scholarly journals Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent

1978 ◽  
Vol 173 (3) ◽  
pp. 723-737 ◽  
Author(s):  
J Carlsson ◽  
H Drevin ◽  
R Axén
Keyword(s):  
Gamma Globulin ◽  
De Novo ◽  
Ester Group ◽  
Alpha Amylase ◽  
Amino Groups ◽  
Protein Conjugates ◽  
Disulphide Bonds ◽  
Concomitant Reduction ◽  
Antibody Conjugate ◽  
Aliphatic Thiols

A heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate, was synthesized. Its N-hydroxysuccinimide ester group reacts with amino groups and the 2-pyridyl disulphide structure reacts with aliphatic thiols. A new thiolation procedure for proteins is based on this reagent. The procedure involves two steps. First, 2-pyridyl disulphide structures are introduced into the protein by the reaction of some of its amino groups with the N-hydroxysuccinimide ester sie of the reagent. The protein-bound 2-pyridyl disulphide structures are then reduced with dithiothreitol. This reaction can be carried out without concomitant reduction of native disulphide bonds. The technique has been used for the introduction of thiol groups de novo into ribonuclease, gamma-globulin, alpha-amylase and horseradish peroxidase. N-Succinimidyl 3-(2-pyridyldithio)propionate can also be used for the preparation of protein-protein conjugates. This application is based on the fact that protein-2-pyridyl disulphide derivatives (formed from the reaction of non-thiol proteins with the reagent) react with thiol-containing proteins (with native thiols or thiolated by, for example, the method described above) via thiol-disulphide exchange to form disulphide-linked protein-protein conjugates. This conjugation technique has been used for the preparation of an alpha-amylase-urease, a ribonuclease-albumin and a peroxidase-rabbit anti-(human transferrin) antibody conjugate. The disulphide bridges between the protein molecules can easily be split by reduction or by thiol-disulphide exchange. Thus conjugation is reversible. This has been demonstrated by scission of the ribonuclease-albumin and the alpha-amylase-urease conjugate into their components with dithiothreitol. N-Succinimidyl 3-(2-pyridyldithio)propionate has been prepared in crystalline form, in which state (if protected against humidity) it is stable on storage at room temperature (23 degrees C).

Triiodothyronine and 3′,5′-cyclic AMP secretion by cultured human thyroid cells in response to thyrotropin and thyroid-stimulating immunoglobulin

1988 ◽  
Vol 119 (4) ◽  
pp. 493-500 ◽  
Author(s):  
Z. Kraiem ◽  
O. Sadeh ◽  
E. Sobel
Keyword(s):  
De Novo ◽  
Testicular Tissue ◽  
Culture Conditions ◽  
Human Thyroid ◽  
Intracellular Camp ◽  
Camp Accumulation ◽  
High Concentration ◽  
Thyroid Cells ◽  
Concomitant Reduction ◽  
Dual Action

Abstract. We have established a relatively simple and sensitive system for measuring T3 as well as cAMP secretion using cryopreserved human thyroid cells in culture. We defined optimal culture conditions and characterized the system. T3 secretion from human thyrocytes (only 1 × 105 cells/well) could be stimulated in a time- and dose-dependent fashion by both TSH (doses as low as 10 mU/l) and thyroid-stimulating immunoglobulin to levels 5- to 10-fold above baseline. The response to the thyroid stimulating agents was preserved for at least 3 weeks. Experiments with inhibitors of iodothyronine synthesis (propylthiouracil and methimazole) indicated that the bulk of the TSH-stimulated T3 secretion measured apparently derives from de novo iodothyronine biosynthesis rather than preformed T3. We utilized the system to investigate some aspects in the regulation of human thyrocyte T3 and cAMP secretion. Maximum stimulation of the thyroid hormone was achieved at TSH doses capable of evoking a further rise in levels of cAMP. A rise in cAMP accumulation was observed as early as 15 min following exposure to TSH, whereas it took 1–4 days to detect a significant increase in T3 secretion. Within 6 h of incubation, the bulk of TSH-stimulated intracellular cAMP was found released into the medium. l-methyl-3-isobutylxanthine (MIX) caused a dose-related decrease (beyond 0.1 mmol/l MIX) in TSH-stimulated T3 secretion which contrasted with a concomitant expected increase in cAMP accumulation. Hence, as also observed in adrenal and testicular tissue, xanthines at high concentration seem to exhibit a dual action: potentiation of cAMP accumulation by inhibiting phosphodiesterase activity and a concomitant reduction of hormone formation.

scholarly journals DELAYED HYPERSENSITIVITY TO HAPTEN-PROTEIN CONJUGATES

1962 ◽  
Vol 115 (5) ◽  
pp. 1037-1051 ◽  
Author(s):  
P. G. H. Cell ◽  
Arthur M. Silverstein
Keyword(s):  
Guinea Pig ◽  
Antigenic Determinant ◽  
Gamma Globulin ◽  
Delayed Hypersensitivity ◽  
Antigenic Determinants ◽  
Carrier Protein ◽  
Protein Conjugates ◽  
Circulating Antibodies ◽  
Human Gamma Globulin ◽  
Delayed Hypersensitivity Reaction

Further data have been presented showing that the specificity of the delayed hypersensitivity reaction in the guinea pig to hapten-protein conjugates involves to a considerable degree a contribution by the protein carrier. The carrier contribution is such that sensitization to guinea pig albumin-m-azobenzenesulfonate, for example, does not result in cross-reaction with conjugates of the same hapten with unrelated proteins such as ovalbumin or human gamma globulin, nor were cross-reactions observed between conjugates prepared with the same hapten, coupled to the same protein, but by two different chemical routes, such that the point of attachment of the hapten to the protein differed. It thus appears that in this system both hapten and carrier protein are necessary, but that neither alone is in general sufficient to stimulate the delayed sensitive cell. Desensitization experiments with cross-reacting hapten-protein conjugates have suggested the presence of a multiplicity of antigenic determinants participating in the elicitation of the delayed lesion, and of a concomitant development of a heterogeneity of specificities in the population of delayed sensitive cells in the sensitized animal. The data are discussed in terms of the apparent requirement of the delayed sensitivity mechanism for a larger functional antigenic determinant than that required for interaction with circulating antibodies. Some possible explanations for this difference, and some of its consequences, are discussed.

scholarly journals STUDIES OF HYPERSENSITIVITY TO LOW MOLECULAR WEIGHT SUBSTANCES

1953 ◽  
Vol 98 (6) ◽  
pp. 533-549 ◽  
Author(s):  
Herman N. Eisen ◽  
Sidney Belman
Keyword(s):  
Skin Surface ◽  
Chromatographic Technique ◽  
Human Beings ◽  
Amino Groups ◽  
Sh Groups ◽  
Protein Conjugates ◽  
Ph Values ◽  
Skin Protein

2,4-dinitrophenylsulfenyl chloride (DSCl) and 2,4-dinitrophenylthiocyanate (DSCN) elicited allergic reactions of the delayed type when applied to the skin of guinea pigs and of human beings who had been sensitized by prior exposure to 2,4-dinitrofluorobenzene (DF). DSCl and DSCN, together with 2,4-dinitrobenzene sulfonate (DSO3), constitute a clearly defined group of allergenic dinitrophenyl compounds in that they all combined with skin protein in vivo through reaction with cysteine or cystine. In vitro, these compounds combine with free SH groups, and with —S—S— groups of hair and epidermis, but not with —S—S— groups of oxidized glutathione or of bovine gamma globulins. DSO3, DSCl, and DSCN did not react with amino groups in vivo, but did react with protein amino groups in vitro at pH values of about 10. Another group of dinitrophenyl compounds (DF, DCl, and DBr) previously had been shown to combine with lysine ϵ-NH2 groups of epidermal proteins. In the present work it was found that these compounds do not react with the disulfide groups of these proteins, either in vivo or in vitro. Moreover, they did not seem to react with SH groups of viable skin, although they are highly reactive with sulfhydryl in vitro. This apparent discrepancy between reactivity with SH groups in vitro and in vivo may be due to the fact that the chromatographic technique employed was relatively insensitive for the sulfhydryl derivative. When a compound of either group was applied to the skin surface, dinitrophenyl-amino acids were recovered from the epidermis but not from the dermis. The results are discussed from the viewpoint of the epidermal localization of dinitrophenyl-protein conjugates.

scholarly journals Characterization of key enzymes involved in triacylglycerol biosynthesis in mycobacteria

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Agostina Crotta Asis ◽  
Franco Savoretti ◽  
Matías Cabruja ◽  
Hugo Gramajo ◽  
Gabriela Gago
Keyword(s):  
Phosphatidic Acid ◽  
Growth Phase ◽  
De Novo ◽  
Cell Envelope ◽  
Exponential Growth Phase ◽  
Genes Encoding ◽  
Concomitant Reduction ◽  
Tag Biosynthesis ◽  
Odd Chain Fatty Acids

AbstractPhosphatidic acid phosphatase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. PAP activity has a key role in the regulation of PA flux towards TAG or glycerophospholipid synthesis. In this work we have characterized two Mycobacterium smegmatis genes encoding for functional PAP proteins. Disruption of both genes provoked a sharp reduction in de novo TAG biosynthesis in early growth phase cultures under stress conditions. In vivo labeling experiments demonstrated that TAG biosynthesis was restored in the ∆PAP mutant when bacteria reached exponential growth phase, with a concomitant reduction of phospholipid synthesis. In addition, comparative lipidomic analysis showed that the ∆PAP strain had increased levels of odd chain fatty acids esterified into TAGs, suggesting that the absence of PAP activity triggered other rearrangements of lipid metabolism, like phospholipid recycling, in order to maintain the wild type levels of TAG. Finally, the lipid changes observed in the ∆PAP mutant led to defective biofilm formation. Understanding the interaction between TAG synthesis and the lipid composition of mycobacterial cell envelope is a key step to better understand how lipid homeostasis is regulated during Mycobacterium tuberculosis infection.

scholarly journals The disulphide bonds of human and rabbit γ-globulins

1965 ◽  
Vol 97 (2) ◽  
pp. 569-572 ◽  
Author(s):  
R Cecil ◽  
GT Stevenson
Keyword(s):  
Heavy Metal ◽  
Gamma Globulin ◽  
Sh Groups ◽  
Predominant Species ◽  
Disulphide Bonds ◽  
Γ Globulins ◽  
Presence And Absence ◽  
Metal Reagents

1. The reaction of the disulphide bonds of the predominant species of human and rabbit gamma-globulins (the 7s gamma-globulins) with sulphite was studied in the presence and absence of denaturing agents and heavy-metal reagents. 2. The total number of bonds reacting/mol. of mol.wt. 160000 was approx. 18 for human and 20 for rabbit gamma-globulin. 3. Six S.S bonds/mol. of human and 6.5 S.S bonds/mol. of rabbit gamma-globulin reacted with sulphite alone at pH6. These appeared to include all the interchain S.S bonds. 4. The number of free SH groups was less than 0.2/mol. of human and less than 0.3/mol. of rabbit gamma-globulin.

scholarly journals Treatment with Succinic Anhydride Improves the Immunogenicity ofShigella flexneri Type 2a O-Specific Polysaccharide–Protein Conjugates in Mice

1999 ◽  
Vol 67 (10) ◽  
pp. 5526-5529 ◽  
Author(s):  
Danka Pavliakova ◽  
Chiayung Chu ◽  
Slavomir Bystricky ◽  
Nathaniel W. Tolson ◽  
Joseph Shiloach ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Succinic Anhydride ◽  
Critical Level ◽  
Shigella Flexneri ◽  
Carrier Protein ◽  
Carrier Proteins ◽  
Amino Groups ◽  
Protein Conjugates ◽  
Native Proteins ◽  
Specific Polysaccharide

ABSTRACT Seroepidemiological data and a clinical trial with a Shigella sonnei O-specific polysaccharide (O-SP)–Pseudomonas aeruginosa recombinant exoprotein A (rEPA) conjugate provide evidence that a critical level of immunoglobulin G (IgG) lipopolysaccharide (LPS) antibodies in serum confers protection against shigellosis. We evaluated the immunogenicity of conjugates whose carrier proteins and O-SPs were treated with succinic anhydride (SA), which reacts with amino groups at neutral pH to form amide-linked carboxyls (succinylation). Conjugates were synthesized with either of two genetically inactivated medically useful toxins, the diphtheria protein CRM9 or rEPA, bound to the O-SP ofShigella flexneri type 2a. Conjugates composed of the succinylated protein, succinylated O-SP, or both succinylated components were administered to mice by a clinically relevant scheme, and their levels of serum IgG anti-LPS and anti-proteins were assayed 7 days after the second and third injections. CRM9 served as a more immunogenic carrier than rEPA. Conjugates composed of succinylated components were more immunogenic than the conjugates composed of the native components. SA treatment of both the carrier protein and the O-SP did not confer an advantage over the succinylated protein alone. Conjugates prepared with native proteins, in general, elicited slightly higher levels of IgG protein antibodies than conjugates composed of the SA-treated proteins.

scholarly journals Masking of protein antigen by modification of amino groups with carbobenzoxychloride (benzyl chloroformate) and demasking by treatment with nonspecific protease.

1978 ◽  
Vol 26 (11) ◽  
pp. 914-920 ◽  
Author(s):  
H Takamiya ◽  
W Bodemer ◽  
A Vogt
Keyword(s):  
Salmonella Typhimurium ◽  
Gamma Globulin ◽  
Bovine Insulin ◽  
Protein Antigen ◽  
Antigenic Determinants ◽  
Amino Groups ◽  
Protein Antigens ◽  
Polysaccharide Antigens ◽  
Cryostat Sections

Cryostat sections of various substrates were treated with carbobenzoxychloride in acetone to modify antigens. By applying specific fluorescent antibodies, it could be shown that the antigenic determinants of rabbit gamma-globulin and bovine insulin were totally masked. The antigenicity of ACTH was markedly reduced, whereas the polysaccharide antigens of Salmonella typhimurium were only partially masked. After masking, antigenicity could be restored by treatment with nonspecific protease. The reversible protection of amino groups by carbobenzoxychloride may be a way to preserve protein antigens during embedding in plastics, as such materials also bind to amino groups, blocking the antigenicity of proteins.

scholarly journals N–H⋯O hydrogen bonding to the alkoxy oxygen of a carboxylic ester group: crystal structures of methyl 2,6-diaminobenzoate and its derivatives

CrystEngComm ◽  
2020 ◽  
Vol 22 (21) ◽  
pp. 3701-3712
Author(s):  
Songjie Yang ◽  
A. Christopher Garner ◽  
John D. Wallis
Keyword(s):  
Hydrogen Bonding ◽  
Hydrogen Bonds ◽  
Oxygen Atom ◽  
Crystal Structures ◽  
Ester Group ◽  
Amino Groups ◽  
Carboxylic Ester

Molecules with hydrogen bonds from amino groups to both oxygens of a carboxylic ester are described, and other examples of hydrogen bonding to an ester's alkoxy oxygen atom are highlighted.

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