Molecular characterization of the non-biotin-containing subunit of 3-methylcrotonyl-CoA carboxylase.

The biotin enzyme, 3-methylcrotonyl-CoA carboxylase (MCCase) (3-methylcrotonyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1. 4), catalyzes a pivotal reaction required for both leucine catabolism and isoprenoid metabolism. MCCase is a heteromeric enzyme composed of biotin-containing (MCC-A) and non-biotin-containing (MCC-B) subunits. Although the sequence of the MCC-A subunit was previously determined, the primary structure of the MCC-B subunit is unknown. Based upon sequences of biotin enzymes that use substrates structurally related to 3-methylcrotonyl-CoA, we isolated the MCC-B cDNA and gene of Arabidopsis. Antibodies directed against the bacterially produced recombinant protein encoded by the MCC-B cDNA react solely with the MCC-B subunit of the purified MCCase and inhibit MCCase activity. The primary structure of the MCC-B subunit shows the highest similarity to carboxyltransferase domains of biotin enzymes that use methyl-branched thiol esters as substrate or products. The single copy MCC-B gene of Arabidopsis is interrupted by nine introns. MCC-A and MCC-B mRNAs accumulate in all cell types and organs, with the highest accumulation occurring in rapidly growing and metabolically active tissues. In addition, these two mRNAs accumulate coordinately in an approximately equal molar ratio, and they each account for between 0.01 and 0.1 mol % of cellular mRNA. The sequence of the Arabidopsis MCC-B gene has enabled the identification of animal paralogous MCC-B cDNAs and genes, which may have an impact on the molecular understanding of the lethal inherited metabolic disorder methylcrotonylglyciuria.

3-Methylcrotonyl-coenzyme A carboxylase is a component of the mitochondrial leucine catabolic pathway in plants

3-Methylcrotonyl-coenzyme A carboxylase (MCCase) is a mitochondrial biotin-containing enzyme whose metabolic function is not well understood in plants. In soybean (Glycine max) seedlings the organ-specific and developmentally induced changes in MCCase expression are regulated by mechanisms that control the accumulation of MCCase mRNA and the activity of the enzyme. During soybean cotyledon development, when seed-storage proteins are degraded, leucine (Leu) accumulation peaks transiently at 8 d after planting. The coincidence between peak MCCase expression and the decline in Leu content provides correlative evidence that MCCase is involved in the mitochondrial catabolism of Leu. Direct evidence for this conclusion was obtained from radiotracer metabolic studies using extracts from isolated mitochondria. These experiments traced the metabolic fate of [U-14C]Leu and NaH14CO3, the latter of which was incorporated into methylglutaconyl-coenzyme A (CoA) via MCCase. These studies directly demonstrate that plant mitochondria can catabolize Leu via the following scheme: Leu --> alpha-ketoisocaproate --> isovaleryl-CoA --> 3-methylcrotonyl-CoA --> 3-methylglutaconyl-CoA --> 3-hydroxy-3-methylglutaryl-CoA --> acetoacetate + acetyl-CoA. These findings demonstrate for the first time, to our knowledge, that the enzymes responsible for Leu catabolism are present in plant mitochondria. We conclude that a primary metabolic role of MCCase in plants is the catabolism of Leu.

Variation of the CGG repeat in FMR-1 gene in normal and fragile X Chinese subjects.

The fragile X syndrome is believed to be caused by an expansion of a CGG trinucleotide repeat segment in the FMR-1 gene on the fragile X site of the long arm of the X-chromosome. To understand the variation of the CGG repeat in the FMR-1 gene in southern Chinese from the Hong Kong and Guangzhou area, we undertook the present study. A total of 83 normal and three fragile X subjects were examined. In the normal group, 16 distinct alleles, ranging in size from 272 bp to 332 bp with 17 to 37 CGG repeats were detected. A repeat size of 29 was the most frequent. Compared with data collected in the USA, the repeat size observed in this population was somewhat smaller. Whether this discrepancy is due to ethnic difference remains to be determined. The three fragile X patients examined in this study did not have a greatly expanded CGG segment. One of them may be a mosaic with one full and one premutation allele. The other two patients, although having clinical and cytological features of fragile X syndrome, had a CGG repeat size within normal range. To explain this, we infer that the mutation in these patients may be caused by other mechanisms, such as other types of FMR-1 mutation or mutation in another site. It is possible that the expansion of the CGG repeats may not be as frequent a cause of fragile X syndrome in southern Chinese as in other ethnic groups.

More sensitive way to determine iron using an iron(II)-1,10-phenanthroline complex and capillary electrophoresis.

Iron is one of the major metal species of concern in many samples, such as in serum, foods, drinking waters, etc. In this paper, we present a more sensitive way to determine the iron concentration in water solutions by using an iron(II)-1,10-phenanthroline complexing system with high-performance capillary electrophoresis, and have applied this method to the determination of the levels of iron in serum samples. The technique uses ammonium acetate-acetic acid (50 mM NH4Ac-HAc, pH 5.0) as a running buffer, and the detection wavelength is set at 270 nm instead of 508 nm. This new approach enhances the molar absorbance of the Fe(II)-1,10-phenanthroline complex by about eight-fold compared with that obtained at 508 nm. By combining the larger light output of the deuterium (D2) lamp and the lower noise level at 270 nm, the sensitivity was improved at least twenty-fold compared to that at 508 nm. The detection limit for iron(II) is lower than 5 x 10(-9) M, which has never been reached by reported spectrophotometric methods or with the recently published HPCE method. The effects of pH, buffer concentration and operation voltages on the sensitivity and resolution are also discussed. The signal response is linear over two orders of magnitude (r2 = 0.995) and the iron recovery for samples reached 99-101%. The technique described here is much more sensitive, fast and simple and is suitable for determining trace amounts of iron in biological, food, water and other samples.

Quantitative determination of serum iron in human blood by high-performance capillary electrophoresis.

A capillary electrophoretic (HPCE) method that can be used to quantitatively determine trace amounts of iron has been developed and applied to determine the iron level in human serum. After precipitation of serum proteins, Fe(III) in the serum is reduced to Fe(II) with hydroxylamine hydrochloride, and a stable Fe(II)-1,10-phenanthroline complex is formed by adding 1,10-phenanthroline to the supernatant containing 2.5 mM ammonium acetate-acetic acid at pH 5.0. The Fe(II)-1,10-phenanthroline complex, [Fe(C12H8N2)3]2+, has a very strong absorbance at 270 nm (with a molar absorptivity of approximately 9.2.10(4)). By measuring the absorbance of [Fe(C12H8N2)3]2+ at 270 nm, the iron level in human serum can be precisely quantified. The interference from copper, a major interference in serum, can be totally eliminated due to the complete separation of [Fe(C12H8N2)3]2+ and the Cu(II)-1,10-phenanthroline complex. In addition, other problems that usually occurred with conventional spectrophotometric methods, such as co-precipitation and occlusion of iron during sample pretreatment, are significantly minimized due to the ability to wash the precipitate and the higher detection sensitivity. With this method, a single drop (10 microliters) of serum would be sufficient to determine the serum iron concentration. The method is reliable, sensitive, rapid and reproducible. Thus it is highly suitable for use in the clinical laboratory.