• Microbiology and Biotechnology Letters
• /
• v.27 no.2
• /
• pp.124-128
• /
• 1999

TK6 was able to produce NAD+ dependent cyclohexanol dehydrogenase(CDH). The production of CDH was increased rapidly at the logarithmic phase and maintained constantly after that. In order to investigate the inductive production of CDH by various substrates, the bacteria were grown in the media containing alicyclic hydrocarbons and various alcohols as a sole crabon souce. CDH was induced most actively by cyclohexanol. Cyclohexanone and cyclohexane-1,2-diol also induced remarkable amount of CDH but it was induced weakly by 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, 2-propanol, and 2-methyl-1-propanol. The dehydrogenase of the bacteria grown in the media containing cyclohexanol were weakly active for various alcohols, but the dehydrogenase activity for cyclohexane-1,2-diol was twice as much as that for cyclohexanol. Activity staining on PAGE of the cell free extract of Rhodococcus sp. TK6 grown in the media containing cyclohexanol reveals at least sever isozyme bands of CDH and we nominated the four major activity bands as CDH I, II, III, and IV. CDH I was strongly induced by cyclohexanol, cyclohexane-1,2-diok, but its activity was specific to cyclohexane-1,2-diol and 1-pentanol. CDH IV was strongly induced by cyclohexanol and cyclohexane-1,2-diol, and its activity was very specific to cyclohexane-1,2-diol.

• Journal of Microbiology and Biotechnology
• /
• v.14 no.1
• /
• pp.197-201
• /
• 2004

A 2-D gel protein analysis of Lactobacillus reuteri ATCC 55739 produced spots corresponding to subunits of the pyruvate dehydrogenase complex, as identified by N-terminal protein sequencing. Oligonucleotide probes specific for the subunits of the pyruvate dehydrogenase complex were synthesized ,md used to screen a L. reuteri genomic library to clone the structural genes. Two positive clones were isolated and identified as having the same 2.2 kb insert. A pdhB encoding the $\beta$-subunit of El subunit (pyruvate dehydrogenase component) of the pyruvate dehydrogenase complex was located in the middle of the insert. Furthermore, a 5' truncated pdhA encoding the $\alpha$-subunit of the E1 subunit and a 3' truncated pdhC encoding the E2 subunit (dihydrolipoamide acetyltransferase) were also located upstream and downstream of the pdhB, respectively.

• Food Science of Animal Resources
• /
• v.24 no.2
• /
• pp.151-155
• /
• 2004

This study was performed to develop the method of differentiation fresh and frozen meat by using the measurement of mitochondrial malate dehydrogenase. The principle of this experiment is based on the fact the enzyme proteins associated with mitochondria membrane could be released by freezing. The methods were studied by measurements of protein concentration of meat press juice, WHC (water-holding capacity), drip loss and mitochondrial malate dehydrogenase enzyme activity. Samples were stored at 4$^{\circ}C$ and -18$^{\circ}C$ during storage period, respectively. Protein concentration of meat press juice was ranged from 8.5 mg/mL to 12.7 mg/mL and increased by freezing below at -18$^{\circ}C$(p<0.05). The WHC was not significantly different between fresh meat and frozen chicken meat (p＞0.05). The amount of drip loss of fresh and frozen chicken meat at 4$^{\circ}C$ and -18$^{\circ}C$ was not significantly different (p＞0.05). Mitochondrial malate dehydrogenase activity of frozen meat (-18$^{\circ}C$) was significantly higher (p＜0.05) than that of fresh meat. Also, enzyme activity of frozen meat was maintained at the same level after 3 minutes reaction. But fresh meat had not this reaction. From these results, it suggests that mitochondrial malate dehydrogenase can be used as a promising enzyme to differentiate between fresh and frozen meat.

• Asian-Australasian Journal of Animal Sciences
• /
• v.15 no.3
• /
• pp.421-426
• /
• 2002

The pyruvate dehydrogenase complex (PDC), a member of $\alpha$-keto acid dehydrogenase complex, catalyzes the oxidative decarboxylation of pyruvate with the formation of $CO_2$, acetyl-CoA, NADH, and $H^+$. This complex contains multiple copies of three catalytic components including pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). Two regulatory components (E1-kinase and phospho-E1 phosphatase) and functionally less-understood protein (protein X, E3BP) are also involved in the formation of the complex. In this study, we have partially cloned the gene for E3BP in human. Nine putative clones were isolated by human genomic library screening with 1.35 kb fragment of E3BP cDNA as a probe. For investigation of cloned genes, Southern blot analysis and the construction of the restriction map were performed. One of the isolated clones, E3BP741, has a 3 kb-SacI fragment, which contains 200 bp region matched with E3BP cDNA sequences. The matched DNA sequence encodes the carboxyl-terminal portion of lipoyl-bearing domain and hinge region of human E3BP. Differences between yeast E3BP and mammalian E3BP coupled with the remarkable similarity between mammalian E2 and mammalian E3BP were confirmed from the comparison of the nucleotide sequence and the deduced amino acid sequence in the cloned E3BP. Cloning of human E3BP gene and analysis of the gene structure will facilitate the understanding of the role(s) of E3BP in mammalian PDC.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 2002.07a
• /
• pp.236-236
• /
• 2002

Pyruvate dehydrogenase phosphatase (PDP) is a mitochondrial protein serine/threonine phosphatase that catalyzes the dephosphorylation and concomitant reactivation of the pyruvate dehydrogenase componant of the pyruvate dehydrogenase complex (PDC). PDP consists of a Mg$\^$+2/ -dependent and Ca$\^$+2)-stimulated catalytic subunit (PDPc) of Mr 52,600 and a FAD-containing regulatory subunit (PDPr) of Mr 95.600. Catalytic subunit of pyruvate dehydrogenase phosphatase (PDPc) has been suggested to have three major functional domains such as dihydrolipoamide acetyltransferase(E$_2$)-binding domain, regulatory subunit of PDP(PDPr)-binding domain, and calcium-binding domain. In order to identify functional domains, recombinant catalytic subunit of pyruvate dehydrogenase phosphatase (rPDPc) was expressed in E. coli JM101 and purified to near homogeneity using the unique property of PDPc: PDPm binds to the inner lipoyl domain (L$_2$) of E$_2$ of pyruvate dehydrogenase complex (PDC) in the presence of Ca$\^$+2/, not under EGTA. PDPc was limited-proteolysed by trypsin, chymotrypsin, Arg-C, and elastase at pH7.0 and 30$^{\circ}C$ and N-terminal analysis of the fragment was done. Chymotrypsin, trypsin, and elastase made two major framents: N-terminal large fragment, approx. 50kD and C-terminal small fragment, approx. 0 kDa. Arg-C made three major fragments: N-terminal fragment, approx. 35 kD, and central fragment, approx. 15 kD, and C-terminal fragment, approx. 10 kD. This study strongly suggest that PDPc consists of three major functional domains. However, further study should be necessary to identify the functional role.

• Journal of Life Science
• /
• v.8 no.1
• /
• pp.8-13
• /
• 1998

The maize mitochondrial mutant (NCS2) is derived from homologous recombination between genes encoding NADH dehydrogenase subunit 4 and subunit 6. Plants from mitochondria mutants exhibited severe related growth and development including dwarfism and striping on the leaves. Aborted embryos from NCS2 mutants have been rescued and cultured on the N6 medium supplemented with 2,4-D 1 mg/l. Most calli from NCS2 aborted embryos showed slow growing pattern at first stage. However, upon continuous culturing them on the medium, those were segregated into mutant and normal callus lines. These segregations could be detected by using PCR method with three primers. Such segregation seems to be resulted from the preferential growth of normal cells over the mutant cells on the normal culture condition. Therefore, this method can be used for determining rate of indirect cytoplasmic segregation by estimating amplified band intensities. When NCS2 mutant callus lines cultured on regeneration medium, no adventitious shoot induction was observed. However, callus lines with more mitochondria induced adventitious shoots. These studies suggest that mitochondria NADH-dehydrogenase for electron transport in the inner membrane of mitochondria is essential for the differentiation and development of plants.

• Biomaterials and Biomechanics in Bioengineering
• /
• v.3 no.1
• /
• pp.37-46
• /
• 2016

To maintain activity in a coenzyme/enzyme mixture system, such as ${\beta}$-nicotinamide adenine dinucleotide (NADH)/dehydrogenase, the water-soluble 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers as an additive were synthesized and investigated for their stabilizing function. The inhibitor for the NADH/dehydrogenase reaction was spontaneously formed when the NADH was stored in the dehydrogenase solution. Therefore, we hypothesized that if the additive polymer could interact with an inhibitor without any adverse effect on the dehydrogenase, the activity in the NADH/dehydrogenase mixture could be maintained. We selected lactose dehydrogenase (LDH) as the enzyme, and the NADH was dissolved and incubated at $37^{\circ}C$ in the LDH solution containing the polymers. The phospholipid polymers used in this study were poly(MPC) (PMPC), poly(MPC-co-3-trimethylammonium-2-hydroxypropyl methacrylate chloride) (PMQ) and poly[MPC-co-potassium 3-methacryloyloxypropyl sulfonate ($MSO_3$)] ($PMMSO_3$). The poly($MSO_3$) was used as a reference. For the PMQ and $PMSO_3$ aqueous solutions, the activity of the NADH/LDH mixture system decreased with incubation time as the same level or lower than that in the Tris buffered solution in the absence of the polymers. However, for the poly($MPC-co-MSO_3$) ($PMMSO_3$) aqueous solution, the activity of the NADH/LDH mixed system was six times higher than that in the buffered solution even after a 3-days incubation. The LDH activity was 1.5-1.8 times higher in the presence of the $PMMSO_3$ compared with that in the $PMSO_3$ solution. The mixture of two polymers, poly(MPC) and poly($MSO_3$), did not produce any stabilization. Thus, both the MPC and $MSO_3$ units in the polymer chain had important and cooperative effects for stabilizing the NADH/LDH mixture.

• Korean Journal of Microbiology
• /
• v.34 no.4
• /
• pp.207-211
• /
• 1998

Melhylovorus sp. strain SS1 grown on methanol was found to show activities of key enzymes of the linear route, $NAD^+$-linked formaldehyde and formate dehydrogenases, and the cyclic route, hexulose-6-phosphate synthase, glucose-6-phosphate isomerase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase, for formaldehyde oxidation. The activities of the cyclic route enzymes were higher than those of the linear route enzymes. The bacterium also exhibited activities of the key enzymes of the ribulose monophosphate and Entner-Doudoroff pathways and transaldolase involved in the formaldehyde assimilation and the enzymes involved in the biosynthesis of extracellular polysaccharide. Cells grown in the presence of 2.3 mM ammonium sulfate were higher in the productivity of extracellular polysaccharide, but lower in the growth yield, than those grown in the presence 7.6 mM ammonium sulfate. The activities of 6-phosphogluconate dehydrogenase, phosphoglucomutase, and UDP-pyrophosphorylase in cells grown under nitrogen-limited condition were higher, but that of 6-phosphogluconate dehydratase/2-keto-3-deoxy-6-phosphogluconate aldolase was lower, than those in cells grown in the presence of sufficient amount of nitrogen source.

• Bulletin of the Korean Chemical Society
• /
• v.34 no.6
• /
• pp.1621-1622
• /
• 2013

• Bulletin of the Korean Chemical Society
• /
• v.35 no.6
• /
• pp.1603-1604
• /
• 2014