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PubMed1986

Identification of the renal Na+/H+ exchanger with N,N'-dicyclohexylcarbodiimide (DCCD) and amiloride analogues.

Dicyclohexylcarbodiimide (DCCD) and the 5-ethyl-isopropyl-6-bromo-derivative of amiloride (Br-EIPA) have been used as affinity and photoaffinity labels of the Na+/H+ exchanger in rat renal brush-border membranes. Intravesicular acidification by the Na+/H+ exchanger was irreversibly inhibited after incubation of vesicles for 30 min with DCCD. The substrate of the antiporter, Na+, and the competitive inhibitor, amiloride, protected from irreversible inhibition. The Na+-dependent transport systems for sulfate, dicarboxylates, and neutral, acidic, and basic amino acids were inhibited by DCCD, but not protected by amiloride. An irreversible inhibition of Na+/H+ exchange was also observed when brush-border membrane vesicles were irradiated in the presence of Br-EIPA. Na+ and Li+ protected. [14C]-DCCD was mostly incorporated into three brush-border membrane polypeptides with apparent molecular weights of 88,000, 65,000 and 51,000. Na+ did not protect but rather enhanced labeling. In contrast, amiloride effectively decreased the labeling of the 65,000 molecular weight polypeptide. In basolateral membrane vesicles one band was highly labeled by [14C]DCCD that was identified as the alpha-subunit of the Na+,K+-ATPase. [14C]-Br-EIPA was mainly incorporated into a brush-border membrane polypeptide with apparent molecular weight of 65,000. Na+ decreased the labeling of this protein. Similar to the Na+/H+ exchanger this Na+-protectable band was absent in basolateral membrane vesicles. We conclude that a membrane protein with an apparent molecular weight of 65,000 is involved in rat renal Na+/H+ exchange.

Amiloride↗
PubMed1978

Nuclear proteins. V. Studies of histone-binding proteins from mouse liver by affinity chromatography.

We examined four extracts of mouse liver for histone-binding proteins using histone affinity chromatography and positively charged resins. The extracts used were cytoplasm and washes from isolated nuclei with buffers containing 0.05 M Tris, 0.15 M NaCl or 0.35 M NaCl. Proteins from the nuclear washes showed greater binding to the columns than proteins from the cytoplasm. The binding fractions were heterogeneous in gel electrophoresis systems. Proteins bound to affinity columns of individual histones were similar to those bound to columns of whole histone, polylysine and DEAE. A 25,000 dalton polypeptide (J2), found only in nuclear washes was a prominent histone-binding protein. It could be competitively eluted from DEAE with histones, suggesting polypeptide J2 may show a specific affinity for histones. Polypeptide J2 has an acidic to basic amino acid ratio of 1.58, and its amino acid composition is not similar to that of the high mobility group 1 protein. Polypeptide J2 binds to hydrophobic columns and may play a role in modifying histone-histone and histone-DNA interactions.

Amino Acids↗
PubMed1979

Isolation of an acidic protein from the armadillo submandibular gland.

An acidic protein fraction with an apparent molecular weight of 34 000 has been isolated from the Cetavlon-treated, mucin-free supernatant of the armadillo submandibular gland 0.01 M NaCl extract. This purified material, which was obtained in a yield of 0.45%/g wet gland, contains 24 mol % acidic amino acids and 4 mol % basic amino acids. Hexosamines, sialic acid, and neutral sugars represent 7% of the dry sample weight. In polyacrylamide gel and cellulose acetate electrophoresis, a single protein band was observed. The acidic protein fraction is highly reactive with the Lowry phenol reagent, giving a protein value 83% higher than that obtained by summation of its anhydrous amino acids, and is explained by the occurrence of peptide linkages peculiar to this material. The presence of other basophilic components besides mucus glycoproteins within the salivary gland of the armadillo may have physiological significance.

Amino Acids↗
PubMed1989

Purification and molecular properties of the toxin coded by Ustilago maydis virus P4.

The toxin from the P4 strain of Ustilago maydis was purified and characterized using a series of gel-filtration and ion-exchange columns. The apparent molecular weight of the purified toxin was estimated from gel electrophoresis to be 11.3 kd in the presence of 2-mercaptoethanol and 10.3 kd in the absence of 2-mercaptoethanol. Amino acid analysis indicated 12% basic amino acids, 14% acidic amino acids and 16% glycine. The toxin was also stable to filtration and repeated freezing at -20 degrees C and thawing.

Amino Acids↗
PubMed1981

Differential effects of hypertonic mannitol and glycerol on rat brain metabolism and amino acids.

Intraperitoneal injections in rats of two different dosages of hypertonic solutions containing mannitol or glycerol caused complex and differential changes in brain amino acids. When plasma osmolalities were elevated to toxic levels of 397--432 mOsm/kg H2O, brain sodium was increased, whereas plasma sodium was decreased. Brain potassium was not affected. Brain water decreased significantly, concomitant with elevation of plasma osmolality. Both brain lactic acid and [125I]albumin space rose significantly. Brain amino acids (mostly aliphatic and basic amino acids) as well as Gaba and glycine (putative inhibitory neurotransmitters) increased after both mannitol and glycerol. Ammonia was stimulated by mannitol but was unaffected by glycerol. Plasma amino acids, which generally increased after mannitol, were decreased by glycerol. When the plasma osmolalities were elevated only to moderate levels (about 350 mOsm/kg H2O), only glycerol induced a significant increase in brain taurine, aspartic acid, alanine, leucine and lysine. Thus, with moderate hyperosmolality, glycerol has striking effects on brain amino acid metabolism that are not observed with mannitol.

Amino Acids↗
PubMed1986

Which electrodic solutions for immobilized pH gradients?

Carrier ampholytes covering a pH range corresponding to, or narrower than, the span of the immobilized pH gradient (IPG) are a most suitable electrodic solution for IPGs. They are able to collect, and completely remove from the gel, much higher amounts of non-buffering ions than are solutions of acidic and basic amino acids. This makes it possible to directly run IPGs just after their polymerization, without the need of a washing step to remove catalysts and unreacted Immobiline monomers. The same applies most advantageously when the gel formulation includes urea and/or detergents. Ions contributed by the sample solution are also prevented from casting high-conductivity ridges around the electrodes, without any need either of a dialysis step or of an increased slab size with pH plateaus. The migration of the sample proteins toward their equilibrium position is faster in the presence of carrier ampholytes. The effective concentrations of the latter are in the range 0.3-1%.

Ampholyte Mixtures↗
PubMed1982

Purification and properties of Streptococcus mutans extracellular glucosyltransferase.

Extracellular glucosyltransferase (sucrose:1,6-alpha-D-glucan 3-alpha- and 6-alpha-glucosyltransferase) was purified about 10 000-fold from the culture supernatant of Streptococcus mutans 6715. The enzyme preparation was homogeneous on polyacrylamide gel electrophoresis, isoelectric focusing and ultracentrifugation analyses. The specific activity of the enzyme was 34.9 I.U. per mg of protein and the carbohydrate content was less than 1% (w/w). The molecular weight was determined to be 149 000 +/- 5000 by sedimentation equilibrium experiment. The acidic and basic amino acids of the enzyme comprised 29 and 8.4% of total amino acid, respectively, and the isoelectric point was pH 4.1. The enzyme had the optimum pH of 5.5 and the Km value of 2.4 mM for sucrose. The water-soluble glucan, which was de novo-synthesized from sucrose by the purified enzyme, was analyzed by a gas-liquid chromatography-mass spectroscopy and was found to be 1,6-alpha-D-glucan with highly (35%) branched structure of 1,3,6-linked glucose residue.

Amino Acids↗
PubMed1987

Purification and some properties of bound cysteine proteinase inhibitor from cornified cells of newborn rat epidermis.

Cysteine proteinase inhibitor exists in two forms in terminally differentiated keratinocytes. One is readily soluble in 20 mM sodium phosphate buffer but the other is bound to the plasma membrane and is poorly soluble. The cysteine proteinase inhibitor (CPI) from the membrane was extracted from cornified epidermal layers of 2-day-old rats and its properties were compared with those of soluble CPI. This CPI (bound CPI) was solubilized in alkaline 8 M urea containing 2-mercaptoethanol from the residual tissue exhaustively treated with buffered 4 M urea. CPI was separated from keratin by ammonium sulfate precipitation and purified by means of papain affinity chromatography, ion exchange column chromatography and gel filtration. Bound CPI had an Mr value of about 16,000, a pI value of 3.8 and was unstable at above 80 degrees C, while soluble CPI was of Mr 13,000 and stable at above 80 degrees C. Both CPIs were stable at 4 degrees C in the range of 3.0-9.0. Bound CPI contained half cystine and the ratio of acidic-to-basic amino acids was 3.18. Bound CPI inhibited rat liver cathepsins B, H, and L but did not inhibit the activity of noncysteine proteinases. Papain activity was inhibited by bound CPI at three sites, noncompetitively, and the Ki value was calculated to be 0.11 nM.

Animals↗
PubMed1995

Increased heparin binding by site directed mutagenesis of a recombinant chimera of bombyxin and insulin-like growth factor II.

BOMIGF, a chimera between bombyxin and insulin-like growth factor II (IGF II) has been modified and extended to a total of 85 amino acids including 7 additional basic amino acids. The new peptide bound heparin as efficiently as GM-CSF and much better than IGF II or BOMIGF. It could be retained on albumin-coated tissue culture wells without loosing its capacity to stimulate thymidine incorporation into erythroid cells of fetal bovine liver and on fibronectin-heparin coated wells keeping its mitogenic activity towards L6 muscle cells.

Amino Acid Sequence↗
PubMed1993

L-[2-18F]fluorophenylalanine and L-[U-14C]phenylalanine: a comparative study of their transport to rat brain.

The transport rates of L-[2-18F]fluorophenylalanine ([18F]Phe) and L-[U-14C]phenylalanine ([14C]Phe) across the blood-brain barrier were compared in saline, large neutral amino acids (NAA) and large basic amino acids (BAA) loaded rats. The fraction of transported tracer (FTT) was expressed as the ratio of brain radioactivity to integrated plasma radioactivity at 5 min after injection of the tracers. Cerebral FTTs of [18F]Phe and [14C]Phe in the control group were 0.042 and 0.045, respectively, and decreased to 0.016 and 0.019 in the NAA loaded group. The ratios of FTT of [18F]Phe and that of [14C]Phe were approx. 0.90, and did not differ among the three groups. These results indicate that the transport of [18F]Phe from the blood to the brain can be considered to be mediated by the NAA transport system and that its transport rate is almost the same as that of [14C]Phe.

Amino Acids↗
PubMed2003

Nuclear localization domains in human thyroid transcription factor 2.

Thyroid transcription factor-2 (TTF2) is a nuclear protein involved in morphogenesis and gene expression in thyroid gland, belonging to the family of the forkhead/winged-helix transcription factors. In the present study we have investigated the sequence determinants for transport and accumulation into the nucleus of the TTF2 protein. By transient expression of fusion proteins constructed by joining different parts of TTF2 to the reporter gene of the jellyfish green fluorescent protein (GFP) and, in a separate set of deleted constructs, the glutathione S-transferase (GST) coding sequence, we have demonstrated that a basic amino acid stretch present at both ends of the DNA-binding domain is a bona fide nuclear localization signal (NLS). We have analyzed the subcellular localization of deleted GFP-GST-TTF2 fusion proteins and have shown that residues inside the forkhead domain (FHD) contributed to the complete nuclear TTF2 protein accumulation. Furthermore, by means of GST binding assays we have shown that distinct TTF2 fragments, containing the NLS, were able to bind the nuclear import receptor importin alpha. Taken together, our results provide the first documentation about nuclear targeting of a forkhead protein containing two identical NLS signal flanking the DNA-binding domain.

Active Transport, Cell Nucleus↗
PubMed2003

Potentiating effect of distant sites in non-phosphorylated cyclic peptide antagonists of the Grb2-SH2 domain.

Without the presence of a phosphotyrosyl group, a phage library derived non-phosphorylated cyclic peptide ligand of Grb2-SH2 domain attributed its high affinity and specificity to well-defined and highly favored interactions of its structural elements with the binding pocket of the protein. We have disclosed a significant compensatory role of the Glu(2-) sidechain for the absence of the phosphate functionality on Tyr(0) in the peptide ligand, cyclo(CH(2)CO-Glu(2-)-Leu-Tyr(0)-Glu-Asn-Val-Gly-Met(5+)-Tyr-Cys)-amide (termed G1TE). In this study, we report the importance of hydrophobic residue at the Tyr+5 site in G1TE. Both acidic and basic amino acid substitutes are disfavored at this position, and replacement of Met with beta-tert-butyl-Ala was found to improve the antagonist properties. Besides, the polarity of the cyclization linkage was implicated as important in stabilizing the favored binding conformation. Oxidation of the thioether linkage into sulfoxide facilitated the binding to Grb2-SH2 markedly. Simultaneous modification of the three distant sites within G1TE provided the best agent with an IC(50) of 220 nM, which is among the most potent non-phosphorous- and non-phosphotyrosine-mimic containing Grb2-SH2 domain inhibitors yet reported. This potent peptidomimetic provides a novel template for the development of chemotherapeutic agents for the treatment of erbB2-related cancer. Biological assays on G1TE(Gla(2-)) in which the original residue of Glu(2-) was substituted by gamma-carboxyglutamic acid (Gla) indicated that it could inhibit the interaction between activated GF receptor and Grb2 protein in cell homogenates of MDA-MB-453 breast cancer cells at the 2 microM level. More significantly, both G1TE(Gla(2-)) alone and the conjugate of G1TE(Gla(2-)) with a peptide carrier can effectively inhibit intracellular association of erbB2 and Grb2 in the same cell lines with IC(50) of 50 and 2 microM, respectively.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Sphingosine kinase-1 is cleaved by cathepsin B in vitro: identification of the initial cleavage sites for the protease.

Previous work has identified sphingosine kinase-1 (SK1) as a substrate for the cysteine protease cathepsin B in vitro. In this study, the mechanism of SK1 cleavage by cathepsin B was investigated. We identified two initial cleavage sites for the protease, the first at histidine 122 and the second at arginine 199. Mutation analysis showed that replacement of histidine 122 with a tyrosine maintained the activity of SK1 while significantly reducing cleavage by cathepsin B at the initial cleavage site. The efficacy of cleavage of SK1 at arginine 199, however, was not affected. These studies demonstrate that SK1 is cleaved by cathepsin B in a sequential manner after basic amino acids, and that the initial cleavages at the two identified sites occur independently of each other.

Amino Acids, Basic↗
PubMed2000

Functional characterization of purified zinc transporter from renal brush border membrane of rat.

Major zinc binding protein purified from renal brush border membrane (BBM) (R. Kumar, R. Prasad, Biochim. Biophys. Acta 1419 (1999) 23) was reconstituted into liposomes and its functional characteristics were investigated. Physical incorporation of the major zinc binding protein into the proteoliposomes was checked by SDS-PAGE, which showed a single band on silver staining. The structural integrity of the proteoliposomes was assessed by phase contrast microscopy, which revealed the proteoliposomes as globular structures and intact boundaries. Further structural integrity/leakiness of the proteoliposomes was checked by monitoring efflux of Zn(2+) from the pre-loaded proteoliposomes in the presence of either 2 mM Ca(2+) or Cd(2+) or Zn(2+). It was observed that even after 2 h of the initiation of efflux, 85-95% of Zn(2+) was retained in the proteoliposomes, thereby indicating that proteoliposomes were not leaky and maintained structural integrity during the uptake study. Zinc uptake into the proteoliposomes followed Michaelis-Menten kinetics with affinity constant (K(m)) of 1.03 mM and maximal velocity (V(max)) of 1333 nmol/mg protein per min. The uptake process followed first-order kinetics with a rate constant (k) of 1. 09x10(-3) s(-1). The specificity of zinc transport system was determined by studying the interaction of divalent cations viz. Ca(2+) and Cd(2+) with the zinc uptake. It was observed that Cd(2+) competitively inhibited the zinc uptake process with inhibitory concentration (K(i)) of 2.9 mM. Kinetic analysis of inhibitory effect of Cd(2+) on zinc uptake revealed an increase in K(m) to 1.74 mM without influencing V(max). Zn(2+) uptake into the proteoliposomes was found to be temperature sensitive and Arrhenius plot showed a breakpoint at 27 degrees C. The apparent energies of activation (E(a)) were found to be 7.09 and 2.74 kcal/mol below and above the breakpoint, respectively. The initial velocity of Zn(2+) uptake increased with the increase in outwardly directed proton gradient ([H](i) greater than [H](o)). The Zn(2+) uptake was inhibited by DCCD, thereby suggesting the involvement of -COOH groups in the translocation of Zn(2+) across the lipid bilayer. The ratio of acidic to basic amino acids (1.26) strongly indicates that it is an acidic protein. The cysteine content in this protein was insignificant, which further corroborates the possibility that the acidic amino acids might be prominent candidates for binding to zinc. The findings of the present study confirms that 40 kDa major zinc binding glycoprotein purified from renal BBM is a zinc transporter involved in the influx of Zn(2+) into the epithelial cells of the renal tubular system.

Amino Acids↗
PubMed2001

The effect of peptide-pectin interactions on the gelation behaviour of a plant cell wall pectin.

The effect of basic peptides on the gelation of a pectin from the cell wall of tomato was examined through the determination of gel stiffness, and swelling behaviour of the gel in water. Poly-L-lysine, poly-L-arginine, and a synthetic peptide, designed to mimic a sequence of basic amino acids found in a plant cell wall extensin, act as crosslinking agents. Circular dichroism studies on the interaction of synthetic extensin peptides with sodium polygalacturonate demonstrated that a conformational change was induced as a result of their complexation. In addition to their effect as crosslinking agents, the polycationic peptides reduced the swelling of the pectin network in water.

Amino Acid Sequence↗
PubMed1998

A galactose-specific lectin from the red marine alga Ptilota filicina.

A lectin from the red marine alga Ptilota filicina (PFL) was isolated by affinity chromatography on cross-linked guar gum. PFL agglutinated native and papain-treated human erythrocytes with preference for type O erythrocytes. The lectin was inhibited by galactose and its derivatives. The most potent inhibitors were p-Nitrophenyl-N-acetyl-alpha- and beta-D-galactosaminide. Porcine stomach mucin, bovine submaxillary gland mucin and asialo bovine mucin were also inhibitory. The M(r) of PFL, determined by gel filtration, was 56,900. SDS-PAGE gave one band with a subunit M(r) of 19,320, indicating the native protein to be a trimer of apparently identical subunits. PFL was shown to be rich in acidic and hydroxyl amino acids but low in basic amino acids. The ten N-terminal amino acids were Asx-Thr-Lys-Thr-Leu-Leu-Ala-.

Amino Acid Sequence↗
PubMed1997

Determination of acid dissociation constants of peptide side-chain functional groups by two-dimensional NMR.

An NMR method is described for determining residue-specific acid dissociation constants for peptides which contain more than one residue of the same acidic or basic amino acid. The method is based on using the differences in C alpha H proton chemical shifts which result from peptide sequence nearest-neighbor and possibly secondary structure effects to resolve resonances for carbon-bonded reporter protons adjacent to each side-chain acidic group in two-dimensional total correlation spectroscopy (TOC-SY) spectra. Acid dissociation constants were determined for each of the four lysine side-chain ammonium groups of the peptide Lys-Asn-Asn-Gln-Lys-Ser-Glu-Pro-Leu-Ile-Gly-Arg-Lys-Lys-Thr-NH2. Resonances for the C epsilon H2 protons adjacent to the four side-chain ammonium groups, which overlap in the one-dimensional spectrum, were resolved using the C alpha H-C epsilon H2 cross peaks in the TOCSY spectrum. Chemical shift-pH titration data were obtained for each lysine side-chain ammonium group from one-dimensional subspectra taken from two-dimensional TOCSY spectra measured as a function of pH. The pKAs of the Lys1, Lys5, Lys13, and Lys14 side-chain ammonium groups were determined to be 11.14 +/- 0.01, 10.95 +/- 0.01, 10.96 +/- 0.02, and 11.09 +/- 0.02, respectively. The chemical shift-pH titration data were also analyzed by a pH-independent procedure to obtain relative acid dissociation constants: KA(Lys1)/KA(Lys5) = 0.663 +/- 0.009, KA(Lys1)/KA(Lys13) = 0.703 +/- 0.014, and KA(Lys1)/KA(Lys14) = 0.910 +/- 0.009, which correspond to relative acidities for the side-chain ammonium groups of Lys1, Lys5, Lys13, and Lys14 of 1:1.508:1.422:1.099. To further demonstrate the utility of this method, acid dissociation constants were determined for the six acidic groups of the peptide Glu-Ala-Cys-Asn-Pro-Ala-Ala-Gly-Arg-His-Tyr-Ser-Cys-NH2. Chemical shift-pH titration curves were obtained for the C beta H2 protons adjacent to the two cysteine thiol groups using one-dimensional subspectra taken from TOCSY spectra measured as a function of pH. The pKAs of the CyS3 and Cys13 thiol groups were determined to be 9.21 +/- 0.07 and 8.60 +/- 0.06, respectively. The relative acid dissociation constants (KA(Cys3)/ (KA(Cys13)) were found to be 0.21 +/- 0.06 by the pH-independent calculation procedure.

Amino Acid Sequence↗
PubMed2000

Influence of the axial ligands on the spectral properties of P700 of photosystem I: a study of site-directed mutants.

Two histidines provide the axial ligands of the two chlorophyll a (Chl a) molecules which form the primary electron donor (P700) of photosystem I (PSI). Histidine 676 in the protein subunit PsaA, His(A676), and histidine 656 in subunit PsaB, His(B656), were replaced in the green algae Chlamydomnas reinhardtii by site-directed mutagenesis with nonpolar, uncharged polar, acidic, and basic amino acid residues. Only the substitutions with uncharged polar residues led to a significant accumulation of PSI in the thylakoid membranes. These PSI complexes were isolated and the physical properties of the primary donor characterized. The midpoint potential of P700(+)(*)/P700 was increased in all mutants (up to 140 mV) and showed a dependence on size and polarizability of the residues when His(B656) was substituted. In the light-minus-dark absorbance spectra, all mutations in PsaB exhibited an additional bleaching band at 665 nm at room temperature comparable with the published spectrum for the replacement of His(B656) with asparagine [Webber, A. N., Su Hui, Bingham, S. E., Käss, H., Krabben, L., Kuhn, M., Jordan, R., Schlodder, E., and Lubitz, W. (1996) Biochemistry 35, 12857-12863]. Substitutions of His(A676) showed an additional shoulder around 680 nm. In the low-temperature absorbance difference spectra of P700(+)(*)/P700, a blue shift of the main bleaching band by 2 nm and some changes in the spectral features around 660 nm were observed for mutations of His(B656) in PsaB. The analogous substitution in PsaA showed only a shift of the main bleaching band. Similar effects of the mutations were found in the (3)P700/P700 absorbance difference spectra at low temperatures (T = 2 K). The zero-field splitting parameters of (3)P700 were not significantly changed in the mutated PSI complexes. The electron spin density distribution of P700(+)(*), determined by ENDOR spectroscopy, was only changed when His(B656) was replaced. In all measurements, two general observations were made. (i) The replacement of His(B656) had a much stronger impact on the physical properties of P700 than the mutation of His(A676). (ii) The exchange of His(B656) with glutamine induces the smallest changes in the spectra or the midpoint potential, whereas the other replacements exhibited a stronger but very similar influence on the spectroscopic features of P700. The data provide convincing evidence that the unpaired electron in the cation radical and the triplet state of P700 are mainly localized on the Chl a of the dimer which is axially coordinated by His(B656).

Amino Acid Substitution↗