Terminal Amino Acid Residues Research Articles

Isolation and properties of sea turtle ( Chelonia mydas) pituitary prolactin

Sea turtle prolactin (PRL) was isolated in a highly purified state from sea turtle pituitary side fractions obtained from other studies and some of its biological, chemical, and immunological properties were determined. Sea turtle PRL is a protein of 22–24 kDa [sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis]. Its sole amino terminal amino acid residue is leucine. The amino acid composition of sea turtle PRL is similar to ovine PRL and is characterized by a high content of aspartic acid (20 residues), glutamic acid (34 residues), serine (19 residues), and leucine (24 residues). It possesses three disulfide bonds and 2 tryptophan residues which is also characteristic of many species of PRL. As with PRLs of other species, it displayed multiple stained bands in disc gel electrophoresis, at pH 8.3. Biologically, sea turtle PRL was active in pigeon crop-sac assay but the doseresponse characteristics were nonparallel when compared to ovine PRL. An antiserum against sea turtle PRL was raised in rabbit and a homologous radioimmunoassay was developed with a sensitivity of 2.8 ng for sea turtle PRL. Sea turtle gonadotropins did not cross-react, but sea turtle growth hormone showed a 5% cross-reactivity. Pituitary extracts from other species of turtles displayed parallel inhibition curves to the sea turtle PRL. Extracts and prolactin preparations from several birds, snakes, alligator, and marsupials cross-reacted, but in a nonparallel fashion. Bullfrog pituitary extract and Tilapia PRL showed no cross-reaction at high doses. Several purified mammalian PRLs (pig, sheep, human, horse, dog) showed minimal or no ability to cross-react in the RIA.

Spectroscopic properties of the alpha fragment of metallothionein.

Absorption, CD, and magnetic circular dichroism spectra are reported for the alpha fragment of rat liver Cd,Zn-metallothionein (MT) 2. The CD and magnetic circular dichroism spectra of the Cd4 cluster unit are particularly well-resolved and are remarkably similar to data of the complete Cd,Zn-MT. It is suggested that the high signal intensity in the 225 nm CD band may be attributed to an interaction between a terminal amino acid residue and the Cd4 cluster. Titration experiments with CdCl2 and [Cu(CH3CN)4]+ show that while no additional Cd2+ can be bound in the presence of excess Cd2+, Cu+ does replace the bound Cd2+ in a complex reaction to form at least two species. One of these species requires the presence of both Cu+ and Cd2+, with a stoichiometry of Cu 3.0, Cd 2.5. Further, Cu+ displaces all the remaining Cd2+, and the spectra recorded now closely resemble Cu-MT formed by titration of Cd,Zn-MT with greater than 8 mol eq of Cu+.

Gene encoding a hybrid OmpF--PhoE pore protein in the outer membrane of Escherichia coli K12.

To study the structure-function relationship of outer membrane pore proteins of E. coli K12, a hybrid gene was constructed in which the DNA encoding amino acid residues 2-73 of the mature PhoE protein is replaced by the homologous part of the related ompF gene. The product of this gene is incorporated normally into the outer membrane. It was characterized with respect to its pore activity and its phage receptor and colicin receptor properties. It is concluded that the preference of the PhoE protein pore for negatively charged solutes is partly determined by the amino terminal 73 amino acids, that part of the receptor site of PhoE protein for phage TC45 is located in this part of the protein, that colicin N uses OmpF protein as (part of) its receptor, that the specificity of OmpF protein as a colicin N receptor is completely located within the 80 amino terminal amino acid residues, whereas the specificity of this protein as a colicin A receptor is completely located within the 260 carboxy terminal amino acid residues, and that the amino terminal 73 amino acid residues of PhoE protein span the membrane at least once.

Properties of a heparin-poly(vinyl alcohol) hydrogel coating.

Some physical, chemical, and biological characteristics of a heparin-PVA hydrogel potentially useful as a thromboresistant coating for the preparation of small-diameter vascular prostheses and blood-sampling catheters have been determined. The molecular weight between crosslinks in the acetal crosslinked gel was approximately 8000, permitting proteins the size of albumin to enter the interior of the gel. The release rate of heparin from a gel containing 7 mg/g gel was 10(-2) micrograms/g min which was significantly lower than the reported minimum required for thromboresistance of ionically heparinized materials. Nevertheless, in vitro biological activity was observed in both thrombin time and plasma recalcification time assays, which could not be attributed to the release of heparin into the incubated plasma. Correlation of final heparin contents with the amount of terminal amino acid residues in three samples of heparin suggests that the heparin is bound to the PVA in the gel through the amino acid terminus; this provides a plausible explanation for the retention of biological activity by the immobilized heparin.

Carboxy-terminal analysis of poliovirus proteins: termination of poliovirus RNA translation and location of unique poliovirus polyprotein cleavage sites.

The carboxy-terminal amino acids of a number of poliovirus proteins were determined by carboxypeptidase A analysis. The nonstructural proteins P3-2, P3-4b and their precursor. P3-1b, were found to be coterminal with a sequence of -Ser-Phe-COOH. As these proteins are coded for at the extreme 3' end of the viral RNA, it is possible to establish the termination site of translation at nucleotide 7,361, 73 nucleotides before the start of the polyadenylic acid tract of the RNA. Two additional nonstructural proteins, P2-X and its precursor, P2-3b, were also found to be coterminal with a sequence of -Phe-Gln-COOH. This result confirms the existence of at least one Gln-Gly proteolytic cleavage site. These Gln-Gly cleavage sites are predicted from the nucleotide sequence to be ubiquitous throughout the poliovirus genome. The only exceptions are the cleavage sites at the carboxy termini of the structural protein VP4 and VP1. Carboxypeptidase A analysis of VP1 establishes a terminal sequence of -Thr-Tyr-COOH, and similar analysis of VP4 shows Asn to be the terminal amino acid residue, observations that prove the existence of the exceptional C-terminal amino acids. In none of the analyzed cases has C-terminal trimming after cleavage been observed.

Immunological probes for bacteriorhodopsin. Identification of three distinct antigenic sites on the cytoplasmic surface.

We have prepared site-specific immunological reagents to study the orientation and surface topography of the integral membrane protein bacteriorhodopsin. Monoclonal and polyclonal antibodies with strong affinity for antigenic determinants on proteolytic and cyanogen bromide fragments of bacteriorhodopsin have been isolated and characterized. Three distinct antibody binding sites have been identified on the cytoplasmic surface of bacteriorhodopsin. The first due is readily accessible in native bacteriorhodopsin and lies close to the COOH terminus. This binding site is lost when only three amino acid residues are removed from the COOH terminus. The second site, which is also near the COOH terminus, is located approximately within the 17 COOH terminal amino acid residues. The third site is in the fragment that comprises Tyr-83 to Met-118 and is probably contained in the short loop connecting the third and fourth helices. The use of COOH terminus-specific antibodies in determination of the orientation of bacteriorhodopsin molecules in the Halobacterium halobium membrane confirms the earlier conclusion that the COOH terminus is on the cytoplasmic side.

Purification, crystallisation and characterisation of carboxypeptidase from wheat bran

A carboxypeptidase was purified and crystallised from wheat bran. Disc gel electrophoresis at pH 4·0 and ultracentrifugal analysis revealed that the enzyme was essentially homogeneous. The sedimentation constant and isoelectric point were determined to be 6·3 S and 6·0, respectively. The molecular weight of the enzyme was estimated to be 118,000 by a gel filtration method. The enzyme liberated carboxyl terminal amino acid residues from a wide range of N-substituted dipeptides and tripeptides which contain l-proline. It had a pH optimum at pH 4·0 for Z-Glu-Tyr ( Z-benzyloxycarbonyl). The K m and k cat values for Z-Glu-Tyr at pH 4·0 and 30°C were 0·19 m m and 20 s −1, respectively. The enzyme hydrolysed Z-Gly-Pro-Leu-Gly-Pro and bradykinin sequentially at pH 4·0 from their carboxyl terminal amino acid residues. The enzyme activity was completely inhibited by DFP.

Studies on Detoxin complex, the selective antagonists of Blasticidin S. The structures of minor congeners of the detoxin complex.

The structures of minor congeners of the detoxin complex, viz., detoxins E1, C1, C2, C3, B1, B3 and Al, have been established on the basis of degradative and spectroscopic evidences.It is of interest that the detoxin B group contains 5-deoxydetoxinine in place of detoxinine, and that a change of the terminal amino acid residue from l-valine to l-isoleucine was first recognized in detoxin E1.The structure-activity relationship of the detoxin compounds is discussed with comparison of their antagonistic activities against blasticidin S.

Kinixys crosa myoglobin isolation and characterization

1. 1. Myoglobin was isolated from the skeletal muscle of the land tortoise, Kinixys crosa. 2. 2. The myoglobin is monomeric with an apparent molecular weight of 18,000 as determed on Sephadex G-75 (with a non-denaturing eluant), 16,700 ± 300 on sodium dodecyl sulfate electrophoresis and 17,741 from the amino acid composition. 3. 3. There is one cysteine residue per mol. wt 18,000. 4. 4. Spectrophotometric characteristics for a variety of ferrous and ferric derivatives have been determined. 5. 5. The N- terminal amino acid residue is glycine.

A comparison of molecular properties of hepatic triglyceride lipase and lipoprotein lipase from human post-heparin plasma.

Hepatic triglyceride lipase was isolated from human post-heparin plasma by the method of Ehnholm et al. using modifications which increased the specific activity 12-fold to approximately 3,000 mumol of free fatty acid/h/mg of protein. Lipoprotein lipase with similar specific activity was prepared from the same plasma samples using heparin and concanavalin A affinity chromatography. The molecular weight of hepatic triglyceride lipase (69,000) was slightly greater than that of lipoprotein lipase (67,000) as determined by polyacrylamide electrophoresis in sodium dodecyl sulfate-containing buffers. These proteins had identical amino acid compositions, terminal amino acid residues, and tryptic peptide maps. However, the differences previously described regarding optima of pH and ionic strength and the requirement for apolipoprotein CII (only for lipoprotein lipase) were maintained in the highly purified state. It was found that both proteins contain approximately 8% carbohydrate. Antisera prepared in goats selectively precipitated each activity. Other antisera prepared in chickens reacted with both enzymes, suggesting a common antigenic determinant.

PURIFICATION AND PARTIAL CHARACTERIZATION OF THE apoA‐I OF RABBIT HIGH DENSITY LIPOPROTEIN

Purification of apoA-I from rabbit high density lipoproteins (HDL) gave one single band in sodium dodecylsulphate-polyacrylamide disc electrophoresis and reacted only with antiserum to apoA-I. The molecular weight was about 25000. The amino acid composition of rabbit ,poA-I gave a difference index of 7.4 compared to human apoA-I and of 6.5 compared to dog apoA-I. Of the three carboxy terminal amino acid residues the rabbit protein has one in common with the dog. The amino acid sequence of twenty-nine amino terminal residues showed 62% homology with the human protein; a minimum of thirteen base changes were required in the DNA sequences that encoded these two proteins. When the same sequence was compared with dog apoA-I, it was found that ten base changes would be sufficient to account for the differences between the proteins.

Hypotensin — a hypotensive peptide isolated from Crotalus atrox venom: Purification, amino acid composition and terminal amino acid residues

Hypotensin — a hypotensive peptide isolated from Crotalus atrox venom: Purification, amino acid composition and terminal amino acid residues

Studies on the structure and mechanism of an exo-(1ₒ3)-β- D-glucanase from basidiomycete qm806

A method for the large-scale production of a (1ₒ3)-β- D-glucan glucohydrolase (EC 3.2.1.58) from the culture filtrate of Basidiomycete QM806 is described. The final preparation is homogeneous by disc electrophoresis under non-dissociating and denaturing conditions, by ultracentrifugation, and by isoelectric focusing. Various physical and chemical characteristics of the enzyme have been determined, including terminal amino acid residues, extinction coefficient, and stability to pH extremes. The N-terminal amino acids are leucine and serine (Sanger's method) and the C-terminal amino acids are alanine, serine, and glycine (hydrazinolysis). pH profile studies show that no group titrating in the region 2.5–8 is directly involved with substrate binding and that a single group having a p K a of 6.5 is involved in the catalysis. Photooxidation of the enzyme caused rapid inactivation. The pH-dependence of this photooxidation, and amino acid analysis of the photooxidized enzyme, indicate that decomposition of histidine is probably responsible for the loss of activity. Other chemical modifications performed were: treatment with hydrogen peroxide under acidic conditions, esterification with diphenyldiazomethane, and oxidation with N-bromosuccinimide. Oxidation with N-bromosuccinimide indicated that a tryptophan side-chain is involved in, but not necessary for, the catalytic activity.

Isolation, amino acid composition and terminal amino acid residues of the vasoactive octacosapeptide from chicken intestine. Partial purification of chicken secretin

FEBS LettersVolume 47, Issue 2 p. 284-289 Full-length articleFree Access Isolation, amino acid composition and terminal amino acid residues of the vasoactive octacosapeptide from chicken intestine. Partial purification of chicken secretin Ann Nilsson, Ann Nilsson Medicinska Nobelinstitutet, Biokemiska Avdelningen, Karolinska Institutet, Stockholm, SwedenSearch for more papers by this author Ann Nilsson, Ann Nilsson Medicinska Nobelinstitutet, Biokemiska Avdelningen, Karolinska Institutet, Stockholm, SwedenSearch for more papers by this author First published: October 15, 1974 https://doi.org/10.1016/0014-5793(74)81031-8Citations: 44AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 S.I. Said, V. Mutt, Science, 169, (1970), 1217– 1218. 2 V. Mutt, S.I. Said, Eur. J. Biochem., 42, (1974), 581– 589. 3 J.E. Jorpes, V. Mutt, S. Magnusson, B.B. Steele, Biochem. Biophys. Res. Comm., 9, (1962), 275– 279. 4 S.I. Said, V. Mutt, Eur. J. Biochem., 28, (1972), 199– 204. 5 V. Mutt, U. Söderberg, Ark. Kemi, 15, (1959), 63– 68. 6 V. Mutt, S. Magnusson, J.E. Jorpes, E. Dahl, Biochemistry, 4, (1965), 2358– 2362. 7 J.T. Potts Jr., H.D. Niall, H.T. Keutmann, H.B. Brewer Jr., L.J. Deftos, Proc. Natl. Acad. Sci. U.S., 59, (1968), 1321– 1328. 8 R.R. Redfield, Biochem. Biophys. Acta, 10, (1953), 344– 345. 9 K. Jones, J.G. Heathcote, J. Chromatog., 24, (1966), 106– 111. 10 D.H. Spackman, W.H. Stein, S. Moore, Anal. Chem., 30, (1958), 1190– 1206. 11 V. Mutt, Ark. Kemi, 15, (1959), 69– 74. 12 A. Ahlroth, V. Mutt, Anal. Biochem., 37, (1970), 125– 128. 13 E.W. Johns, Biochem. J., 104, (1967), 78– 82. 14 B. Blombäck, M. Blombäck, P. Edman, B. Hessel, Biochim. Biophys. Acta, 115, (1966), 371– 396. 15 J.R. Spies, D.C. Chambers, Anal. Chem., 21, (1949), 1249– 1266. 16 S. Moore, Biol. Chem., 238, (1963), 235– 237. 17 M.O. Olowo-Okurn, B.O. Amuru, Nature, 246, (1973), 424– 425. 18 J. Markussen, E. Frandsen, L.G. Hedig, F. Sundby, Horm. Metab. Res., 4, (1972), 360– 363. 19 F. Sundby, E.K. Frandsen, J. Thomsen, K. Kristiansen, K. Brunfeldt, FEBS Letters, 26, (1972), 289– 293. 20 G.J. Dockray, Experientia, 29, (1973), 1510– 1511. Citing Literature Volume47, Issue2October 15, 1974Pages 284-289 ReferencesRelatedInformation

ChemInform Abstract: COBALT(II) COMPLEXES OF DIETHYLENETRIAMINE‐N,N′,N′′‐TRIACETIC ACID

AbstractDer Titelligand wurde auf folgendem Wege synthetisiert: Die endständigen Aminogruppen der Säure (I) werden mit Brommalonsäure in alkalischer Lösun zu einem Pentacarboxylderivat umgesetzt, das in verdünnter Salzsäure zu der Triessigsäure (II) decarboxyliert wird.

Cobalt(II) Complexes of Diethylenetriamine-N,N′,N″-triacetic Acid

Abstract Cobalt(II) complexes of diethylenetriamine-N,N′,N″-triacetic acid (DTTA) were prepared. From spectrophotometric and magnetic data, diaquo-DTTA-Co(II) was found to have an octahedral arrangement in which the N,N′-diacetate moiety and two water molecules are in the coordination sphere, and the terminal amino acid residue dangles outside. On heating the diaquo-complex, thermal rearrangement occurred with loss of water and the complex took a tetrahedral configuration. This was confirmed by its electronic spectrum and magnetic moment. The tetrahedral complex turned to another octahedral complex with absorption of water, the configuration of which seemed to differ from the original as judged by spectral difference. Cobalt(II) complexes of ethylenediamine-N,N′-diacetic acid (EDDA) were also prepared and investigated for the sake of comparison.

Studies on the amino and carboxyl terminal amino acid sequences of reovirus capsid polypeptides.

Abstract Techniques are described for isolating several reovirus capsid polypeptides in amounts sufficient for determination of amino and carboxyl terminal amino acid sequences and fingerprinting. Among them are chromatography on CM- and DEAE-Sephadex in the presence of urea, and gel filtration on agarose A-15 m in the presence of sodium dodecyl sulfate. Using a combination of these procedures, and starting with either virions or cores, polypeptides σ3, σ2, and μ2 have been obtained essentially pure. Polypeptides λ1 and λ2 have been obtained as a mixture which has so far not been resolved. All reovirus capsid polypeptides except μ2 possess blocked amino terminal amino acid residues. The amino terminal amino acid sequence of polypeptide μ2 is H2N-Pro-Gly-Gly-Val-Pro-. This suggests that polypeptide μ2 is derived from its precursor, polypeptide μ1, by cleavage of the amino terminal portion of the polypeptide chain. The carboxyl terminal regions of at least three of the five major reovirus capsid polypeptides are different. Polypeptide σ3 ends in -(val, val, leu)-COOH; polypeptide μ2 in -leu-(arg, tyr, tyr)-Arg-COOH; and either one or both of the two polypeptides λ1 and λ2 terminate(s) in -Arg-COOH, the adjacent amino acid sequence being different from that of μ2.

Chemical properties of equine pituitary follicle-stimulating hormone.

Purified equine pituitary follicle stimulating hormone (FSH) was analyzed for protein and carbohydrate contents, amino acid composition, and NH2- and COOH—terminal amino acid residues. The results indicate that equine FSH is a glycoprotein having 74.9% protein, 9.3% hexose, 9.1% hexosamine, 4.7% sialic acid and 1.3% fucose. The amino acid composition was characterized by high levels of half—cystine, threonine, lysine and aspartic and glutamic acids, and a low level of methionine. Phenylalanine and aspartic acid were identified as NH2—terminal amino acids while the COOH—terminal residues were found to be isoleucine and glutamic acid. (Endocrinology 91: 1418, 1972)

Characterization of Three Feline Paraproteins

Abstract Serum protein electrophoretic studies on three cats, two with lymphosarcoma and one with multiple myeloma, revealed M components in the γ region. These proteins were isolated by zone electrophoresis and were characterized immunologically, physically and chemically. Antisera to two feline isolates demonstrated individual antigenic specificity. All three proteins were classified as γG lambda immunoglobulins on the basis of cross-reactivities with human antisera. Cross-reactions were also documented between each of the feline proteins and previously studied canine light chains and intact human γG proteins. By analytical ultracentrifugation these feline immunoglobulins had Svedberg constants of approximately 6.8S. Molecular weight determinations of the intact γG proteins by SDS acrylamide gel analysis indicated an average m.w. of 154,000. The amino terminal amino acid residue of one of the feline heavy chains was glutamic acid and that of another aspartic acid. Thus these chains could be partially sequenced by an automatic protein sequencer. Comparison of the results with sequences of human VHIII heavy chains showed a striking sequence homology between the two species.

End group and carbohydrate analyses of equine LH

Summary The terminal amino acid residues and carbohydrate composition of a highly purified equine luteinizing hormone (LH) preparation were determined. The N-terminal amino acids were serine and phenylalanine while the C-termini were determined to be serine and isoleucine. The carbohydrate analyses showed high amounts of sialic acid.