Zinc Enzymes in Crassostrea virginica

1970 ◽  
Vol 27 (1) ◽  
pp. 59-69 ◽  
Author(s):  
Douglas A. Wolfe
Keyword(s):  
Alkaline Phosphatase ◽  
Metal Binding ◽  
Zinc Enzymes ◽  
Tissue Extracts ◽  
Metabolic Functions ◽  
Binding Agents ◽  
High Molecular Weight Proteins ◽  
Cellular Components ◽  
Inhibition Studies

Nearly all the zinc in oysters is bound, either to soluble high-molecular weight proteins or to structural cellular components such as cell membranes. Oyster alkaline phosphatase is a zinc metalloenzyme, as indicated by in vitro inhibition studies with various metal-binding agents. Dialysis of soluble tissue extracts at pH 7–9 removes up to 96% of the total zinc without effect on alkaline phosphatase. If alkaline phosphatase is considered representative of the metabolic functions of zinc in oysters, most of the zinc accumulated by oysters must be superfluous to the animal's requirements.

INHIBITION OF IN VITRO ACETOACETATE FORMATION: AN ACID PHOSPHOHYDROLASE

1965 ◽  
Vol 43 (6) ◽  
pp. 731-745 ◽  
Author(s):  
Ian C. Caldwell ◽  
George I. Drummond
Keyword(s):  
Metal Binding ◽  
Protein Concentration ◽  
Coenzyme A ◽  
Chicken Liver ◽  
Phosphate Esters ◽  
Acetyl Phosphate ◽  
Tissue Extracts ◽  
Binding Agents ◽  
Enzymatic Inactivation

The rate of formation of acetoacetate in vitro by tissue extracts from acetyl phosphate and coenzyme A in the presence of phosphate transacetylase and β-ketothiolase is proportional to protein concentration with extracts of most tissues. However, studies with chicken liver extracts are complicated by the presence in these extracts of a factor which interferes with acetoacetate formation. This factor has been identified as an acid phosphohydrolase which hydrolyzes the phosphate monoester bond of coenzyme A, forming the inactive 3′-dephosphocoenzyme A. This enzyme has been purified 300-fold from chicken liver extracts, and some of its properties have been examined. The rate of inactivation of coenzyme A by the enzyme is neither enhanced by divalent cation nor inhibited by metal-binding agents. Enzymatic inactivation of coenzyme A is optimal at pH 3.6, with half-maximal rates observed at pH 5.5 and below pH 2.5. The most highly purified enzyme fraction exhibited phosphohydrolase activity against a wide variety of phosphate esters. Some evidence was obtained to suggest that the coenzyme A phosphohydrolase could be separated from nonspecific acid phosphohydrolase.

Dithiol metal binding agents — Efficacies and properties in vivo and in vitro

1983 ◽  
Vol 18 ◽  
pp. 161
Author(s):  
R. Maiorino ◽  
C.A. Hsu ◽  
E.R. Stine ◽  
T.D. Hoover ◽  
H.V. Aposhian
Keyword(s):  
Metal Binding ◽  
Binding Agents

scholarly journals Influence of reagents reacting with metal, thiol and amino sites of catalytic activity and l-phenylalanine inhibition of rat intestinal alkaline phosphatase

1967 ◽  
Vol 105 (3) ◽  
pp. 1163-1170 ◽  
Author(s):  
William H. Fishman ◽  
Nimai K. Ghosh
Keyword(s):  
Alkaline Phosphatase ◽  
Metal Binding ◽  
Amino Group ◽  
Intestinal Alkaline Phosphatase ◽  
Ample Evidence ◽  
Metal Ligands ◽  
Binding Agents ◽  
Optimum Ph ◽  
Starch Gel

1. Studies on the inactivation of rat intestinal alkaline phosphatase by several metal-binding agents, namely EDTA, 8-hydroxyquinoline, pyridine-2,6-dicarboxylic acid, αα′-bipyridyl, o-phenanthroline and sodium cyanide, indicated the functional role of a metal, probably zinc, in the catalysis. The metal ligands lowered stereospecific uncompetitive inhibition of the enzyme by l-phenylalanine by an extent that paralleled the decline in enzyme activity. 2. The thiol reagents p-hydroxymercuribenzoate, iodoacetamide and iodine inactivated rat intestinal phosphatase. The enzyme could be protected from inactivation by either cysteine or substrate. The l-phenylalanine inhibition remained unchanged only in the presence of moderately inactivating concentrations of the thiol reagents. 3. Inactivation of the enzyme by the amino-group-blocking reagent, O-methylisourea, provided ample evidence for the participation in the catalysis of the ∈-amino group of lysine. At the same time, l-phenylalanine inhibition remained unaltered even when the enzyme was strongly inactivated. This ∈-amino-group-blocked enzyme exhibited no change in migration in starch gel, in contrast with enzyme treated with acetic anhydride, formaldehyde or succinic anhydride. The Michaelis constant of the enzyme was enhanced by such modifications, but the optimum pH remained the same. 4. d-Phenylalanine acted as a competitive or ‘co-operative’ activator for intestinal alkaline phosphatase after it had been modified by acetylation.

In Situ Localization of PPAR Gamma and Uncoupling Protein in Mouse Embryo Sections Using Digoxigenin-Labeled Riboprobes

1996 ◽  
Vol 54 ◽  
pp. 32-33
Author(s):  
C. Jennermann ◽  
S. A. Kliewer ◽  
D. C. Morris
Keyword(s):  
Alkaline Phosphatase ◽  
Room Temperature ◽  
Uncoupling Protein ◽  
Ppar Gamma ◽  
Nuclear Hormone Receptor ◽  
Full Length ◽  
Northern Analysis ◽  
Peroxisome Proliferator

Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor superfamily and has been shown in vitro to regulate genes involved in lipid metabolism and adipocyte differentiation. By Northern analysis, we and other researchers have shown that expression of this receptor predominates in adipose tissue in adult mice, and appears first in whole-embryo mRNA at 13.5 days postconception. In situ hybridization was used to find out in which developing tissues PPARg is specifically expressed.Digoxigenin-labeled riboprobes were generated using the Genius™ 4 RNA Labeling Kit from Boehringer Mannheim. Full length PPAR gamma, obtained by PCR from mouse liver cDNA, was inserted into pBluescript SK and used as template for the transcription reaction. Probes of average size 200 base pairs were made by partial alkaline hydrolysis of the full length transcripts. The in situ hybridization assays were performed as described previously with some modifications. Frozen sections (10 μm thick) of day 18 mouse embryos were cut, fixed with 4% paraformaldehyde and acetylated with 0.25% acetic anhydride in 1.0M triethanolamine buffer. The sections were incubated for 2 hours at room temperature in pre-hybridization buffer, and were then hybridized with a probe concentration of 200μg per ml at 70° C, overnight in a humidified chamber. Following stringent washes in SSC buffers, the immunological detection steps were performed at room temperature. The alkaline phosphatase labeled, anti-digoxigenin antibody and detection buffers were purchased from Boehringer Mannheim. The sections were treated with a blocking buffer for one hour and incubated with antibody solution at a 1:5000 dilution for 2 hours, both at room temperature. Colored precipitate was formed by exposure to the alkaline phosphatase substrate nitrobluetetrazoliumchloride/ bromo-chloroindlylphosphate.

Electron microscopic study of the membrane glycoproteins in the rat kidney after cisplatin treatment

1989 ◽  
Vol 47 ◽  
pp. 912-913
Author(s):  
S.K. Aggarwal
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Kidney ◽  
Light And Electron Microscopy ◽  
Kidney Tissue ◽  
Membrane Glycoproteins ◽  
Electron Microscopic ◽  
Before And After ◽  
Interstrand Cross Links ◽  
Cross Links

The proposed primary mechanism of action of the anticancer drug cisplatin (Cis-DDP) is through its interaction with DNA, mostly through DNA intrastrand cross-links or DNA interstrand cross-links. DNA repair mechanisms can circumvent this arrest thus permitting replication and transcription to proceed. Various membrane transport enzymes have also been demonstrated to be effected by cisplatin. Glycoprotein alkaline phosphatase was looked at in the proximal tubule cells before and after cisplatin both in vivo and in vitro for its inactivation or its removal from the membrane using light and electron microscopy.Outbred male Swiss Webster (Crl: (WI) BR) rats weighing 150-250g were given ip injections of cisplatin (7mg/kg). Animals were killed on day 3 and day 5. Thick slices (20-50.um) of kidney tissue from treated and untreated animals were fixed in 1% buffered glutaraldehyde and 1% formaldehyde (0.05 M cacodylate buffer, pH 7.3) for 30 min at 4°C. Alkaline phosphatase activity and carbohydrates were demonstrated according to methods described earlier.

In Vitro Immunodetection of Prothymosin Alpha in Normal and Pathological Conditions

2020 ◽  
Vol 27 (29) ◽  
pp. 4840-4854 ◽  
Author(s):  
Chrysoula-Evangelia Karachaliou ◽  
Hubert Kalbacher ◽  
Wolfgang Voelter ◽  
Ourania E. Tsitsilonis ◽  
Evangelia Livaniou
Keyword(s):  
Mammalian Cells ◽  
Therapeutic Potential ◽  
Prothymosin Alpha ◽  
Disease States ◽  
Leading Role ◽  
Tissue Extracts ◽  
Biologic Response ◽  
Health And Disease ◽  
Almost All

Prothymosin alpha (ProTα) is a highly acidic polypeptide, ubiquitously expressed in almost all mammalian cells and tissues and consisting of 109 amino acids in humans. ProTα is known to act both, intracellularly, as an anti-apoptotic and proliferation mediator, and extracellularly, as a biologic response modifier mediating immune responses similar to molecules termed as “alarmins”. Antibodies and immunochemical techniques for ProTα have played a leading role in the investigation of the biological role of ProTα, several aspects of which still remain unknown and contributed to unraveling the diagnostic and therapeutic potential of the polypeptide. This review deals with the so far reported antibodies along with the related immunodetection methodology for ProTα (immunoassays as well as immunohistochemical, immunocytological, immunoblotting, and immunoprecipitation techniques) and its application to biological samples of interest (tissue extracts and sections, cells, cell lysates and cell culture supernatants, body fluids), in health and disease states. In this context, literature information is critically discussed, and some concluding remarks are presented.

Mycobacterial DNA GyrB Inhibitors: Ligand Based Pharmacophore Modelling and In Vitro Enzyme Inhibition Studies

2014 ◽  
Vol 14 (17) ◽  
pp. 1990-2005 ◽  
Author(s):  
Shalini Saxena ◽  
Janupally Renuka ◽  
Variam Jeankumar ◽  
Perumal Yogeeswari ◽  
Dharmarajan Sriram
Keyword(s):  
Enzyme Inhibition ◽  
Pharmacophore Modelling ◽  
Inhibition Studies
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