Terminal NH2 Groups Research Articles

Steady-state kinetics of plasmin- and trypsin-catalysed hydrolysis of a number of tripeptide- p-nitroanilides

The steady-state kinetics of plasmin- (EC 3.4.21.7) and trypsin-catalysed (EC 3.4.21.4) hydrolysis of Bz- l-Phe-Val-Arg-pNA, Bz- d-Phe-Val-Arg-pNA, l-Phe-Val-Arg-pNA, d-Phe-Val-Arg-pNA and d-Val-Leu-Lys-pNA were investigated in the pH range 6–9. The pH dependences of the kinetic parameters correspond with the effects of catalytically essential ionizations in the enzymes, except for reactions with l- and d-Phe-Val-Arg-pNA, in which protonation of the NH 2-terminal α-amino groups (p K = 7.0) shows some inhibitory effect. The reactions of plasmin and trypsin with the p-nitroanilides k c values similar to those normally found with specific ester substrates, indicating that the deacylation steps of the reactions are rate determining.

Cyanate modification of essential lysyl residues in the catalytic subunit of tobacco ribulosebisphosphate carboxylase

Crystalline ribulose-1,5-bisphosphate carboxylase (3-phospho- d-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) isolated from tobacco ( Nicotiana tabacum L.) leaf homogenates is irreversibly inactivated by incubation with potassium cyanate at pH 7.4. The rate of inactivation is pseudo first-order and linearly dependent on reagent concentration. In the presence of ribulosebisphosphate or high levels of CO 2 and Mg 2+ the rate constant for inactivation is reduced, suggesting that chemical modification occurs in the active site region of the enzyme. In contrast, neither the effector NADPH nor the activator Mg 2+ alone significantly affect the rate of inactivation by cyanate; however, NADPH markedly enhances the protective effect of CO 2 and Mg 2+. Incubation of the carboxylase with potassium [ 14C]cyanate in the absence or presence of ribulosebisphosphate revealed that the substrate specifically reduces cyanate incorporation into the large catalytic subunits of the enzyme. Analysis of acid hydrolysates of the radioative carboxylase indicated that the reagent carbamylates both NH 2-terminal groups and lysyl residues in the large and small subunits. Comparison ot the substrate-protected enzyme with the inactivated carboxylase revealed that ribulosebisphosphate preferentially reduces lysyl modification within the large subunit. The data here presented indicate that inactivation of ribulosebisphosphate carboxylase by cyanate or its reactive tautomer, isocyanic acid, results from the modification of lysyl residues within the catalytic subunit, presumably at the activator and substrate CO 2 binding sites on the enzyme.

An adenosine 3′:5′-monophosphate-adenosine binding protein from mouse liver: some physicochemical properties

A number of physiochemical properties of the cyclic AMP-adenosine binding protein of mouse liver (Ueland, P.M. and Døskeland, S.O. (1977) J. Biol. Chem. 252, 677–686) have been studied. 1. 1.|The specific extinction coefficient, E 280nm 1%, was estimated to 13.0. 2. 2.|Amino acid and amide group analyses confirmed the acidic properties of the protein as determined by electrofocusing (p I = 5.7). Based on the estimated partial specific volume ( υ = 0.74 cm 3/g ) the minimum molecular weight of the native, tetrameric protein was recalculated to be 185 000 ( s 20,w = 8.8 · 10 −13 s and Stokes radius = 48 A ̊ ). 3. 3.|No NH 2-terminal amino acid was found by the dansyl method using [ 14C]-dansyl chloride, indicating that the NH 2-terminal groups are blocked. 4. 4.|Amino acid analyses gave 6 half-cystine residues per subunit, and the same number of free sulfhydryl groups was found by titration of the denatured protein with 5,5′-dithiobis (2-nitrobenzoic acid). 5. 5.|The reactivity of the SH groups in the native protein with 5,5′-dithiobis (2-nitrobenzoic acid) revealed rapidly reacting (SH I), sluggishly reacting (SH II) and “masked” (SH III) SH groups. ATP, adenosine, Mg 2+ and KCl, factors known to affect the activation of cyclic AMP binding sites (Ueland, P.M. and Døskeland, S.O. (1978) Arch. Biochem. Biophys., in press) changed the reactivity of separate SH groups.

Morphological, histochemical, and biochemical studies on the gut ofHaemonchus contortus Rud., 1803

A study has been made on the structure and chemical composition of the gut of Haemonchus contortus (Rud., 1803). The oesophagus has typically a triradiate, cuticle-lined lumen. The intestinal epithelium is provided with a well-developed brush border which contains periodic acid-Schiff-positive mucoproteins. The intestinal epithelium stores glycogen and lipids. It stains diffusely for phospholipids and general proteins and also for terminal-NH2 group. The presence of Fe2+ and Fe3+ containing pigments and activities of acid and alkaline phosphatases, glucose-6-phosphatase, and 5'-nucleotidase have been observed in the intestinal epithelium. Biochemically pH optimum for intestinal acid phosphatase has been found to be 4.8. The brush border shows positive reactions for acid phosphatase and glucose-6-phosphatase, and negative reactions for alkaline phosphatase and 5'-nucleotidase, and negative reactions for alkaline phosphatase and 5'-nucleotidase. The presence of enzymes in the brush border is related to extracellular digestion and absorption of nutrients.

Demonstration of pyroglutamylpeptidase and amidase activities toward thyrotropin-releasing hormone in hamster hypothalamus extracts.

Using a radioimmunoassay method for thyrotropin-releasing hormone, the presence of thyrotropin-releasing hormone-metabolizing activity in various hamster tissues was demonstrated. While there was substantial activity degrading thyrotropin-releasing hormone in hypothalamus, there was a notable absence of such activity in pituitary. The enzymatic activity in the hypothalamus was shown to be soluble and separable into two fractions. Analysis of the metabolic products formed by the two enzymes indicated that one possessed an amidase activity (less than Glu-His-Pro-NH2 leads to less than Glu-His-Pro) and the other possessed pyroglutamylpeptidase activity (less than Glu-His-Pro-NH2 leads to less than Glu+His-Pro-NH2). Other peptides containing NH2-terminal pyroglutamic acid or COOH-terminal amide groups did not block the hydrolysis of thyrotropin-releasing hormone, suggesting that the enzymes were specific. Some inhibitors preferentially blocked the activity of one or the other enzymes. Of possible biological significance is the observation that thyroid-stimulating hormone inhibited the amidase activity while hydrocortisone inhibited the pyroglutamylpeptidase activity.

Isolation, purification and some properties of a lectin and abrin from Abrus precatorius linn

An alternative and simple procedure has been described for the simultaneous separation of the lectin and abrin from the seeds of Abrus precatorius and their purification free from each other. Both the lectin and abrin have been crystallized, the latter as salt-free crystals. One variety of abrin which was nonhemagglutinating and did not bind d-galactose was obtained. The lectin found homogeneous by immunodiffusion, immunoelectrophoresis and sedimentation had a molecular weight of 132,000 which underwent pH-dependent reversible association-dissociation at pH 7 and 2, dissociating into non-covalently bound subunits of approximately 64,000 molecular weight. The protein was stable in the pH range 2–10. The abrin molecule did not undergo any change at low pH values. The C- and NH 2-terminal groups of the lectin were found to be Ala-Leu (or Leu-Ala) and valine, respectively. Crystalline lectin showed the presence of three isolectins in isoelectric focusing.

Contribution of the peptide backbone to the action of oxytocin analogs.

This investigation was undertaken to evaluate the functional contribution of the peptide backbone of oxytocin in its interaction with receptor. Corey-Pauling-Koltun models of (Gly-7) deaminooxytocin, deaminotocinamide, and their respective retro-D-analogs built in any specific conformation (e.g., the Walter-Urry model for oxytocin) have a quai-equivalent topochemical arrangement of amino-acid side chains; however, the CO and NH elements of the peptide backbone of the retro-D-analog are reversed. The retro-D-analogs of deaminotocinamide and (Gly-7) deaminooxytocin (prepared using D-alle for L-Ile) and their respective N-formyl derivatives were assayed for uterotonic activity relative to related L-peptides. All retro-D-analogs (tested at concentrations ranging from 10-10 to 10-5 M) were devoid of angonistic (or antagonistic) activity in the isolated rat uterus, except for the retro-D-(D-alle-3, Gly-7) deaminooxytocinamine, which retains a terminal NH-2 group on the tail; the latter is a partial agonist with very low affinity. The results obtained with retro-D-analogs indicate that one or more of the elements of the peptide backbone of the tocinamide ring are essential for "occupation" and "activation" of uterine receptors. Oxytocin action may be the resultant of multiple hydrogen-bonding interactions between CO, NH, NH-2, and OH groups of the hormone with complementary groups on receptor, made possible by appropriate hydrophobic bonding.

Adhesive interaction with lignin and various cellulose monolayers

Monolayers of ethylcellulose, cellulose acetate, cellulose acetate-butyrate and lignin were spread on a Langmuir trough and their force against area curves determined. While the cellulose chains are extended, there is definite evidence for a folded structure for the lignin chains in the water surface. Films of these polymers were adjusted to an internal pressure of 2 mN m–1 and the increases of pressure at constant area followed, after injection of solutions of (a) bovine plasma albumin and (b) epoxy resin. The resultant curves were convex to “amount injected” axis for (a) and concave for (b). It is suggested for (a) the polypeptide chains are unravelled and adsorb flat against the monolayer, while for (b) the epoxy chains adsorb by their terminal NH2 groups and hang down as random coils vertically under the monolayer, the adsorbed chains exerting a lateral entropic repulsion on each other.

Evidence for Localization of Polymerization Sites in Fibrinogen

Abstract It was previously reported (Kudryk, B., Reuterby, J., and Blomback, B. (1973) Thromb. Res. 2, 297) that conjugated fibrinogen, after activation with thrombin, has affinity for Fragment D (Fg-D) obtained by digestion of fibrinogen with plasmin. NH2-terminal analysis of the conjugates suggests that fibrinopeptides are released during activation. Conjugates activated with Reptilase have similar affinity characteristics but show a lower content of glycine NH2-terminal groups, suggesting release of only fibrinopeptide A. Most significant is the finding that a conjugated NH2-terminal CNBr fragment of fibrinogen (N-DSK; (Aα1-51, Bβ1-115, γ1-78)2), after thrombin activation and apparent release of fibrinopeptides, has comparable affinity for Fg-D. All of these different conjugates have the strongest affinity for the high molecular weight subspecies of Fg-D (Fg-Ds). These findings are in favor of the existence of two binding domains in fibrinogen which are important for the fibrinogen-fibrin transition. One domain is in Fg-D and the other is in N-DSK. Experiments with an analog of N-DSK showed that certain structures in N-DSK may be of particular importance for binding or activation, or both.

Studies on Human β-d-N-Acetylhexosaminidases: II. KINETIC AND STRUCTURAL PROPERTIES

Abstract β-d-N-Acetylhexosaminidase A and B purified to homogeneity from human placenta have pH optima of 4.4 using 4-methylumbelliferyl-β-d-N-acetylgalactosaminide as substrate. The Km of both hexosaminidase A and hexosaminidase B for this synthetic substrate was found to be 0.5 mm and heat of activation for both the enzymes was 10,500 cal. No effect of any divalent ions studied was observed except for a 25% decrease in the enzyme activity of both hexosaminidase A and B in the presence of 5 mm cupric ion. p-Hydroxymercuribenzoate, 1 mm, almost completely inhibited both hexosaminidase A and hexosaminidase B activity. N-Ethylmaleimide, arsenate, and iodoacetate had no effect on the hexosaminidases. No inhibition of hexosaminidase A or hexosaminidase B was observed in the presence of 30 mm concentration of various anions except for acetate which brought about 75% inhibition of both hexosaminidase A and hexosaminidase B activity. Using Sephadex G-200 filtration, the molecular weight of hexosaminidase A and B was estimated at 140,000. However, using the sedimentation equilibrium technique the molecular weight was found to be 100,000 ± 3,000 and 101,400 ± 4,700 for hexosaminidase A and B, respectively. After S-carboxymethylation, the subunit molecular weight of hexosaminidase A and B was determined using various concentrations of polyacrylamide in disc electrophoresis in the presence of sodium dodecyl sulfate. Hexosaminidase B dissociated into one major subunit corresponding to a molecular weight of 17,000 to 18,000, whereas hexosaminidase A dissociated into one major subunit corresponding to a molecular weight of 17,000 to 18,000 and two more protein bands corresponding to about 35,000 and to 55,000. Similar results were obtained when hexosaminidase A and B were dissociated into subunits by treatment with guanidine hydrochloride or by treatment with maleic anhydride in the presence of sodium dodecyl sulfate and the subunits separated by polyacrylamide gel electrophoresis. In urea-starch gel electrophoresis hexosaminidase A dissociated into three major bands and hexosaminidase B into two bands. In urea-starch gel the electrophoretic mobility of one of the bands of dissociated hexosaminidase A and hexosaminidase B seemed to be very similar or identical. Both hexosaminidase A and hexosaminidase B have blocked NH2-terminal groups. The COOH-terminal amino acids in hexosaminidase A was found to be serine and in hexosaminidase B was found to be aspartic acid or asparagine. The amino acid composition of hexosaminidase A and hexosaminidase B was determined and found to differ.

"Holly wreath" morphology of feline erythrocytes--the effects of cyanate and 4,4'-dipyridyldisulfide.

SummaryFeline erythrocytes develop into “holly wreath” or acanthocytic forms upon deoxygenation. Prior reaction with KCNO or with the sulfhydryl modifying reagent, PDS, prevents the development of abnormal morphology. Cyanate causes irreversible modification of the terminal NH2-groups and PDS modifies the reactive -SH groups in the hemoglobins to profoundly alter the properties of the feline hemoglobins. A possible analogy with the human “sickle cell” system is indicated.We thank Mrs. S. L. Tsai, Mrs. Y. P. Huang, and Miss Colleen Counihan for technical assistance. We also thank Mr. Gene Putz for helpful discussions and Mr. Anthony Kuzma for expert help in preparations of the photomicrographs.

The composition of sea urchin sperm and embryo histones

Four of the five pluteus stage histone fractions from Strongylocentrotus purpuratus were isolated by preparative electrophoresis. The amino acid composition of these embryonal histones resembles that of the corresponding calf thymus fractions except that two lysine-rich F1 fractions were found in the pluteus, both containing significantly more glutamic acid. The NH 2-terminal groups of embryonal histones corresponding to the Fractions F1, F2a1 and F2a2 are blocekd, probably by acetyl. Proline and alanine are the NH 2-terminal amino acids of the Fractions F2b and F3, respectively. The five electrophoretically distinct sperm histone fractions were chemically and electrophoretically fractionated into seven components. Of the two slowly migrating proteins the composition of the slower fraction is similar to the arginine-rich histone F3 (Band 1) from pluteus. The lysine-rich fraction of sperm chromatin (Fraction 3) is quite different from the embryonal lysine-rich histone F1 and has a greater net positive charge. The large δ fraction (Fractions 4, 5 and 6) is heterogeneous consisting of several proteins. One component (Fraction 4) is moderately rich in lysine and resembles the F2b histone in pluteus; another component (Fraction 6) is arginine-rich and differs chemically from the embryonal F2a1 and F2a2 histones. The fastest migrating fraction (Fraction 7) is similar to the arginine-rich histone F2a1 of calf thymus. The NH 2-terminal groups of sperm histones are mostly blocked, probably by acetyl with the exception of alanine NH 2-terminal in the arginine-rich subfraction of the δ histone.

Human amyloid protein: chemical variability and homogeneity.

The morphology of the fibril of amyloid derived from different individuals is similar, but occasionally significant differences are noted. All human amyloid filaments have a "β-pleated sheet" conformation as revealed by x-ray diffraction, and those examined after orientation show a "cross-β" pattern. All amyloid fibril concentrates studied so far can be fractionated to obtain the major amyloid protein component(s) by sequential gel filtration with 5 M guanidine-HCl in 1 N acetic acid on Sepharose 4B and Sephadex G-100 or G-75 columns with the removal of over 28% of proteins representing minor constituents. The major amyloid protein(s) obtained from the spleen and/or liver of six patients is found to contain tryptophan, to be deficient in hydroxylysine and hydroxyproline and usually at least one commonly occurring amino acid and to have a high content of dicarboxylic acid and short chain amino acids and unreactive (blocked) NH2-terminal groups or aspartic acid-asparagine (Asx). However, the amyloid protein(s) from each individual differs from that of the others in molecular weight, in amino acid composition and in the presence or absence of specific tryptic peptides. Amyloid protein(s) from the liver and spleen of the same individual is identical. No chemical characteristics distinguish amyloid proteins derived from cases classified clinically as "primary" from those classified as "secondary." There is a striking chemical similarity between amyloid proteins and the NH2-terminal variable fragment of the light and heavy chain of immumoglobulin proteins.

Hemoglobin synthesis during amphibian metamorphosis: I. Chemical studies on the hemoglobins from the larval and adult stages of Rana catesbeiana

The hemoglobins of Rana catesbeiana tadpoles were resolved into five characteristic components by polyacrylamide gel electrophoresis. Characterization of the peptide chains of the major and of one other component showed each to have two types of chain, none of which is in common. The major tadpole hemoglobin with two valine NH 2-terminal groups has no peptide chain in common with the major frog hemoglobin. The latter has two chains beginning with glycine. No other amino acid groups appear to be available to the Edman end group reagent.

Thiogalactoside Transacetylase: AMINO- AND CARBOXYL-TERMINAL STUDIES

Abstract Carboxypeptidase digestion of denatured thiogalactosidase transacetylase showed that the COOH-terminal sequence is Ser-Val and that 2 moles of valine are released per mole of protein. Although this is consistent with the previous finding that the protein is composed of two identical chains, studies of the NH2 terminus show heterogeneity and lack of stoichiometry. Instead of 2 moles of a single amino acid per mole of protein, 0.5 mole of aspartic acid (or asparagine) and 0.4 mole of methionine were found by both the Sanger and the Stark and Smyth procedures. The same result was obtained after treatment of the protein under conditions expected to cleave α-N-formyl groups. Various explanations to account for these findings are discussed, including blocked NH2-terminal end groups, proteolytic hydrolysis, and random cleavage as a consequence of a step in protein biosynthesis.

Chicken gizzard myosin

The Ca ++-ATPase activity of chicken gizzard myosin is increased 2- to 3-fold by tryptic treatment and by various substances, urea, guanidine-HCl, sodium dodecylsulfate, KCNS, LiBr, ethanol, or ethylene glycol. These substances also activate the Ca ++-ATPase of rabbit uterus myosin but have no effect on the Ca ++-ATPase of chicken breast myosin. Maximal Ca ++-ATPase activity of gizzard myosin is observed when 38 free NH 2-terminal groups in 600,000 gm of this myosin are liberated by trypsin. At this stage most of the myosin remains intact in the analytical ultracentrifuge. The addition of 1.0–2.0 M urea to gizzard myosin, pH 7.0, results in a 3-fold decrease in the Michaelis constant. The effect of urea on the activation of Ca ++-ATPase of gizzard myosin is reversible. Twelve cysteine residues per 600,000 gm of gizzard myosin are reacting with iodoacetate in the presence of 1.5 M urea in contrast to the nonreactivity of the SH groups without urea. Gizzard myosin retains all of its Ca ++-ATPase activity after 12 of its SH groups have been alkylated. From comparative experiments with iodoacetate and iodoacetamide it is concluded that the ATPase site of gizzard myosin undergoes an alteration in the presence of urea as a result of conformational changes in regions of the myosin molecule removed from the ATPase site. The Ca ++-ATPase and NTPase activities of gizzard myosin are much lower than those of breast myosin even in the presence of modifiers. However, the ability of the two different types of myosin to combine with rabbit skeletal F-actin is the same. Amino acid analyses show differences in the composition of gizzard and breast myosin. Gizzard myosin contains cystine and has a slightly lower cysteine content than does breast myosin.

Comparative studies of amino acid composition and N-terminal amino acids of the protein from ribosomes of Escherichia coli, yeast and rat liver

The structure of proteins from ribosomes of Escherichia coli, yeast and rat liver was compared. Analyses of the amino acids composition of proteins revealed remarkable differences among the protein moieties of ribosomes. However, the NH2-terminal groups of three different ribosomal proteins were similar.

Studies on the serial extraction of pituitary proteins.

Pituitary glands of beef, sheep and pig origin were successively extracted with water at pH 5.5, M/10 (NH4)2SO4 at pH 4.0, M/4 (NH4)2SO4 at cither pH 5.5 or 7.5, and finally with 60 percent ethanol at pH 9.8 to 10. The extracts were bioassayed for the anterior pituitary hormones and for the proteinases I and II. With certain exceptions, the first extract contained mainly FSH and proteinase I; the second, LH and TSH, the third, GH and proteinase II; and the fourth, prolactin. FSH and GH were further purified to establish the amenability of these extracts to fractionation. Growth hormone isolated from beef pituitary extracts prepared at pH 5.5 appears to be a mixture of two biologically active proteins: one with alanyl and phenylalanyl NH2-terminal groups and another with methionyl and alanyl NH2-terminal groups. Small amounts of NH2-terminal serine and glutamic acid were also present. By varying the conditions of extraction appropriately, the amounts of the two types of hormones could be varied inversely. ...

Studies on mucoproteins III. The accessibility to trypsin of the susceptible bonds in ovine submaxillary gland mucoprotein

Ovine submaxillary gland mucoprotein was found to be susceptible to trypsin. The evidence is a marked increase in terminal NH 2-groups and a corresponding rise in filterability through cellulose acetate membranes and an increase in electrophoretic mobility after trypsin treatment. Enzymic removal of the terminal neuraminic acid units of the prosthetic groups (6-α- D-N-acetylneuraminyl (2→6) N-acetylgalactosamine) increased the trypsin effect on ovine submaxillary gland mucoprotein by about 45%. This means that at least one-third of the trypsin-susceptible bonds in ovine submaxillary gland mucoprotein are inaccessible to the enzyme molecule. This figure may represent only a minimum value since any steric hindrance exerted by the remaining N-acetylgalactosamine unit cannot be assessed at present. The complete loss of virus haemagglutinin inhibitory capacity of ovine submaxillary gland mucoprotein on trypsin action is interpreted in terms of a critical molecular size below which a mucoprotein ceases to act as an inhibitor.