Terri Gelbart

The human glucocerebrosidase gene and pseudogene: structure and evolution.

We report the sequence of the entire human gene encoding beta-glucocerebrosidase and that of the associated pseudogene. The gene contains 11 exons extending from base pair 355 to base pair 7232 in the overall sequence. The gene promoter contains TATA- and CAT-like boxes upstream of the major 5 end of the glucocerebrosidase RNA. The two TATA boxes lie between nucleotides (-23)-(-27) and (-33)-(-39) and the two possible CAT boxes reside between nucleotides (-90)-(-94) and (-96)-(-99) in relation to the major 5 end of the mRNA. The functionality of the promoter region was monitored by coupling it to the bacterial gene coding for chloramphenicol acetyltransferase (CAT) and assaying the expression of the enzyme in cells transfected with this vector. The glucocerebrosidase promoter not only directs synthesis of the bacterial enzyme but also exhibits the same pattern of tissue-specific expression as that of the endogenous gene. An apparently tightly linked pseudogene is approximately 96% homologous to the functional gene. However, introns 2, 4, 6, and 7 have large deletions consisting of Alu sequences 313, 626, 320, and 277 bp in length, respectively. It is entirely possible that the ancestral gene lacks these sequences and that they have been inserted into the introns of the functioning gene. There is also a 55-bp deletion from a part of exon 9 flanked by a short inverted repeat. The sequence data should facilitate development of methods for diagnosis of Gaucher disease at the molecular level.

The facile detection of the nt 1226 mutation of glucocerebrosidase by ‘mismatched’ PCR

The most common Gaucher disease-producing mutation among Ashkenazi Jews is an A----G substitution at cDNA nt 1226 (genomic nt 5841). We describe a simple method for detecting this mutation both in genomic DNA and in cDNA by performing polymerase chain reaction (PCR) using a 5-primer mismatched at one nucleotide so as to create an Xho I restriction site. When the mutation is present. the 105 bp fragment formed is cleaved to 89 and 16 nt fragments. The 89 bp fragment is easily visualized on a gel making it possible to distinguish individuals who do not have the mutation from heterozygotes and homozygotes.

Improved assay of the enzymes of glutathione synthesis: γ-glutamylcysteine synthetase and glutathione synthetase

New methods for the estimation of red cell gamma-glutamylcysteine synthetase and glutathione synthetase have been developed. gamma-32P ATP is allowed to equilibrate until the gamma and beta phosphate groups are equally labelled. The amount of 32Pi released in the presence of glutamic acid and cysteine, the substrates for GC-S or in the presence of gamma-glutamylcysteine and glycine, the substrates of GSH-S, is measured. This is accomplished by extraction of the phosphomolybdate complex into isobutanol-benzene. The methods are linear with time and hemolysate concentration. Normal values are presented.

Blood cell phosphogluconolactonase: assay and properties.

Summary. 6‐Phosphogluconolactonase (6‐PGL) catalyses the second reaction of the hexosemonophosphate pathway. Although the ‐Iactone of 6‐phosphogluconic acid is the natural substrate for this enzyme, the more stable γ‐lactone may also be used. We prepared the γ‐lactone of 6‐phosphogluconic acid from 6‐phosphogluconate. When stored in dimethylsulfoxide, this material was found to be stable in liquid nitrogen for several months. A method for measuring 6‐phosphogluconolactonase (6‐PGL) using the γ‐lactone as substrate has been developed, after defining conditions under which spontaneous hydrolysis of the lactone is relatively slow and the enzymatic velocity is relatively rapid.

The Molecular Biology of Gaucher Disease

Several decades of investigation of Gaucher disease by conventional biochemical techniques has greatly increased our understanding of this disorder. Its cause is a deficiency of the lysosomal enzyme glucocerebrosidase leading to the accumulation of glucocerebroside in the tissues. However, our understanding of some aspects of Gaucher disease remains incomplete. For example, its presentation may vary from a neuronopathic disorder with a fatal outcome in the first year of life to a benign storage disorder first diagnosed in old age, and the causes of the marked differences in clinical severity have eluded us. Moreover, efforts to devise a treatment strategy have not been successful.

Mechanism of improved maintenance of 2,3-diphosphoglycerate in stored blood by the xanthone compound 2-(2-hydroxyethoxy)-6-(1-H-tetrazole-5-yl)xanthen-9-one (BW A440C).

The effect of the xanthone derivative 2-(2-hydroxyethoxy)-6-(1-H-tetrazole-5-yl)xanthen-9-one (BW A440C) on red cells was studied. When added to stored red cells at a concentration of 6 mM, greatly improved preservation of 2,3-diphosphoglycerate (2,3-DPG) was observed. There was no effect on internal pH of the erythrocyte. At a concentration 0.500 mM, many red cell enzyme activities were inhibited completely. At a 0.500 mM concentration, however, inhibition of pyruvate kinase and diphosphoglycerate phosphatase was most striking. Inhibition of either of these enzymes could result in elevation of 2,3-DPG levels. BW A440C in concentrations which elevated 2,3-DPG levels in humans caused a decrease in 2,3-DPG levels in rabbits and markedly impaired the viability of 21-day stored rabbit erythrocytes.