activity of plasmin-modified human growth hormone and its two peptide fragments has been investigated. The activity was completely retained after plasmin treatment. The NH2-terminal fragment [Cys (Cam)53-HGH-(1-134)] retained 10% of the activity, whereas the COOH-terminal fragment [Cys (Cam) 165, 182, 189-(141-191)] was not active. The lipolytic activity of human growth hormone was greatly reduced after plasmin treatment, as examined in isolated rabbit adipocytes. It is suggested that the structural requirements for the lipolytic activity of the hormone are different from those required for stimulation of ornithine decarboxylase activity.     

Action of thrombin on ovine, bovine and human pituitary growth hormones.

1. This communication reports the action of bovine thrombin on ovine, bovine and human growth hormones. Thrombin cleavage was shown to be restricted to a single homologous peptide bond in all three growth hormones (at sequence positions 133--134 of the ovine and bovine hormones). 2. Ovine growth hormone was the most sensitive to the action of thrombin, bovine growth hormone was attacked to a relatively less extent, and human growth hormone was the most resistant to the enzyme. 3. After reduction and carbamidomethylation of the disulfide bonds in thrombin modified ovine growth hormone, the two fragments (residues 1--133 and 134--191) were isolated. The large NH2-terminal thrombin fragment of the hormone (residues 1--133) was found to be inactive in the rat tibia test, whereas a tryptic fragment (residues 96--133) isolated in an independent way gave measurable responses.     

Human somatotropin: Noncovalent interactions of the natural NH2-terminal 1–134 segment with synthetic analogs of the COOH-terminal fragment

Three analogs of the COOH-terminal fragments of human somatotropin (HGH), namely [Nle¹⁷⁰,Ala^165,182]-HGH-(150–187), [Nle¹⁷⁰,Ala^165,182]-HGH-(152–187), and [Nle¹⁷⁰,Ala^165,182]-HGH-(154–187), have been synthesized by the solid-phase method. The synthetic analogs were complemented with the natural NH₂-terminal fragment [Cys(Cam)⁵³]-HGH-(1–134) to form recombinants with HGH activities, as revealed by the rabbit liver membrane receptor binding and the Nb2 lymphoma cell assays.     

Solid phase synthesis of somatostatin-28

The synthesis of ovine hypothalamic somatostatin-28 (Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-$\text{Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys}$-OH) has been accomplished by solid phase methodology. The structure of the synthetic material was verified by: (1) direct sequence analysis with a Beckman 89°C sequencer, (2) correlation of the amino acid analyses of the isolated tryptic peptide fragments with their theoretical compositions, and (3) comparison, using high performance liquid chromatography, of the synthetic methionine-sulfoxide and methionine-sulfone modified NH₂-terminal peptides (residues 1–11) with the corresponding tryptic fragment from somatostatin-28.     

Peptides with 6-Aminohexanoic Acid: Synthesis and Evaluation as Plasmin Inhibitors

Fifteen new peptide derivatives of ?-aminocaproic acid (EACA) containing the known fragment –Ala–Phe–Lys– with an affinity for plasmin were synthesised in the present study. The synthesis was carried out a solid phase. The following compounds were synthesised: H–Phe–Lys–EACA–X, H–d-Ala–Phe–Lys–EACA–X, H–Ala–Phe–Lys–EACA–X, H–d-Ala–Phe–EACA–X and H–Ala–Phe–EACA–X, where X = OH, NH₂ and NH–(CH₂)₅–NH₂. All peptides, except for those containing the sequence H–Ala–Phe–EACA–X, displayed higher inhibitory activity against plasmin than EACA. The most active and selective inhibitor of plasmin was the compound H–d-Ala–Phe–Lys–EACA–NH₂ which inhibited the amidolytic activity of plasmin (IC₅₀ = 0.02 mM), with the antifibrinolytic activity weaker than EACA. The resulting peptides did not affect the viability of fibroblast cells, colon cancer cell line DLD-1, breast MCF-7 and MDA-MB-231 cell lines.     

Human growth hormone: 1974–1981

Summary Chemical investigations of the human growth hormone (HGH) molecule were briefly reviewed for the period between 1974 and 1981. These include chemical modification, selective enzymic cleavage, the 20K HGH, synthetic peptide fragments, complementation of the natural NH₂ terminal 134-amino acid fragment with natural or synthetic COO H-terminal fragments of various chain lengths, covalent reconstitution of two contiguous fragments of HGH with thrombin and bacterially synthesized methionyl-HGH.Abbreviations GH growth hormone or somatotropin - HGH human GH - CD circular dichroism - NMR nuclear magnetic resonance - DEAE diethylaminoethyl - CMC carboxymethyl cellulose - HPLC high performance liquid chromatography - RA-HGH reduced-carbamidomethylated HGH - PL-HGH plasmin-modified HGH - Cam Carbamidomethyl - BGH bovine GH - SDS sodium dodecylsulfate     

Studies on prolactin 48: Isolation and properties of the hormone from horse pituitary glands

Isolation of prolactin from equine pituitary glands has been described. It has a potency of 42 IU/mg in the pigeon crop-sac test and consists of 199 amino acids. The hormone has only four half-cystine residues in contrast to other mammalian prolactins which have six residues. From NH₂-terminal sequence analysis and amino acid composition of cyanogen bromide fragments, the NH₂-terminal disulfide loop is missing in the equine prolactin molecule. Circular dichroism spectra indicate that the α-helical content of equine prolactin appears to be lower (50%) than that found in the ovine hormone (65%).     

Structure and function relationship of staphylocoagulase

The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (K_d=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (K_d=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (K_d=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH₂-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH₂-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH₂-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

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.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.     

A specific radioimmunoassay for the novel opioid peptide dynorphin

Dynorphin was recently isolated from porcine pituitary extracts and shown to be the most potent known opioid peptide. Antisera were prepared to synthetic dynorphin-(1–13), the biologically active NH₂-terminal fragment of the peptide. A high-titer, sensitive antiserum was characterized with fragments from dynorphin-(1–13). Leucine-enkephalin, which is contained in dynorphin, is not recognized at all by the antiserum. To study distribution in tissue, a procedure using hot acidified methanol extraction of rat pituitary neurointermediate lobe preparations was developed and validated. ¹²⁵I-labelled dynorphin-(1–13), when added to tissue, remained intact throughout this extraction procedure, and added dynorphin-(1–13) was almost completely recovered. There was no destruction of radiolabelled peptide during incubation in the radioimmunoassay. Serial dilutions of pituitary extracts yielded curves that were parallel to the dynorphin-(1–13) standard curve. The immunoreactivity from tissue was completely destroyed by papain treatment.     

Response of human and chick kidney adenylate cyclase to biologically active synthetic fragments of human parathyroid hormone

The 34-amino acid NH₂-terminal fragment of human parathyroid hormone synthesized according to the sequence described by Niall $\text{et al.}$ (1) is approximately 140 times more potent than the fragment synthesized according to Brewer $\text{et al}$ (2) in activating human renal cortex adenylate cyclase. The potencies of the two peptides, relative to the effect of MRC standard bovine parathyroid hormone preparation $\text{67}\text{342}$ in this system, were 5600 ± 600 (S.E.M.) units/mg and 40 ± 5 units/mg respectively. The potencies of the more active peptide and the corresponding bovine parathyroid hormone sequence were similar in this system and also in assays based upon the production of cyclic AMP by chick kidney both $\text{in vivo}$ and $\text{in vitro}$.     

Antimicrobial function of short amidated peptide fragments from the tick‐derived OsDef2 defensin

Previously Os, a 22 amino acid sequence of a defensin from the soft tick Ornithodoros savignyi, was found to kill Gram‐positive and Gram‐negative bacteria at low micromolar concentrations. In this study, we evaluated synthetic peptide analogues of Os for antibacterial activity with an aim to identify minimalized active peptide sequences and in so doing obtain a better understanding of the structural requirements for activity. Out of eight partially overlapping sequences of 10 to 12 residues, only Os(3–12) and Os(11–22) exhibit activity when screened against Gram‐positive and Gram‐negative bacteria. Carboxyamidation of both peptides increased membrane‐mediated activity, although carboxyamidation of Os(11–22) negatively impacted on activity against Staphylococcus aureus. The amidated peptides, Os(3–12)NH₂ and Os(11–22)NH₂, have minimum bactericidal concentrations of 3.3 μM against Escherichia coli. Killing was reached within 10 minutes for Os(3–12)NH₂ and only during the second hour for Os(11–22)NH₂. In an E. coli membrane liposome system, both Os and Os(3–12)NH₂ were identified as membrane disrupting while Os(11–22)NH₂ was less active, indicating that in addition to membrane permeabilization, other targets may be involved in bacterial killing. In contrast to Os, the membrane disruptive effect of Os(3–12)NH₂ did not diminish in the presence of salt. Neither Os nor its amidated derivatives caused human erythrocyte haemolysis. The contrasting killing kinetics and effects of amidation together with structural and liposome leakage data suggest that the 3–12 fragment relies on a membrane disruptive mechanism while the 11–22 fragment involves additional target mechanisms. The salt‐resistant potency of Os(3–12)NH₂ identifies it as a promising candidate for further development.     

A Gene Encoding a Cytochrome c Oxidase-Like Protein Is Located Closely to the Cytochrome c-553 Gene in the Anaerobic Bacterium,Desulfovibrio vulgaris (Miyazaki F)

The gene encoding cytochrome c-553 from Desulfovibrio vulgaris (Miyazaki F) was cloned using a synthetic oligodeoxyribonucleotide probe. The nucleotide sequence indicated that cytochrome c-553 was synthesized as a precursor protein with an NH₂-terminal signal sequence of 23 residues. In the cloned DNA fragment, there are three other open reading frames whose products have 191, 157, 541 amino acid residues, respectively. The putative ORF-4 product is highly homologous with the cytochrome c oxidase subunit I from various organisms.     

Action of plasmin on ovine beta-lipotropin: revision of the carboxyl terminal sequence

Human plasmin was found to cleave the lysylasparagine bond specifically in positions 79 and 80 of the ovine beta-lipotropin molecule. Structural studies of the COOH-terminal fragment revealed the amino acid sequence to be: Asn-Ala-Ile-Ile-Lys-Asn-Ala-His-Lys-Lys-Gly-Gln which is slightly different from the structure originally proposed. Together with an earlier revision of the NH₂-terminal sequence, the corrected structure of ovine beta-lipotropin is shown in Figure 2.     

Evidence that two zinc fingers in the methionine aminopeptidase from Saccharomyces cerevisiae are important for normal growth

Limited proteolysis of intact yeast methionine aminopeptidase (MAP1) with trypsin releases a 34 kDa fragment whose NH₂-terminal sequence begins at Asp70, immediately following Lys69. These results suggest that yeast MAP may have a two-domain structure consisting of an NH₂-terminal zinc finger domain and a C-terminal catalytic domain. To test this, a mutant MAP lacking residues 2–69 was generated, overexpressed, purified and analyzed. Metal ion analyses indicate that 1 mol of wild-type yeast MAP contains 2 mol of zinc ions and at least 1 mol of cobalt ion, whereas 1 mol of the truncated MAP lacking the putative zinc fingers contains only a trace amount of zinc ions but still contains one mole of cobalt ion. These results suggest that the two zinc ions observed in the native yeast MAP are located at the Cys/His rich region and the cobalt ion is located in the catalytic domain. The k.at and Km values of the purified truncated MAP are similar to those of the wild-type MAP when measured with peptide substrates in vitro and it appears to be as active as the wild-type MAP in vivo. However, the truncated MAP is significantly less effective in rescuing the slow growth phenotype of map mutant than the wild-type MAP. These findings suggest that the zinc fingers are essential for normal MAP function in vivo, even though the in vitro enzyme assays indicate that they are not involved in catalysis. In addition, a series of single mutations were generated by changing the cysteines and the histidines in the zinc finger region to serines and arginines, respectively. Analyses of these point mutations provide further evidence that the cysteines and histidines are important for the growth promotion function of yeast MAP.     

Engineering streptokinase for generation of active site-labeled plasminogen analogs

We previously demonstrated that streptokinase (SK) can be used to generate active site-labeled fluorescent analogs of plasminogen (Pg) by virtue of its nonproteolytic activation of the zymogen. The method is versatile and allows stoichiometric and active site-specific incorporation of any one of many molecular probes. The limitation of the labeling approach is that it is both time-consuming and low yield. Here we demonstrate an improved method for the preparation of labeled Pg analogs by the use of an engineered SK mutant fusion protein with both COOH- and NH₂-terminal His₆ tags. The NH₂-terminal tag is followed by a tobacco etch virus proteinase cleavage site to ensure that the SK Ile¹ residue, essential for conformational activation of Pg, is preserved. The SK COOH-terminal Lys⁴¹⁴ residue and residues Arg253–Leu260 in the SK β-domain were deleted to prevent cleavage by plasmin (Pm) and to disable Pg substrate binding to the SK·Pg^∗/Pm catalytic complexes, respectively. Near elimination of Pm generation with the SKΔ(R253–L260)ΔK414–His₆ mutant increased the yield of labeled Pg 2.6-fold and reduced the time required more than 2-fold. The versatility of the labeling method was extended to the application of Pg labeled with a near-infrared probe to quantitate Pg receptors on immune cells by flow cytometry.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.     

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.