Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme

Terlecki, Grzegorz; Czapińska, Elżbieta; Hotowy, Katarzyna

doi:10.2478/s11658-007-0010-5

Cited by 5 publications

(2 citation statements)

References 42 publications

(49 reference statements)

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“…Native enzymes GOx, AP, Lip and LDH move towards the positive electrode due to the net negative charge on these enzymes. 36 The staining of HRP in the gel is very poor and its electrophoretic properties could not be established. The MECs will have high molecular weight and may remain in the loading well without any movement through the gel.…”

Section: Resultsmentioning

confidence: 99%

Nanoarmoring: strategies for preparation of multi-catalytic enzyme polymer conjugates and enhancement of high temperature biocatalysis

et al. 2017

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We report a general and modular approach for the synthesis of multi enzyme–polymer conjugates (MECs) consisting of five different enzymes of diverse isoelectric points and distinct catalytic properties conjugated within a single universal polymer scaffold. The five model enzymes chosen include glucose oxidase (GOx), acid phosphatase (AP), lactate dehydrogenase (LDH), horseradish peroxidase (HRP) and lipase (Lip). Poly(acrylic acid) (PAA) is used as the model synthetic polymer scaffold that will covalently conjugate and stabilize multiple enzymes concurrently. Parallel and sequential synthetic protocols are used to synthesise MECs, 5-P and 5-S, respectively. Also, five different single enzyme–PAA conjugates (SECs) including GOx–PAA, AP–PAA, LDH–PAA, HRP–PAA and Lip–PAA are synthesized. The composition, structure and morphology of MECs and SECs are confirmed by agarose gel electrophoresis, dynamic light scattering, circular dichroism spectroscopy and transmission electron microscopy. The bioreactor comprising MEC functions as a single biocatalyst can carry out at least five different or orthogonal catalytic reactions by virtue of the five stabilized enzymes, which has never been achieved to-date. Using activity assays relevant for each of the enzymes, for example AP, the specific activity of AP at room temperature and 7.4 pH in PB is determined and set at 100%. Interestingly, MECs 5-P and 5-S show specific activities of 1800% and 600%, respectively, compared to 100% specific activity of AP at room temperature (RT). The catalytic efficiencies of 5-P and 5-S are 1.55 × 10−3 and 1.68 × 10−3, respectively, compared to 9.11 × 10−5 for AP under similar RT conditions. Similarly, AP relevant catalytic activities of 5-P and 5-S at 65 °C show 100 and 300%, respectively, relative to native AP activity at RT as the native AP is catalytically inactive at 65 °C The catalytic activity trends suggest: (1) MECs show enhanced catalytic activities compared to native enzymes under similar assay conditions and (2) 5-S is better suited for high temperature biocatalysis, while both 5-S and 5-P are suitable for room temperature biocatalysis. Initial cytotoxicity results show that these MECs are non-lethal to human cells including human embryonic kidney [HEK] cells when treated with doses of 0.01 mg mL−1 for 72 h. This cytotoxicity data is relevant for future biological applications.

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Section: Resultsmentioning

confidence: 99%

Nanoarmoring: strategies for preparation of multi-catalytic enzyme polymer conjugates and enhancement of high temperature biocatalysis

et al. 2017

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“…This was also spectrometry. Although several proteins were claimed by Terlecki et al (47) for lactate identified, their individual binding characteristics dehydrogenase-phosphatidylserine interaction. and affinity for the phosholipid have not been The mammalian M2 type pyruvate kinase determined in each case.…”

Section: Pyruvate Kinase Forms a Complex With Clathrinmentioning

confidence: 98%

Detection of Serum Lysophosphatidic Acids Using Affinity Binding and Surface Enhanced Laser Desorption/Ionization (SELDI) Time of Flight Mass Spectrometry

Mills¹

2005

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Put ic reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Washington Headquarters Services, Directorate ABSTRACT (Maximum 200 Words)We proposed to apply two novel technologies to the development of an approach suitable for screening for ovarian cancer in high and low risk women. The first of these is a novel approach to the development of antibodies which will recognize specific phospholipids and lysophospholipids present in ovarian cancer patients and the second of these is SELDI tof mass spectroscopy. These two technologies will be merged with powerful computing tools to develop approaches capable of detecting ovarian cancer at an early, curable stage. This approach will further benefit from the expertise of the Mills laboratory (LPA screening, SELDI tof) with that of the Prestwich laboratory (lipid synthesis and antibody development). Progress We have demonstrated that SELDI mass spectroscopy has the ability to detect model lysophospholipids present in plasma and serum. The sensitivity of the assay using standard capture chips is low and would require relatively large volumes of sera to detect the different lysophospholipid isoforms. This will require prepurification approaches to concentrate the lipids which we have now developed. We have obtained a pan sphingosine 1 phosphate antibody as a capture reagent. This antibody is able to bind all isoforms of S I P and when combined with SELDI should allow detection of isoforms of this lysophospholipid. We have shown that the antibodie can detect Si P levels in complex ascites mixtures. We are currently evaluating different CHIP forms for SELDI and developing LPA antibodies as well as obtaining LPA binding proteins to increase the ability to capture lysophospholipids through collaborative approaches.

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Glycation of the Muscle-Specific Enolase by Reactive Carbonyls: Effect of Temperature and the Protection Role of Carnosine, Pirydoxamine and Phosphatidylserine

et al. 2011

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Reactive carbonyls such as 4-hydroxy-2-nonenal (4-HNE), trans-2-nonenal (T2 N), acrolein (ACR) can react readily with nucleophilic protein sites forming of advanced glycation end-products (AGE). In this study, the human and pig muscle-specific enolase was used as a protein model for in vitro modification by 4-HNE, T2 N and ACR. While the human enolase interaction with reactive α-oxoaldehyde methylglyoxal (MOG) was demonstrated previously, the effect of 4-HNE, T2N and ACR has not been identified yet. Altering in catalytic function were observed after the enzyme incubation with these active compounds for 1-24 h at 25, 37 and 45 °C. The inhibition degree of enolase activity occurred in following order: 4-HNE > ACR > MOG > T2N and inactivation of pig muscle-specific enolase was more effective relatively to human enzyme. The efficiency of AGE formation depends on time and incubation temperature with glycating agent. More amounts of insoluble AGE were formed at 45 °C. We found that pyridoxamine and natural dipeptide carnosine counteracted AGE formation and protected enolase against the total loss of catalytic activity. Moreover, we demonstrated for the first time that phosphatidylserine may significantly protect enolase against decrease of catalytic activity in spite of AGE production.

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Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme

Cited by 5 publications

References 42 publications

Nanoarmoring: strategies for preparation of multi-catalytic enzyme polymer conjugates and enhancement of high temperature biocatalysis

Nanoarmoring: strategies for preparation of multi-catalytic enzyme polymer conjugates and enhancement of high temperature biocatalysis

Detection of Serum Lysophosphatidic Acids Using Affinity Binding and Surface Enhanced Laser Desorption/Ionization (SELDI) Time of Flight Mass Spectrometry

Glycation of the Muscle-Specific Enolase by Reactive Carbonyls: Effect of Temperature and the Protection Role of Carnosine, Pirydoxamine and Phosphatidylserine

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