scholarly journals Chemical modification of Penicillium 1,2-α-d-mannosidase by water-soluble carbodi-imide: identification of a catalytically important aspartic acid residue

1994 ◽  
Vol 303 (1) ◽  
pp. 97-103 ◽  
Author(s):  
T Yoshida ◽  
K Maeda ◽  
M Kobayashi ◽  
E Ichishima
Keyword(s):  
Chemical Modification ◽  
Aspartic Acid ◽  
Competitive Inhibitor ◽  
Reversed Phase ◽  
Penicillium Citrinum ◽  
Water Soluble ◽  
Edman Degradation ◽  
Aspartic Acid Residue ◽  
Partial Protection ◽  
Glycine Ethyl Ester

1,2-alpha-D-Mannosidase from Penicillium citrinum was inactivated by chemical modification with 1-ethyl-3-(3-dimethylamino-propyl)carbodi-imide (EDC). Most of the activity was lost after modification in the absence of a nucleophile, glycine ethyl ester. 1-Deoxymannojirimycin (dMM), a competitive inhibitor of the enzyme, showed partial protection against the inactivation. After the modification by EDC without the presence of a nucleophile, proteolytic digests of the enzyme were analysed by reversed-phase h.p.l.c. and a unique peptide was shown to decrease when dMM was present during the modification. The peptide was absent from the digests of unmodified enzyme. The amino acid sequence of the peptide (A; Ile-Gly-Pro) was identical in part with that of the adjacent peptide (B; Ile-Gly-Pro-Asp-Ser-Trp-Gly-Trp-Asp-Pro-Lys). When cholecystokinin tetrapeptide (Trp-Met-Asp-Phe-NH2) was modified by EDC alone, the modified peptide could be separated from unmodified peptide by reversed-phase h.p.i.c., and Edman degradation was stopped before the modified aspartic acid residue. This suggested that, in the enzyme, peptide A was derived from peptide B by the modification. Consequently, Asp-4 in peptide B was assumed to be masked by dMM during the modification, and to be involved in the interaction of the enzyme with its substrate.

scholarly journals Chemical modification studies on a lectin from Saccharomyces cerevisiae (baker's yeast)

1987 ◽  
Vol 244 (3) ◽  
pp. 579-584 ◽  
Author(s):  
M Kundu ◽  
J Basu ◽  
A Ghosh ◽  
P Chakrabarti
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chemical Modification ◽  
Binding Site ◽  
Structural Changes ◽  
Thiol Groups ◽  
Lectin Activity ◽  
Partial Protection ◽  
Sugar Binding ◽  
Arginine Residues ◽  
Glycine Ethyl Ester

The effect of chemical modification on a galactose-specific lectin isolated from a fatty acid auxotroph of Saccharomyces cerevisiae was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of 50 free amino groups with succinic anhydride or citraconic anhydride led to an almost complete loss of activity. This could not be protected by the inhibitory sugar methyl alpha-D-galactopyranoside. Treatment with N-bromosuccinimide and N-acetylimidazole, for the modification of tryptophan and tyrosine residues, did not affect lectin activity. Modification of carboxy groups with glycine ethyl ester greatly affected lectin activity, although sugars afford partial protection. Modification of four thiol groups with N-ethylmaleimide was accompanied by a loss of 85% of the agglutinating activity, and two thiol groups were found to be present at the sugar-binding site of the lectin. Modification of 18 arginine residues with cyclohexane-1,2-dione and 26 histidine residues with ethoxyformic anhydride led to a loss of lectin activity. However, in these cases, modification was not protected by the abovementioned inhibitory sugar, suggesting the absence of these groups at the sugar-binding site. In all the cases, immunodiffusion studies with modified lectin showed no gross structural changes which could disrupt antigenic sites of the lectin.

Determination of Glutamate and GABA Released by Mouse Embryonic Stem Cells Using HILIC-ESI-MS/MS

2020 ◽  
Vol 10 (2) ◽  
pp. 122-129
Author(s):  
Haoyu Lv ◽  
Yabin Tang ◽  
Fan Sun ◽  
Shimin An ◽  
Xinjie Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Reversed Phase ◽  
Water Soluble ◽  
Substitution Matrix ◽  
Neural Cells ◽  
Esi Ms

Background:In recent years, more and more researches have shown that neurotransmitters can also be synthesized and released by peripheral non-neural cells. However, specificity and high sensitivity detection means were required for confirming ESCs autocrine glutamate and γ - aminobutyric acid (GABA). Glutamate and GABA are water-soluble and polar compounds which cannot be retained on a reversed phase C18 column, and their contents are often at a trace level. On the other hand, the biological matrix such as cell culture fluid contains a large number of amino acids, vitamins, carbohydrates, inorganic ions and other substances. Therefore, the main problem is the selection of the chromatographic column to avoid matrix interference.Objective:To establish a rapid and reliable method for the simultaneous determination of glutamate and GABA released from embryonic stem cells based on analytical chemistry.Methods:Glutamate and GABA released from mouse embryonic stem cells were determined on the basis of hydrophilic interaction chromatography coupled with electrospray ionization tandem Mass Spectrometry (HILIC- ESI- MS/MS), using isotope internal standards and substitution matrix method.Results:Undifferentiated embryonic stem cells autocrine glutamate and GABA and will reach releasing- reuptacking dynamic equilibriums at different time points. In contrast, neither glutamate nor GABA releasing could be detected from the MEFs, indicating the specificity release of the mESCs in the applied analytic method.Conclusion:A novel, simple, sensitive, selective and quantitative method was developed for determination of the glutamate and GABA from mouse embryonic stem cells.

Chemical modification of simian virus 40 DNA by reaction with a water-soluble carbodiimide.

1976 ◽  
Vol 18 (1) ◽  
pp. 205-210 ◽  
Author(s):  
J Lebowitz ◽  
C G Garon ◽  
M C Chen ◽  
N P Salzman
Keyword(s):  
Chemical Modification ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Water Soluble

scholarly journals Determination of Vitamin B3 Vitamer (Nicotinamide) and Vitamin B6 Vitamers in Human Hair Using LC-MS/MS

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4487
Author(s):  
Sundus M. Sallabi ◽  
Aishah Alhmoudi ◽  
Manal Alshekaili ◽  
Iltaf Shah
Keyword(s):  
Human Hair ◽  
Vitamin B6 ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Reversed Phase ◽  
Water Soluble ◽  
Vitamin B3 ◽  
Hair Samples ◽  
Pyridoxal Hydrochloride ◽  
Vitamin B3 Deficiency

Water-soluble B vitamins participate in numerous crucial metabolic reactions and are critical for maintaining our health. Vitamin B deficiencies cause many different types of diseases, such as dementia, anaemia, cardiovascular disease, neural tube defects, Crohn’s disease, celiac disease, and HIV. Vitamin B3 deficiency is linked to pellagra and cancer, while niacin (or nicotinic acid) lowers low-density lipoprotein (LDL) and triglycerides in the blood and increases high-density lipoprotein (HDL). A highly sensitive and robust liquid chromatography–tandem mass spectroscopy (LC/MS-MS) method was developed to detect and quantify a vitamin B3 vitamer (nicotinamide) and vitamin B6 vitamers (pyridoxial 5′-phosphate (PLP), pyridoxal hydrochloride (PL), pyridoxamine dihydrochloride (PM), pridoxamine-5′-phosphate (PMP), and pyridoxine hydrochloride (PN)) in human hair samples of the UAE population. Forty students’ volunteers took part in the study and donated their hair samples. The analytes were extracted and then separated using a reversed-phase Poroshell EC-C18 column, eluted using two mobile phases, and quantified using LC/MS-MS system. The method was validated in human hair using parameters such as linearity, intra- and inter-day accuracy, and precision and recovery. The method was then used to detect vitamin B3 and B6 vitamers in the human hair samples. Of all the vitamin B3 and B6 vitamers tested, only nicotinamide was detected and quantified in human hair. Of the 40 samples analysed, 12 were in the range 100–200 pg/mg, 15 in the range 200–500 pg/mg, 9 in the range of 500–4000 pg/mg. The LC/MS-MS method is effective, sensitive, and robust for the detection of vitamin B3 and its vitamer nicotinamide in human hair samples. This developed hair test can be used in clinical examination to complement blood and urine tests for the long-term deficiency, detection, and quantification of nicotinamide.

scholarly journals Capsid assembly in a family of animal viruses primes an autoproteolytic maturation that depends on a single aspartic acid residue.

1994 ◽  
Vol 269 (18) ◽  
pp. 13680-13684
Author(s):  
A. Zlotnick ◽  
V.S. Reddy ◽  
R. Dasgupta ◽  
A. Schneemann ◽  
W.J. Ray ◽  
...  
Keyword(s):  
Aspartic Acid ◽  
Capsid Assembly ◽  
Aspartic Acid Residue

scholarly journals Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase

1994 ◽  
Vol 301 (2) ◽  
pp. 577-583 ◽  
Author(s):  
K Oda ◽  
J Cheng ◽  
T Saku ◽  
N Takami ◽  
M Sohda ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Endoplasmic Reticulum ◽  
Aspartic Acid ◽  
Chimeric Protein ◽  
Attachment Site ◽  
In Vitro Translation ◽  
Placental Alkaline Phosphatase ◽  
Wild Type ◽  
Aspartic Acid Residue

Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.

Stability of oxidized Escherichia coli thioredoxin and its dependence on protonation of the aspartic acid residue in the 26 position

Biochemistry ◽  
1993 ◽  
Vol 32 (29) ◽  
pp. 7526-7530 ◽  
Author(s):  
John E. Ladbury ◽  
Richard Wynn ◽  
Homme W. Hellinga ◽  
Julian M. Sturtevant
Keyword(s):  
Escherichia Coli ◽  
Aspartic Acid ◽  
Aspartic Acid Residue

scholarly journals Formation of complement subcomponent C1q-immunoglobulin G complex. Thermodynamic and chemical-modification studies

1982 ◽  
Vol 205 (2) ◽  
pp. 361-372 ◽  
Author(s):  
E J Emanuel ◽  
A D Brampton ◽  
D R Burton ◽  
R A Dwek
Keyword(s):  
Immunoglobulin G ◽  
Water Soluble ◽  
Small Peptide ◽  
Experimental Conditions ◽  
Salt Ions ◽  
Salt Range ◽  
Lysine Residues ◽  
Arginine Residues ◽  
Equilibrium Process ◽  
Glycine Ethyl Ester

The interaction between the complement subcomponent C1q and immunoglobulin G was investigated under a variety of experimental conditions. Formation of the subcomponent C1q-immunoglobulin G complex was shown to be an equilibrium process. Thermodynamic studies of the effect of varying the ionic strength indicate that over the salt range 0.15-0.225 M-NaCl the binding of subcomponent C1q to immunoglobulin aggregates releases 9-12 salt ions (Na+ and/or Cl-), illustrating the importance of ionic interactions for the formation of the complex. The effects of small peptide and organic ion inhibitors support this conclusion. Chemical modifications of carboxylate residues on immunoglobulin G by glycine ethyl ester/water-soluble carbodi-imide (up to 12 residues modified per whole molecule of immunoglobulin G) and of lysine residues by acetic anhydride (3 residues per whole molecule of immunoglobulin G) or methyl acetimidate (19 residues per whole molecule of immunoglobulin G) lowered the binding affinity of immunoglobulin for subcomponent C1q. Modification of arginine residues by cyclohexane-1,2-dione-1,2 (14 residues per whole molecule of immunoglobulin G) and of tryptophan by hydroxynitrobenzyl bromide (2 residues per whole molecule of immunoglobulin G), however, had little or no effect. The results are consistent with the proposal that the subcomponent-C1q-binding site on immunoglobulin G is to be found on the last two beta-strands of the Cv2 domain [Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek (1980) Nature (London) 288, 338-344].

Increased Urinary Excretion of Iohexol after Enteral Administration in Patients with Ileal Crohn's Disease

1993 ◽  
Vol 34 (3) ◽  
pp. 237-241 ◽  
Author(s):  
L. Halme ◽  
J. Edgren ◽  
K. von Smitten ◽  
H. Linden
Keyword(s):  
Crohn’S Disease ◽  
Small Bowel ◽  
Crohn's Disease ◽  
Urinary Excretion ◽  
High Performance ◽  
Cell Damage ◽  
Oral Intake ◽  
Reversed Phase ◽  
Water Soluble ◽  
Mucosal Cell

Iohexol is a water-soluble contrast medium that is partly absorbed/permeated through mucosa of the small bowel and excreted unchanged in the urine. Iohexol was administered orally to 12 patients with Crohn's disease of the ileum and to 10 healthy controls to measure its excretion in the urine. The location and activity of Crohn's disease were determined by barium double-contrast radiography in all patients and by ileoscopy and biopsy in 9 patients. Iohexol concentrations in serum and 24-hour urine were measured using reversed phase high-performance liquid chromatography. Urinary excretion of iohexol was significantly greater in patients with active Crohn's disease than in controls. We suggest this method as a new way of measuring Crohn's disease activity and mucosal damage in the small bowel. Bowel inflammation and mucosal cell damage are strongly indicated if the iohexol excreted in the urine is over 1% of the oral intake.

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