Glucosyl transferase activity of bovine galactosyl transferase

Bovine galactosyl transferase was found to utilize UDPglucose as a substrate and elicit disaccharide biosynthesis with glucose and N-acetylglucosamine as acceptors. The relative rate of glycosyl transferase with N-acetylglucosamine as acceptor was 0.3%, the rate for N-acetyllactosamine biosynthesis. This activity was also evidenced indirectly from NMR water proton relaxation experiments, and from Mn(II) ESR experiments. In direct experiments with radioactive UDPglucose, paper chromatography showed a product which migrated with cellobiose when glucose was the acceptor and a new, glucose-containing product which resulted when GlcNAc was the acceptor.Despite this marginally expanded specificity of the donor site, spin-label experiments with a covalently bound UDPgalactose analog reaffirmed the restrictive nature of the donor site against this non-glycosyl-like analog.     

Glucosyl transferase activity of bovine galactosyl transferase.

Bovine galactosyl transferase was found to utilize UDPglucose as a substrate and elicit disaccharide biosynthesis with glucose and N-acetylglucosamine as acceptors. The relative rate of glucosyl transferase with N-acetylglucosamine as acceptor was 0.3%, the rate for N-acetyllactosamine biosynthesis. This activity was also evidenced indirectly from NMR water proton relaxation experiments, and from Mn(II) ESR experiments. In direct experiments with radioactive UDPglucose, paper chromatography showed a product which migrated with cellobiose when glucose was the acceptor and a new, glucose-containing product which resulted when GlcNAc was the acceptor. Despite this marginally expanded specificity of the donor site, spin-label experiments with a covalently bound UDPgalactose analog reaffirmed the restrictive nature of the donor site against this non-glycosyl-like analog.     

Palmitoyl transferase activity of lecithin retinol acyl transferase

Lecithin retinol acyl transferase (LRAT) has the essential role of catalyzing the transfer of an acyl group from the sn-1 position of lecithin to vitamin A to generate all-trans-retinyl esters (tREs). In vitro studies had shown previously that LRAT also can exchange palmitoyl groups between RPE65, a tRE binding protein essential for vision, and tREs. This exchange is likely to be of regulatory significance in the operation of the visual cycle. In the current study, the substrate specificity of LRAT is explored with palmitoylated amino acids and dipeptides as RPE65 surrogates. Both O- and S-substituted palmitoylated analogues are excellent substrates for tLRAT, a readily expressed and readily purified form of LRAT. Using vitamin A as the palmitoyl acceptor, tREs are readily formed. The cognate of these reactions occurs in crude retinal pigment epithelial (RPE) membranes as well. RPE membranes containing LRAT transfer palmitoyl groups from radiolabeled [1-(14)C]-l-alpha-dipalmitoyl diphosphatidylcholine (DPPC) to RPE65. Palmitoyl transfer is abolished by preincubation with a specific LRAT antagonist both in membranes and with purified tLRAT. These experiments are consistent with an expanded role for LRAT function as a protein palmitoyl transferase.     

Endo-xyloglucan transferase,a new class of transferase involved in cell wall construction

Cell shape in plants is constrained by cell walls, which are thick yet dynamic structures composed of crystalline cellulose microfibrils and matrix polymers. Xyloglucans are the principal component of the matrix polymers and bind tightly to the surface of cellulose microfibrils and thereby cross-link them to form an interwoven xyloglucan-cellulose network structure. Thus, cleavage and reconnection of the cross-links between xyloglucan molecules are required for the rearrangement of the cell wall architecture, the process essential for both cell wall expansion and the wall deposition occurring during cell growth and differentiation. Endoxyloglucan transferase (EXT) is a newly identified class of transferase that catalyzes molecular grafting between xyloglucan molecules. This enzyme catalyzes both endo-type splitting of a xyloglucan molecule and reconnection of a newly generated reducing terminus of the xyloglucan to the non-reducing terminus of another xyloglucan molecule, thereby mediating molecular grafting between xyloglucan cross-links in plant cell walls. Molecular cloning and sequencing of EXT-cDNAs derived from five different plant species includingA. thaliana andV. angularis has revealed that the amino acid sequence of the mature protein is extensively conserved in the five different plant species, indicating that EXT protein is ubiquitous among higher plants. This structural study has also disclosed the presence of a group of xyloglucan related proteins (XRPs) with transferase activity in higher plants. Current data strongly suggest that these proteins are involved in a wide spectrum of physiological activities including cell wall expansion and deposition in growing cell walls. Recipient of the Botanical Sociaty Award of Young Scientists, 1993.     

Molecular biology of terminal transferase

Terminal transferase is an unusual deoxynucleotide polymerizing enzyme found only in prelymphocytes. The protein was purified to homogeneity from calf thymus glands in 1971 as a 32 kDa protein with a two peptide structure. Subsequent biochemical and immunological analyses of terminal transferase protein in crude extracts from a number of animal species showed a single peptide with a molecular weight of about 58,000. The two peptide structure found earlier was caused by proteolysis. Homogeneous 58 kDa terminal transferase has now been produced from human lymphoblastoid cells and calf thymus glands by immunoaffinity chromatography. In vitro phosphorylation studies showed that the terminal transferase protein contains one phosphorylation site near one end of the polypeptide chain, and the phosphorylation of the enzyme has been confirmed by in vivo labeling experiments. Unambiguous demonstration of the molecular weight of the human terminal transferase was obtained by translation of the cloned human terminal transferase DNA sequence to a 58,308 Da protein. The translated amino acid sequence also provided a possible phosphorylation site near the amino-terminus of the protein. Preliminary analysis of the genomic structure shows a simple intron/exon pattern with the total human terminal transferase gene spanning at least 65 Kb.     

The ribosomal peptidyl transferase

Peptide bond formation on the ribosome takes place in an active site composed of RNA. Recent progress of structural, biochemical, and computational approaches has provided a fairly detailed picture of the catalytic mechanism of the reaction. The ribosome accelerates peptide bond formation by lowering the activation entropy of the reaction due to positioning the two substrates, ordering water in the active site, and providing an electrostatic network that stabilizes the reaction intermediates. Proton transfer during the reaction appears to be promoted by a concerted proton shuttle mechanism that involves ribose hydroxyl groups on the tRNA substrate.     

Hypoxanthine-guanine phosphoribosyl transferase deficiency

Summary In man congenital lack of an enzyme of the purine salvage system, hypoxanthineguanine phosphoribosyl transferase (HG-PRT E.C. 2.4.2.8), is mostly accompanied by a picture known as the Lesch-Nyhan syndrome. The degree of deficiency may vary from zero to a few percent of normal activity but a correlation between the severity of HG-PRT deficiency and the clinical picture has not been observed, no more than a correlation between HG-PRT deficiency and neurological dysfunction. But individuals with undetectable HG-PRT activity but without the Lesch-Nyhan syndrome have been described. Patients with partial HG-PRT deficiency have clinically distinctive findings. Sometimes mild neurological abnormalities are observed. Because of marked overproduction of uric acid severe gouty arthritis and renal dysfunction are often encountered in both complete and partial deficiency.There is considerable molecular heterogeneity in HG-PRT deficiency in man. Mutant enzymes may exhibit different kinetic and electrophoretic properties, indicating that there might be a mutation on the structural gene coding for HG-PRT.Lack of HG-PRT disturbs purine interconversions profoundly. In addition to an important function of HG-PRT in the uptake of the purine bases hypoxanthine and guanine into the cell, the effective uptake of inosine, guanosine and adenosine also seems to be dependent on HG-PRT. Uptake of purine bases into intact red blood cells occurs according to a two component mechanism, one component probably involving a phosphoribosyl transferase system.The inheritance of HG-PRT deficiency is X-linked recessive and it is transmitted by asymptomatic carrier females. Several methods have been introduced for carrier detection. As a consequence of X chromosome inactivation, in these females a mosaicism of HG-PRT positive and HG-PRT negative fibroblasts can be demonstrated after cloning or after selection of HG-PRT negative cells in a selective medium. A more rapid method involves direct measurements of HG-PRT activities in single hair roots from the scalp. Because hair roots develop more or less clonally, in heterozygote females HG-PRT positive and negative hair roots are encountered. HG-PRT deficiency can be detected antenatally by demonstrating the presence or absence of enzyme activity in ammiotic fluid derived fibroblasts qualitatively by autoradiography and quantitatively by ultramicrochemical measurements of enzyme activities in single or small numbers of cells.In studies with isolated cells the metabolic defect can be corrected in several ways. Metabolic cooperation between HG-PRT positive and HG-PRT negative cells leads to apparently normal phenotype of all cells, provided there is cell to cell contact. There is evidence that a missing enzyme product or a derivative might be transferred from the normal to the mutant cells. Apparent correction of the enzyme defect is also observed when HG-PRT deficient lymphocytes are stimulated with phytohaemagglutinin.The first data suggestive of genetic complementation between two human HG-PRT deficient cell strains by which hybrid cells can synthesize a functionally active HG-PRT, are consistent with the view that HG-PRT deficiency in man is due to a structural gene mutation. Recent results show that other interesting findings might come from experiments in which HG-PRT deficient cells are treated with exogenous genetic material (isolated DNA or metaphase chromosomes) to reactivate or induce HG-PRT activity.Supported by grants from FUNGO (Foundation for Medical Scientific Research in the Netherlands) and the Medical Prevention Fund.     

Pineal hydroxyindole-0-methyl transferase and N-acetyl transferase during sexual maturation of coturnix quail

The enzymic activities of hydroxyindole-o-methyl transferase (HIOMT) and N-acetyl transferase (NAT) were determined in pineals of female and male Coturnix during sexual maturation. Optimum assay conditions for HIOMT was incubation of 5 μl of homogenate for 30 min at pH 7.9, while NAT was 5 μl homogenate for 15 min at pH 6.9. These enzymes were determined in pineals of Coturnix which were immature (30 d), rapidly maturing (40 d), mature (50 d) and discontinued breeders (270 d). HIOMT was significantly decreased in pineals of Coturnix undergoing rapid sexual maturation at 40 d, compared to other stages investigated. However, NAT was significantly greater in rapidly maturing and mature Coturnix compared to immatures and discontinued breeders. These results suggest that the decline in HIOMT and increase in NAT during rapid sexual maturation may be a consequence of increased gonadal activity in this species.     

A comparison of the reversibility of phosphoethanolamine transferase and phosphocholine transferase in rat brain microsomes

The reversibility of phosphoethanolamine transferase (EC 2.7.8.1) in rat brain is demonstrated in this paper. Microsomal ethanolamine glycerophospholipids were prelabeled with an intracerebral injection of [3H]ethanolamine 4 h before killing young rats. Labeled CDPethanolamine was produced by incubation of the microsomes with CMP, although to a lesser extent than for the previously observed release of CDPcholine. Ethanolamine and choline glycerophospholipids were labeled with [2-3H]glycerol by incubation with primary cultures of rat brain. Microsomes from rat brains, with diisopropyl phosphofluoridate for inhibition of lipases, were incubated with the labeled glycerophospholipids separately, and labeled diacylglycerols were produced. The kinetic parameters of phosphoethanolamine transferase and phosphocholine transferase (EC 2.7.8.2) were compared by incubating rat brain microsomes with [3H]CMP. Inclusion of AMP in the reaction mixture was necessary in order to inhibit the hydrolysis of CMP by an enzyme with the properties of 5'-nucleotidase (EC 3.1.3.5). For phosphoethanolamine transferase and phosphocholine transferase respectively, the Km values for CMP were 40 and 125 microM and the V values were 2.3 and 21.6 nmol/h per mg protein. The reversibility of both enzymes permits the interconversion of the diacylglycerol moieties of choline and ethanolamine glycerophospholipids. During brain ischemia, a principal pathway for degradation of ethanolamine glycerophospholipids may be by reversal of phosphoethanolamine transferase followed by hydrolysis of diacylglycerols by the lipase.     

Glutathione transferase of human breast is closely related to transferase of human placenta and erythrocytes

An acidic form (pI 4.6) of glutathione transferase has been purified to homogeneity from normal and tumor specimens of human breast. The two proteins did not differ significantly in their molecular and catalytic properties. The enzyme has a molecular weight of 46,000 and is composed of two identical subunits. The data presented, including amino acid composition, substrate specificity and immunological studies, give strong evidence that the glutathione transferase of human breast, placenta and erythrocytes are similar if not identical proteins.     

Pseudo-isoenzyme forms of liver prenyl transferase

Prenyl transferase of pig liver exists in two forms, A and B, the latter being more acidic than form A. The ratio of these two forms in initial extracts of the organ varies according to the presence or absence of a thiol reducing agent in the extraction medium. In the presence of 10 mM β-mercaptoethanol the two forms exist as an equilibrium mixture of ca. 70% form A and 30% form B. The latter, when separated and rechromatographed with β-mercaptoethanol reverts to approximate the same 7:3 ratio of forms A and B. In the absence of a thiol reducing agent form B does not change, but form A may be converted almost completely to form B after treatment with oxidized glutathione.     

Diaryl ether inhibitors of farnesyl-protein transferase

Imidazolemethyl diaryl ethers are potent inhibitors of farnesyl-protein transferase. The SNAr displacement reaction used to prepare these diaryl ethers was amenable to rapid parallel synthesis of FPTase inhibitors. The use of a broad range of commercially available phenols quickly identified compounds which proved active in cells.