Mechanism of inhibition of cytochrome P450 C21 enzyme activity by autoantibodies from patients with Addison's disease.

OBJECTIVE: To study possible mechanisms for the inhibition of cytochrome P450 C21 (steroid 21-hydroxylase) enzyme activity by P450 C21 autoantibodies (Abs) in vitro. DESIGN: Two possible mechanisms for the inhibition of P450 C21 enzyme activity by P450 C21 Abs were studied: (a) conformational changes in the P450 C21 molecule induced by Ab binding and (b) the effects of Ab binding to P450 C21 on the electron transfer from the nicotinamide adenine dinucleotide phosphate reduced (NADPH) cytochrome P450 reductase (CPR) to P450 C21. METHODS: The effect of P450 C21 Ab binding on the conformation of recombinant P450 C21 in yeast microsomes was studied using an analysis of the dithionite-reduced CO difference spectra. The effect of P450 C21 Abs on electron transfer was assessed by analysis of reduction of P450 C21 in the microsomes in the presence of CO after addition of NADPH. RESULTS: Our studies confirmed the inhibiting effect of P450 C21 Abs on P450 C21 enzyme activity. Binding of the Abs did not induce significant change in the P450 C21 peak at 450nm (native form) and did not produce a detectable peak at 420 nm (denatured form) in the dithionite-reduced CO difference spectra. This indicated that conformation of P450 C21 around the heme was not altered compared with the native structure. However, incubation of the P450 C21 in yeast microsomes with P450 C21 Ab inhibited the fast phase electron transfer from the CPR to P450 C21. CONCLUSIONS: Our observations suggested that the mechanism by which P450 C21 Abs inhibit P450 C21 enzyme activity most likely involves inhibition of the interaction between the CPR and P450 C21.

Gene expression of Corynebacterium glutamicum in response to the conditions inducing glutamate overproduction.

AIM: The ultimate aim is to elucidate the molecular mechanisms for glutamate overproduction by Corynebacterium glutamicum. METHODS AND RESULTS: Gene expression in response to the conditions inducing glutamate overproduction was investigated by using a DNA microarray technique. Most genes involved in the EMP pathway, the PPP, and the TCA cycle were downregulated, while five genes that were highly upregulated (NCgl0917, NCgl2944, NCgl2945, NCgl2946, and NCgl2975) were identified under all the three conditions for overproduction that are studied here. Gene products of NCgl2944, NCgl2945, and NCgl2946 were highly homologous to each other, did not resemble any other protein, and have remained uncharacterized thus far. The product of NCgl0917 showed a similarity to a few hypothetical and uncharacterized proteins. NCgl2975 was homologous to metal-binding proteins. CONCLUSIONS: The decrease in the activity of 2-oxoglutarate dehydrogenase complex, a key enzyme that is downregulated during glutamate overproduction, can be mainly attributed to the downregulation of odhA and sucB. Five highly upregulated genes were also identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Although fermentative production of glutamate has been carried out for more than 45 years, information on the molecular mechanisms of glutamate overproduction is still limited. This study further elucidates these mechanisms.

Isolation and characterisation of a human monoclonal autoantibody to the islet cell autoantigen IA-2.

A hybridoma secreting a human monoclonal autoantibody to the islet cell autoantigen IA-2 was prepared from peripheral lymphocytes of a patient with type 1 diabetes and Graves' disease using EBV infection followed by fusion with a mouse/human hybrid cell line. The monoclonal antibody (M13) is an IgG1/kappa and in an immunofluorescence test M13 at 1 microg/mL showed islet cell antibody reactivity equivalent to 40 JDF units. M13 IgG bound (35)S-labelled IA-2 (26% at 100 microg/mL) and (125)I-labelled IA-2 (34% at 100 microg/mL) in an immunoprecipitation assay and reacted well with IA-2 in western blotting analysis. Amino acids 777-808 in the PTP domain of IA-2 were found to be important for M13 binding in an analysis using modified (35)S-labelled IA-2 proteins. M13 V region genes were from VH1-3, D3-22, JH4b, VKI DPK8/Vd+ and JK3 genes and showed a high replacement/silent mutation ratio for both the heavy (11.0) and the light (6.0) chain genes. Mouse monoclonal antibodies (mMAbs) reactive with at least three different epitopes within IA-2 aa 604-686 corresponding to the juxtamembrane domain were also obtained. F(ab')(2) or Fab from the mMAbs inhibited serum IA-2 autoantibody binding to IA-2 in 20/22 diabetic sera whereas M13 F(ab')(2) caused inhibition in only 6/22 sera. M13 is representative of some patient serum IA-2 autoantibodies and as such provides a useful tool to study autoimmune responses to IA-2.

Molecular cloning of a novel gene, dtsR, which rescues the detergent sensitivity of a mutant derived from Brevibacterium lactofermentum.

Several strains of Corynebacterium and Brevibacterium are known for their ability to secrete large amounts of amino acids, especially L-glutamate. We focused on the mechanism of L-glutamate secretion triggered by a detergent, namely polyoxyethylenesorbitan monopalmitate (PESP). A mutant strain, AJ11060, derived from Brevibacterium lactofermentum ATCC 13869 indicates the sensitivity to PESP. A multicopy suppresser gene that compliments the sensitivity of AJ11060 to the detergent was derived from a gene library of B. lactofermentum AJ12036. A 2855-bp DNA fragment was cloned and sequenced. An open reading frame was found that coded for the rescuer gene of the sensitivity to PESP of AJ11060 and was designated dtsR. The expression of the dtsR gene in B. lactofermentum was confirmed by using anti-DtsR antibody. The deduced DtsR protein indicated significant homology with some biotin enzymes such as the beta chain of propionyl-CoA carboxylase from rat (48.3%) and human (48.7%), or a 12S chain of methylmalonyl-CoA carboxyltransferase from Propionibacterium freudenreichii (43.1%).

A new synthesis of purine arabinosides by a combination of chemical and enzymatic reaction.

The synthesis of hypoxanthine, 2-chlorohypoxanthine, and 2-methylhypoxanthine arabinoside by an enzymatic transarabinosylation and their chemical conversion to biologically interesting purine arabinosides are described. Some of the synthesized compounds were tested for the inhibition of DNA synthesis in cultured tumor cells.

Relationship between the glutamate production and the activity of 2-oxoglutarate dehydrogenase in Brevibacterium lactofermentum.

Enzyme activities of 2-oxoglutarate dehydrogenase complex and glutamate dehydrogenase of wild type Brevibacterium lactofermentum, one of the typical glutamate-producing coryneform bacteria, were investigated by using cells cultured under glutamate-productive and glutamate-non-productive conditions. Significant reduction of the former enzyme activity was observed in the cells under the several glutamate-productive conditions, namely, in the cells cultured in media containing a) limited concentrations of biotin, b) sub-lethal amounts of penicillin, and c) sub-optimal amounts of a surface-active agent, as compared with those under the non-productive conditions. The activity of the latter enzyme was essentially unchanged in every condition. The relationship between glutamate production and the enzyme activities as well as permeability of glutamate through cell membrane was discussed from the results obtained.

A dtsR gene-disrupted mutant of Brevibacterium lactofermentum requires fatty acids for growth and efficiently produces L-glutamate in the presence of an excess of biotin.

A dtsR gene encoding a homolog of the beta subunit of some biotin-containing enzymes suppresses a detergent-sensitive mutation of Brevibacterium lactofermentum (E. Kimura et al., 1996, Biosci. Biotech. Biochem. 60, 1565-1570), which has been used for the fermentative production of L-glutamate. When the dtsR gene was disrupted, the organism exhibited strict fatty acid auxotrophy; oleate or oleate ester, but not palmitate ester or stearate ester, supported the growth of the delta dtsR mutant. Immunoblotting with an anti-DtsR antibody revealed that no intact DtsR was present in the cytosol of the delta dtsR mutant. In the presence of an excess of biotin, the wild type strain did not produce L-glutamate whereas the delta dtsR mutant efficiently produced it. The mechanism underlying the efficient production of L-glutamate by the delta dtsR mutant is discussed as to the possible role of dtsR in fatty acid metabolism.

A new and facile synthesis of purine 2'-amino-2'-deoxyribosides by a combination of chemical and enzymatic reactions.

An enzymatic synthesis of 2'-amino-2'-deoxy-2-chloroinosine and its chemical conversion to purine 2'-amino-2'-deoxyribosides are described. In addition, some of 2'-amino-2'-deoxy-ribosides of 6-substituted purine were also prepared from 2'-amino-2'-deoxyinosine.

Glutamate Overproduction in Corynebacterium glutamicum Triggered by a Decrease in the Level of a Complex Comprising DtsR and a Biotin-containing Subunit.

Glutamate overproduction in Corynebacterium glutamicum is induced by Tween 40, biotin-limitation, or sublethal amounts of penicillin. Disruption of the dtsR gene, which encodes a putative component of a biotin-containing enzyme complex involved in fatty acid synthesis, causes constitutive overproduction of glutamate. We report here that overexpression of dtsR inhibits the induction of glutamate overproduction. In contrast, the level of DtsR in the wild type strain was found to decrease in the presence of Tween 40 or limited amounts of biotin. Tween 40, biotin-limitation, or dtsR disruption also reduced the activity of 2-oxoglutarate dehydrogenase complex (ODHC), which is involved in the synthesis of succinate from 2-oxoglutarate. These results indicate that decrease in the level of DtsR or a complex containing DtsR triggers the increased synthesis of glutamate from 2-oxoglutarate by lowering the ODHC activity.