Affinity Purification and Determination of Enzymatic Activity of Recombinantly Expressed Aldehyde Dehydrogenases

Abstract A simple and rapid method is described for determining the enzymatic activity of microbial phytase. The method is based on the determination of inorganic orthophosphate released on hydrolysis of sodium phytate at pH 5.5.

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Two automated procedures for N-acetyl-beta-D-glucosaminidase determination evaluated for detection of drug-induced tubular nephrotoxicity.

Clinical Chemistry ◽

10.1093/clinchem/32.11.2052 ◽

1986 ◽

Vol 32 (11) ◽

pp. 2052-2055 ◽

Cited By ~ 8

Author(s):

M P Goren ◽

R K Wright ◽

S Osborne

Keyword(s):

Enzymatic Activity ◽

Methotrexate Therapy ◽

Tubular Damage ◽

Sample Treatment ◽

Urine Ph ◽

Drug Induced ◽

Alkaline Urine ◽

Automated Procedures ◽

Drugs And Metabolites

Abstract We automated two procedures for determination of urinary N-acetyl-beta-D-glucosaminidase (NAG; EC 3.2.1.30) concentrations and evaluated their reliability for detecting drug-induced tubular damage in children receiving cisplatin, methotrexate, or ifosfamide. Results for 174 patient specimens correlated well (r = 0.98), but NAG concentrations determined by the m-cresolsulfonphthaleinyl (MCP) procedure were about 40% lower than those obtained with p-nitrophenyl-N-acetyl-beta-D-glucosaminide substrate. Dialysis and assay of 50 specimens disclosed no evidence of activators or inhibitors of enzymatic activity. Drugs and metabolites added to urine had negligible effect on NAG determinations; however, NAG was unstable in alkaline urine (pH greater than 8) associated with methotrexate therapy. Both procedures detect tubular damage equally well and neither requires laborious sample treatment. The MCP procedure, being more sensitive and not requiring a sample blank, is better suited for rapid automated assays. Comparisons of clinical data obtained by the two procedures require standardization against human NAG.

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Determination of serum aminotransferases: activation by pyridoxal-5'-phosphate in relation to substrate concentration.

Clinical Chemistry ◽

10.1093/clinchem/25.1.55 ◽

1979 ◽

Vol 25 (1) ◽

pp. 55-59 ◽

Cited By ~ 14

Author(s):

J C Hafkenscheid ◽

C C Dijt

Keyword(s):

Enzymatic Activity ◽

Serum Sample ◽

Aspartate Aminotransferase ◽

Substrate Concentration ◽

Enzymatic Reaction ◽

Reaction Medium ◽

Aminotransferase Activity ◽

Serum Aminotransferases ◽

Stimulation Of

Abstract To investigate the activation of aspartate- and alanine aminotransferases by pyridoxal-5'-phosphate, we determined the enzymatic activity in serum in two different ways: (a) Preincubation of the serum alone or the serum with pyridoxal-5'-phosphate and starting the reaction by the addition of the serum sample or the serum sample + coenzyme, respectively. (b) Preincubation of the serum or the serum with pyridoxal-5'-phosphate in the reaction medium and starting the reaction by adding 2-oxoglutarate. There are only small differences in activities of both aminotransferases determined according to these two different methods. The stimulation by pyridoxal-5'-phosphate is also of the same order, when both methods are compared. Further, these enzymatic activities were measured with use of various concentrations of substrates. From our experiments we conclude that the degree of stimulation of the apoenzyme of the two enzymes is independent of which way the enzymatic reaction is carried out or the substrate concentration, except that aspartate aminotransferase activity is more stimulated by the coenzyme at higher 2-oxoglutarate concentrations.

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A Simple Protocol for the Determination of Lysostaphin Enzymatic Activity

Antibiotics ◽

10.3390/antibiotics9120917 ◽

2020 ◽

Vol 9 (12) ◽

pp. 917

Author(s):

Alexander V. Grishin ◽

Svetlana V. Konstantinova ◽

Irina V. Vasina ◽

Nikita V. Shestak ◽

Anna S. Karyagina ◽

...

Keyword(s):

Enzymatic Activity ◽

Catalytic Efficiency ◽

Bacterial Cells ◽

Binding Domains ◽

Simple Protocol ◽

Specialized Equipment ◽

Osmotic Lysis ◽

Cross Bridges ◽

Bacterial Peptidoglycan

Antibacterial lysins are enzymes that hydrolyze bacterial peptidoglycan, which results in the rapid death of bacterial cells due to osmotic lysis. Lysostaphin is one of the most potent and well-studied lysins active against important nosocomial pathogen Staphylococcus aureus. Similarly to most other lysins, lysostaphin is composed of enzymatic and peptidoglycan-binding domains, and both domains influence its antibacterial activity. It is thus desirable to be able to study the activity of both domains independently. Lysostaphin cleaves pentaglycine cross-bridges within the staphylococcal peptidoglycan. Here, we report the protocol to study the catalytic activity of lysostaphin on the isolated pentaglycine peptide that is based on the chromogenic reaction of peptide amino groups with ninhydrin. Unlike previously reported assays, this protocol does not require in-house chemical synthesis or specialized equipment and can be readily performed in most laboratories. We demonstrate the use of this protocol to study the effect of EDTA treatment on the lysostaphin enzymatic activity. We further used this protocol to determine the catalytic efficiency of lysostaphin on the isolated pentaglycine and compared it to the apparent catalytic efficiency on the whole staphylococcal cells. These results highlight the relative impact of enzymatic and peptidoglycan-binding domains of lysostaphin on its bacteriolytic activity.

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