X-linked dominant inheritance of partial phosphorylase kinase deficiency in mice

A new mouse strain, the V strain, with a partial deficiency of phosphorylase kinase has been established. The deficiency is caused by an X-linked dominant gene (Phk ^c ). Muscle extracts of homozygous and heterozygous females and hemizygous males have about 25% of the activity found in extracts of normal (C3H/HeHan) mice. This dominant phosphorylase kinase deficiency of the new V strain is different from that of the I-strain mice with the X-linked recessive deficiency of skeletal muscle phosphorylase kinase. The muscle extracts of V-strain and normal mice contain the same phosphorylase phosphatase activity of about 1 U/mg. Heart and liver extracts from V mice contained about 50% and 66%, respectively, of the phosphorylase kinase activity compared to that found in the same organs from the normal mice. The glycogen content of the skeletal muscle of the V strain was normal, i.e., 0.9 mg/g. Phosphorylase kinase was purified from the skeletal muscle of the V strain by (a) hydrophobic chromatography on methylamine Sepharose, (b) ammonium sulfate precipitation, and (c) gel filtration of Sepharose 4B. The enzyme has a similar structure to the normal murine and rabbit skeletal muscle enzyme, except that the proportion of the subunits differs. The molar ratio of the subunits of the V strain mice is (+)::=0.54:1:1.169, in comparison with that of the rabbit (+)::=1.1:1.0:1.0 and that of normal murine enzyme 0.9:1.0:0.7.This work was supported by the Minister für Wissenschaft und Forschung des Landes Nordrhein-Westfalen, West Germany and of the Fonds der Chemie, West Germany, and forms part of the md thesis of A. Vrbica.     

Characterization of the subunits of purine nucleoside phosphorylase from cultured normal human fibroblasts

In previous communications we have demonstrated that the subunits of normal human erythrocyte purine nucleoside phosphorylase can be resolved into four major (1–4) and two minor (1p and 2p) components with the same molecular weight but different apparent isoelectric points (and net ionic charge). The existence of subunits with different charge results in a complex isoelectric focusing pattern of the native erythrocytic enzyme. In contrast, the isoelectric focusing pattern of the native enzyme obtained from cultured human fibroblasts is simpler. The multiple native isoenzymes obtained from human erythrocytes and human brain have isoelectric points ranging from 5.0 to 6.4 and from 5.2 to 5.8, respectively, whereas cultured human fibroblasts have two major native isoenzymes with apparent isoelectric points of 5.1 and 5.6.Purine nucleoside phosphorylase has been purified at least a hundredfold from ³⁵S-labeled cultured human fibroblasts. A two-dimensional electrophoretic analysis of the denatured purified normal fibroblast enzyme revealed that it consists mainly of subunit 1 (90%) with small amounts of subunits 2 (10%) and 3 (1%). This accounts for the observed differences between the native isoelectric focusing and the electrophoretic patterns of the erythrocyte and fibroblast enzymes. The purine nucleoside phosphorylase subunit 1 is detectable in the autoradiogram from a two-dimensional electrophoretic analysis of a crude, unpurified extract of ³⁵S-labeled cultured normal human fibroblasts. The fibroblast phosphorylase coincides with the erythrocytic subunit 1 of the same enzyme, and the cultured fibroblasts of a purine nucleoside phosphorylase deficient patient (patient I) lack this protein component, genetically confirming the identity of the purine nucleoside phosphorylase subunit in cultured fibroblasts.This work was supported by a grant from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, United States Public Health Service. L. J. G. is supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.     

Purine nucleoside phosphorylase variation in the brook lamprey,Lampetra planeri (Bloch) (Petromyzone,Agnatha): Evidence for a trimeric enzyme structure

Genetic evidence for a trimeric structure for purine nucleoside phosphorylase in the brook lamprey is presented. This enzyme is encoded by a single locus with two alleles segregating at frequencies of 0.98 and 0.02 in a Welsh population. It is suggested that this enzyme is likely to be a trimer in all classes of vertebrates.B. J. McAndrew and G. P. Wallis were supported by NERC Research Studentships.     

Multiple regulation of nucleoside catabolizing enzymes in Escherichia coli: effects of 3:5'' cyclic AMP and CRP protein.

Summary The regulation of the synthesis of nucleoside metabolizing enzymes has been studied in cya and crp mutant strains of Escherichia coli.The synthesis of the cyt-enzymes, cytidine deaminase and uridine phosphorylase regulated by the cytR gene product, is activated by the cAMP-CRP complex. On the other hand the synthesis of the deoenzymes: deoxyriboaldolase, thymidine phosphorylase, phosphodeoxyribomutase and purine nucleoside phosphorylase, appears to be increased if an active cAMP-CRP complex cannot be formed.It also seems that nucleosides serve as poor carbon sources for cya and crp mutants; this could not solely be explained by low levels of nucleoside metabolizing enzymes nor by a deficiency in nucleoside uptake. Addition of casamino acids stimulated the growth of cya and crp mutants, with nucleosides as carbon sources. When grown on glucose and casamino acids growth could be stimulated by adenine and hypoxanthine nucleosides; these results suggest an impaired nitrogen metabolism in cya and crp mutants.Abbreviations and Symbols cAMP cyclic adenosine 3:5-monophosphate - CRP cAMP receptor protein. Genes coding for: adenyl cyclase - cya cAMP receptor protein - crp cytidine deaminase - cdd uridine phosphorylase - udp thymidine phosphorylase - tpp purine nucleoside phosphorylase - pup; cytR regulatory gene for cdd, udp, dra, tpp, drm, and pup - deoR regulatory gene for dra, tpp, drm, and pup     

Activity of phosphorylase in Solanum tuberosum during low temperature storage

Changes in the activity of phosphorylase were measured during storage of potatoes at + 2° when the sugar content rises rapidly and subsequently at + 10° when the accumulated sugar is converted mainly to starch. The observed changes were relatively small and could not be related to any of the components of the phosphorylase system, which was shown to be complex.     

Isolation of Amylopectin Granules and Identification of Amylopectin Phosphorylase in the Oocysts of Eimeria tenella

SYNOPSIS. Amylopectin granules were purified from Eimeria tenella oocysts following digestion with sodium dodecyl sulfate and pronase. The oval granules had a uniform size of 0.5 × 0.7 μm, and consisted of only glucose polymers. α-Amylase treatment yielded 235 nmoles of maltose from the granules from 10⁶ unsporulated oocysts and 93 nmoles maltose from those from 10⁶ sporulated oocysts.
Amylopectin phosphorylase activity was detected in the cytoplasm of unsporulated oocysts of E. tenella. It had a specific activity of 13 U/mg protein in crude extracts, and a pH optimum of 6.0. The K m values determined were 9.1 mM for glucose-1-phosphate and 5.6 mM for glucose end groups in potato amylopectin. Enzyme activity declined at a linear rate during sporulation, sporulated oocysts containing less than 8% of the activity of unsporulated oocysts. No amylase-type activity was found in the parasite.     

Neurotrophic Effects of Extracellular Guanosine

Central nervous system (CNS) astrocytes release guanosine extracellularly, that exerts trophic effects. In CNS, extracellular guanosine (GUO) stimulates mitosis, synthesis of trophic factors, and cell differentiation, including neuritogenesis, is neuroprotective, and reduces apoptosis due to several stimuli. Specific receptor-like binding sites for eGUO in the nervous system may mediate its effects through both MAP kinase and PI3-kinase signalling pathways. Extracellular guanine (eGUA) also exerts several effects; the trophic effects of eGUO are likely regulated by conversion of eGUO to eGUA by a membrane located purine nucleoside phosphorylase (ecto-PNP) and by conversion of eGUA to xanthine by guanine deaminase.     

Quality evaluation of Saudi honey harvested from the Asir province by using high-performance liquid chromatography (HPLC)

Sugar profile and hydroxymethylfurfural (HMF) of Saudi honey were examined through high-performance liquid chromatography (HPLC) system equipped with refractive index and diode array detectors. The work was designed to assess the quality of various types of blossom honey i.e. Sider (Ziziphus spina-christi), Dhuhyana (Acacia asak), Sumra (Acacia tortilis), Qatada (Acacia hamulosa), Dhurum (Lavandula dentata), multiflora with majra (Hypoestes forskaolii), multiflora with herbs, Keena (Eucalyptus spp.) produced in the southwestern areas of the kingdom. Hierarchical cluster analysis (HCA), principal cluster analysis (PCA), and similarity and difference indices (SDI) were also applied to examine the possible grouping based on the studied quality parameters. Four main sugars (two monosaccharides i.e. fructose and glucose, two disaccharides i.e. sucrose and maltose) and HMF were investigated . The average values of fructose and glucose were in the range 33.10%–44.77% and 26.68%–37.91%, respectively. The maltose was present in all types of honey and its mean values were in the range of 0.37%–2.97%, while sucrose was absent in six types of honey, 0.25% in one unifloral honey, and 3.25% in one multi-floral honey. HMF was not detected in seven types of honey but was below the limit of quantification (0.13 mg/kg) in one type of honey. PCA displayed the accumulative variance of 79.96% for the initial two PCs suggesting that honey samples were not well distinguished by their sugar profile. Based on the sucrose and HMF contents, it was concluded that all types of blossom honey from the Asir province were of the best quality in the kingdom and met the international quality parameters.     

蔗糖磷酸化酶的研究进展

蔗糖磷酸化酶属于糖苷水解酶13家族,能够催化蔗糖的可逆磷酸解。利用其广泛的底物混杂性,蔗糖磷酸化酶可以将葡萄糖基转移至不同的受体合成熊果苷、甘油葡萄糖苷、低聚糖及多酚化合物的衍生物等产物,这些催化产物可广泛应用于食品、药品、化妆品等行业。随着酶催化技术和蛋白质工程的发展,蔗糖磷酸化酶受到了越来越多的关注,该酶的应用范围也得到了扩大。本文综述了近年来蔗糖磷酸化酶在酶的来源、结构、功能及应用领域等的研究进展,同时讨论了该酶的蛋白质工程改造方法与局限性,并展望了该酶可能的研究方向。     

Enzymatic synthesis of amylose

Amylose is a linear polymer of α-1,4-linked glucose and is expected to be used in various industries as a functional biomaterial. However, pure amylose is currently not available for industrial purposes, since the separation of natural amylose from amylopectin is difficult. It is known that amylose has been synthesized using various enzymes. Glucan phosphorylase, together with its substrate, glucose-1-phosphate, is the most suitable system for the production of amylose since the molecular size of amylose can be controlled precisely. However, the problem with this system is that glucose-1-phosphate is too expensive for industrial purposes. This review summarizes our work on the enzymatic synthesis of essentially linear amylose, together with recent progress in the production of synthetic amylose using sucrose or cellobiose through the combined actions of phosphorylases.