Zymoblot, a new microtechnique used to detect enzyme activity in spiroplasmas and bacteria

1993 ◽  
Vol 39 (5) ◽  
pp. 543-547 ◽  
Author(s):  
Elsayed E. Wagih ◽  
Jacqueline Fletcher
Keyword(s):  
Enzyme Activity ◽  
Pseudomonas Syringae ◽  
Spiroplasma Citri ◽  
Phosphogluconate Dehydrogenase ◽  
Enzyme Substrate ◽  
Spiroplasma Kunkelii ◽  
Coomassie Blue ◽  
Colored Product ◽  
K 12 ◽  
Spiroplasma Melliferum

A new microtechnique that detects enzyme activity in prokaryotes is described. The technique, designated zymoblot, is based on the immobilization of negatively charged enzymes from an alkaline extract spotted onto a nitrocellulose membrane. The presence of specific enzyme activity in the extract is selectively assayed with a reaction mixture containing the corresponding substrate. The enzyme–substrate reaction produces an insoluble colored product that accumulates at the site. The zymoblot technique offers the advantages of simplicity, sensitivity, reproducibility, speed, and the use of microquantities of reactants. The protein in the spot can be visualized by a technique termed "proteinblot," in which the protein is stained with Coomassie blue. Esterase and tyrosinase were detected by the zymoblot method in six spiroplasmas including four strains of Spiroplasma citri, one of Spiroplasma kunkelii, and one of Spiroplasma melliferum, and two bacteria, Pseudomonas syringae pv. syringae B301D and Escherichia coli K-12. Acid phosphatase, alkaline phosphatase, alanine dehydrogenase, peroxidase, and 6-phosphogluconate dehydrogenase were not detected in any of the spiroplasmas, but were each detected in one or both of the walled bacteria.Key words: spiroplasma, enzyme, protein, zymoblot.

scholarly journals Engineering enzyme catalysis: an inverse approach

2019 ◽  
Vol 39 (2) ◽  
Author(s):  
Clare F. Megarity
Keyword(s):  
Enzyme Activity ◽  
Enzyme Catalysis ◽  
Recent Article ◽  
Inverse Approach ◽  
Enzyme Substrate ◽  
Reaction Solution ◽  
Fundamental Research ◽  
Covalently Linked ◽  
Scaffold Molecule ◽  
Polyamine Oxidases

Abstract Enzymes’ inherent chirality confers their exquisite enantiomeric specificity and makes their use as green alternatives to chiral metal complexes or chiral organocatalysts invaluable to the fine chemical industry. The most prevalent way to alter enzyme activity in terms of regioselectivity and stereoselectivity for both industry and fundamental research is to engineer the enzyme. In a recent article by Keinänen et al., published in Bioscience Reports 2018, ‘Controlling the regioselectivity and stereoselectivity of FAD-dependent polyamine oxidases with the use of amine-attached guide molecules as conformational modulators’, an inverse approach was presented that focuses on the manipulation of the enzyme substrate rather than the enzyme. This approach not only uncovered dormant enantioselectivity in related enzymes but allowed for its control by the use of guide molecules simply added to the reaction solution or covalently linked to an achiral scaffold molecule.

scholarly journals Multiple forms of acetylcholinesterase from human erythrocytes

1973 ◽  
Vol 133 (3) ◽  
pp. 521-527 ◽  
Author(s):  
David L. Wright ◽  
David T. Plummer
Keyword(s):  
Enzyme Activity ◽  
Electrophoretic Pattern ◽  
Human Erythrocytes ◽  
Serum Cholinesterase ◽  
Main Peak ◽  
Multiple Forms ◽  
Molecular Weights ◽  
Enzyme Substrate ◽  
Triton X ◽  
Naphthyl Acetate

1. Acetylcholinesterase from human erythrocytes was solubilized with Triton X-100 in strong salt solution and partially purified by (NH4)2SO4 fractionation. This preparation showed three main bands of enzyme activity after electrophoresis on polyacrylamide gel and incubation with either α-naphthyl acetate or acetylthiocholine as enzyme substrate. Two of the multiple forms were completely inhibited by 10μm-eserine and one only partially. Treatment with neuraminidase had no effect on the electrophoretic pattern; therefore sialic acid does not appear to determine or affect the ratios of the acetylcholinesterase multiple forms, unlike those of the serum cholinesterase. 2. Chromatography of the preparation on Sephadex G-200 revealed one major peak of enzyme activity and a suggestion of two minor zones of mol.wt. 546000, 184000 and 93000 (i.e. in the proportion 6:2:1). The main peak was almost completely separated from the Triton X-100 and the overall purification was about 600-fold. Further attempts to purify the enzyme by absorption on calcium phosphate gels were unsuccessful. 3. Electrophoresis of the enzyme preparation on a polyacrylamide gradient for 24h revealed three main bands that corresponded to the three values for molecular weights obtained by column chromatography. After 70h of electrophoresis a further three zones of activity developed making six molecular entities, the molecular weights of which were simple multiples of a monomer, thus resembling the cholinesterase found in serum.

scholarly journals The effect of pH on the kinetics of arylsulphatases A and B

1983 ◽  
Vol 213 (3) ◽  
pp. 603-607 ◽  
Author(s):  
C O'Fagain ◽  
B M Butler ◽  
T J Mantle
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Substrate Binding ◽  
Acid Base ◽  
Effect Of Ph ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
General Acid ◽  
Kinetics Of

The effect of pH on the kinetics of rat liver arylsulphatases A and B is very similar and shows that two groups with pK values of 4.4-4.5 and 5.7-5.8 are important for enzyme activity. Substrate binding has no effect on the group with a pK of 4.4-4.5; however, the pK of the second group is shifted to 7.1-7.5 in the enzyme-substrate complex. An analysis of the effect of pH on the Ki for sulphate inhibition suggests that HSO4-is the true product. A model is proposed that involves the two ionizing groups identified in the present study in a concerted general acid-base-catalysed mechanism.

scholarly journals Extracellular endosulfatase Sulf-2 harbours a chondroitin/dermatan sulfate chain that modulates its enzyme activity

2021 ◽  
Author(s):  
Rana El Masri ◽  
Amal Seffouh ◽  
Caroline Roelants ◽  
Ilham Seffouh ◽  
Evelyne Gout ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Dermatan Sulfate ◽  
Post Translational Modification ◽  
New Paradigm ◽  
Extracellular Milieu ◽  
Enzyme Substrate ◽  
Enzyme Substrate Recognition ◽  
Substrate Binding Domain

AbstractSulfs represent a class of unconventional sulfatases, which differ from all other members of the sulfatase family by their structures, catalytic features and biological functions. Through their specific endosulfatase activity in extracellular milieu, Sulfs provide an original post-synthetic regulatory mechanism for heparan sulfate complex polysaccharides and have been involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their in vivo functions. Here we show that human Sulf-2 (HSulf-2) features a unique polysaccharide post-translational modification. We identified a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate binding domain. We found that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity in vitro and in vivo using a mouse model of tumorigenesis and metastasis. In addition, we showed that mammalian hyaluronidase acted as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a unique proteoglycan enzyme and its newly-identified GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute in clarifying the conflicting data on the activities of the Sulfs and introduce a new paradigm into the study of these enzymes.

Essential Arginine Residues in Lactate Dehydrogenase from Germinating Soybean

1994 ◽  
Vol 59 (2) ◽  
pp. 467-472 ◽  
Author(s):  
Jana Barthová ◽  
Irena Hulová ◽  
Miroslava Birčáková
Keyword(s):  
Enzyme Activity ◽  
Glycine Max ◽  
Lactate Dehydrogenase ◽  
Chemical Modification ◽  
Active Site ◽  
Enzyme Modification ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Arginine Residues ◽  
Blue Sepharose

The lactate dehydrogenase was isolated from soybean (Glycine max. L.) by a procedure that employed biospecific chromatography on a column of Blue-Sepharose CL-6B. The participation of the guanidine group of arginine residues in the mechanism of enzyme action was determined through kinetic and chemical modification studies. The dependence of enzyme activity on pH was followed in the alkaline region (pH 8.6 - 12.8). The pK values found were 12.4 for the enzyme substrate complex and 11.1 for the free enzyme. The enzyme was inactivated by phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and p-hydroxyphenylglyoxal reagents used in modification experiments. Kinetic analysis of the modification indicated that one arginine residue is modified when inactivation occurs. No effect was observed on the rate of inactivation upon addition of coenzyme. The extent of enzyme modification by p-hydroxyphenylglyoxal was determined. It appears there are at least two arginine residues in the active site of the enzyme.

THE INFLUENCE OF ADRENALECTOMY UPON THE ACTIVITY OF THE HEXOSEMONOPHOSPHATE SHUNT IN THE LIVERS AND MAMMARY GLANDS OF LACTATING RATS

1960 ◽  
Vol 38 (1) ◽  
pp. 1265-1273 ◽  
Author(s):  
J. S. Willmer
Keyword(s):  
Enzyme Activity ◽  
Considerable Increase ◽  
Mammary Glands ◽  
Mammary Tissue ◽  
Hepatic Tissue ◽  
Phosphogluconate Dehydrogenase ◽  
Growth Changes ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Adrenalectomized Rats ◽  
Hepatic Level

The development of the hexosemonophosphate shunt in mammary tissue and liver of lactating rats has been studied. A sixfold increase in mammary glucose-6-phosphate dehydrogenase levels between parturition and weaning was accompanied by a considerable increase in 6-phosphogluconate dehydrogenase activity. The hepatic level of the former enzyme was also elevated 11-fold during this period. Adrenalectomy at parturition, or on the 3rd, 6th, 9th, or 14th days of lactation, depressed the activity of this pathway in mammary gland, a lowered level being observed in all cases after operation. A slight increase in enzyme activity was found in hepatic tissue in the immediate postoperative period; this was succeeded by a decrease.These results are discussed in relation to the growth changes observed in groups of unoperated and adrenalectomized rats.

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