Protein Phosphorylase Research Articles

Cell models for McArdle disease and aminoglycoside-induced read-through of a premature termination codon

McArdle disease results from mutations in the gene encoding muscle glycogen phosphorylase (PYGM) protein and the two most common mutations are a premature termination codon (R50X) and a missense mutation (G205S). Myoblasts from patients cannot be used to create a cell model of McArdle disease because even normal myoblasts produce little or no PYGM protein in cell culture. We therefore created cell models by expressing wild-type or mutant (R50X or G205S) PYGM from cDNA integrated into the genome of Chinese hamster ovary cells. These cell lines enable the study of McArdle mutations in the absence of nonsense-mediated decay of mRNA transcripts. Although all cell lines produced stable mRNA, only wild-type produced detectable PYGM protein. Our data suggest that the G205S mutation affects PYGM by causing misfolding and accelerated protein turnover. Using the N-terminal region of PYGM containing the R50X mutation fused to green fluorescent protein, we were able to demonstrate both small amounts of truncated protein production and read-through of the R50X premature termination codon induced by the aminoglycoside, G418.

Characterization of human melanoma cell lines and melanocytes by proteome analysis

We have analyzed the proteomes of two human melanoma cell lines (A375 and 526), and of the human melanocytes, (FOM 78), by two-dimensional electrophoresis (2D-PAGE) and liquid chromatography – tandem mass spectrometry (LC-MS/MS). Our comparative proteomic analysis revealed that six proteins were over-expressed in both melanoma cell lines as compared to melanocytes: galectin-1, inosine-5'-monophosphate dehydrogenase 2, serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform, protein DJ-1, cyclophilin A and cofilin-1. We show, for the first time, that only specific isoforms of these molecules are over-expressed in melanoma. Different protein profiles were also found between each individual melanoma cell line and the melanocytes. s-Methyl-5-thioadenosine phosphorylase, ubiquitin and ribosomal protein S27 a precursor, the basic form of protein DJ-1, annexin a1, proliferation associated protein 2g4, isoform alfa-enolase of alfa-enolase, protein disulfide-isomerase precursor, and elongation factor 2 were more strongly expressed in A375 cells compared to melanocytes. In 526 cells, 60s acidic ribosomal protein p1 and calreticulin precursor were more highly expressed than in melanocytes. These molecular differences may help in better understanding melanoma development and its different responsiveness to therapies. The identified proteins could be exploited as biomarkers or therapeutic targets for melanoma.

Abstract 3519: Histone deacetylase inhibitors upregulates thymidine phosphorylase gene and protein expression and synergize with capecitabine in breast cancer cells

Abstract Capecitabine is a pro-drug which was designed to take advantage of the increased levels of thymidine phosphorylase (TP), a key enzyme for its conversion to 5-florouracil (5-FU), observed in tumours as opposed to normal tissues, potentially allowing for selective toxicity. Capecitabine is a valuable substitute for bolus or infusion 5-FU either as monotherapy or in combination with other cytotoxic drugs in the treatment of several tumor types including breast cancer. We have recently demonstrated for the first time, that the histone deacetylase-inhibitor (HDAC-I) vorinostat induces synergistic antitumour effects in combination with capecitabine by up-regulating, in vitro and in vivo, in colorectal cancer cells but not in ex vivo treated peripheral blood lymphocytes, the mRNA and protein expression of TP. We have also demonstrated that TP knockdown by a specific siRNA significantly impairs the synergistic apoptotic cell death induced by vorinostat/capecitabine combination. In this study we demonstrated a time and dose-dependent induction of TP mRNA and protein expression by several HDAC-Is in different breast cancer cell lines such MCF-7, SKBR3, MDA-MB-231and MDA-MB-468 cells. Interestingly, this effect was shared by pan-HDAC-Is such as panobinostat (LBH589), trichostatin A (TSA) or vorinostat and by class I HDAC-Is such as valproic acid (VPA) and entinostat (MS275), but not by the specific inhibitor of HDAC-6 tubacin, indicating that targeting class I HDACs is crucial for TP induction. Moreover, the mechanism of TP induction is dependent on a transcriptional effect since preliminary data showed that HDAC-Is did not interfere with TP protein stability. On these bases we investigated potential antitumor interaction between capecitabine and either vorinostat or panobinostat or VPA, demonstrating synergistic antiproliferative effects in all breast cancer cells independently of p53, ER or Her2 status. Overall, this study suggests that HDAC-Is, by upregulating TP expression, may improve the therapeutic index of capecitabine representing an innovative antitumour strategy for the treatment of breast cancer that warrants further clinical evaluation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3519. doi:10.1158/1538-7445.AM2011-3519

A novel nonstop mutation in TYMP does not induce nonstop mRNA decay in a MNGIE patient with severe neuropathy

The cellular quality control systems enable surveillance and selective degradation of nonsense, nonstop, and no-go mRNAs. In the case of nonstop mRNA, different mechanisms of nonstop-mediated decay (NSD) have been described for bacteria, yeast and mammals, but the molecular consequences of nonstop mutations have been examined in only few cases of human disease. We describe a novel homozygous nonstop mRNA mutation (c.1416delC) in the TYMP gene encoding thymidine phosphorylase, in a patient with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In contrast to previous reports showing selective decay of pathogenic nonstop mRNAs, quantitative real-time PCR and 3'-RACE-RFLP analysis revealed unreduced nonstop mRNA levels in our patient and 2 heterozygous carriers of the mutation. The absence of thymidine phosphorylase protein in the homozygous patient, together with the partial decrease in levels of this protein in 2 carriers suggest that the main control system in this case resides at the translational or post-translational levels rather than through NSD. This is the first report showing an absence of NSD in a human disease, revealing that this surveillance mechanism has exceptions in vivo.

The cytosol activity of thymidine phosphorylase in endometrial cancer.

BackgroundThymidine phosphorylase (TP) is identical with platelet-derived endothelial cell growth factor (PD-ECGF) which promotes angiogenesis. The aim of this study was to evaluate the cytosol activity of TP in tumor samples from patients with endometrial cancer.MethodsThe activity of TP was measured by the spectrophotometric method in the cytosol of endometrial tumor samples from 43 patients. Moreover, the expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) protein and microvessel density (MD) were examined in the same endometrial tumor samples by immunohistochemical staining. Normal endometrium from 16 women, treated surgically due to nononcological reasons served as a control.A relationship between the cytosol TP activity, PD-ECGF/TP protein expression, MD and clinicopathologic features was investigated.ResultsA significantly higher the cytosol TP activity, PD-ECGF/TP protein expression and MD was stated in malignant tumor samples when compared to the control (samples of normal endometrium). A positive statistically significant correlation between the cytosol enzyme activity and PD-ECGF/TP protein expression and MD was found, but weaker from the remaining ones between PD-ECGF/TP protein expression and MD was observed.Besides no correlation between the cytosol TP activity, PD-ECGF/TP protein expression as well as MD and grading or histopatological type of endometrial cancer was stated.ConclusionThe cytosol TP activity in endometrial cancer is significantly higher than in normal endometrium, with no relation as to the stage and grade of tumors, but correlates with the PD-ECGF/TP protein expression and MD may therefore be associated with favorable prognosis in patients treated with chemo- or radiotherapy after surgery.

Prognostic significance of thymidylate synthase, dihydropyrimidine dehydrogenase and thymidine phosphorylase protein expression in colorectal cancer patients treated with or without 5-fluorouracil-based chemotherapy

BackgroundLow tumour expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and thymidine phosphorylase (TP) have been linked with improved outcome for colorectal cancer (CRC) patients treated with 5-fluorouracil (5-FU). It is unclear whether this occurs because such tumours have better prognosis or they are more sensitive to 5-FU treatment. Patients and methodsAssociations between TS, DPD and TP levels, determined by tissue microarrays and immunohistochemistry, and survival was evaluated in 945 CRC patients according to treatment status. ResultsLow TS and DPD expression associated with worse prognosis in stage II [hazard ratio (HR)=1.69, 95% confidence interval (CI) (1.09–2.63) and HR=1.92 (95% CI 1.23–2.94), respectively] and stage III CRC patients treated by surgery alone [HR=1.39 (95% CI 0.92–2.13) and HR=1.49 (95% CI 1.02–2.17), respectively]. Low TS, DPD and TP associated with trends for better outcome in stage III patients treated with 5-FU [HR=0.81 (95% CI 0.49–1.33), HR=0.70 (95% CI 0.42–1.15) and HR=0.66 (95% CI 0.39–1.12), respectively]. ConclusionLow TS and DPD expression are prognostic for worse outcome in CRC patients treated by surgery alone, whereas low TS, DPD and TP expression are prognostic for better outcome in patients treated with 5-FU chemotherapy. These results provide indirect evidence that low TS, DPD and TP protein expression are predictive of good response to 5-FU chemotherapy.

Glycogen phosphorylase in glycogen-rich cells is involved in the energy supply for ion regulation in fish gill epithelia

The molecular and cellular mechanisms behind glycogen metabolism and the energy metabolite translocation between mammal neurons and astrocytes have been well studied. A similar mechanism is proposed for rapid mobilization of local energy stores to support energy-dependent transepithelial ion transport in gills of the Mozambique tilapia (Oreochromis mossambicus). A novel gill glycogen phosphorylase isoform (tGPGG), which catalyzes the initial degradation of glycogen, was identified in branchial epithelial cells of O. mossambicus. Double in situ hybridization and immunocytochemistry demonstrated that tGPGG mRNA and glycogen were colocalized in glycogen-rich cells (GRCs), which surround ionocytes (labeled with a Na(+)-K(+)-ATPase antiserum) in gill epithelia. Concanavalin-A (a marker for the apical membrane) labeling indicated that GRCs and mitochondria-rich cells share the same apical opening. Quantitative real-time PCR analyses showed that tGPGG mRNA expression levels specifically responded to environmental salinity changes. Indeed, the glycogen content, glycogen phosphorylase (GP) protein level and total activity, and the density of tGPGG-expressing cells (i.e., GRCs) in fish acclimated to seawater (SW) were significantly higher than those in freshwater controls. Short-term acclimation to SW caused an evident depletion in the glycogen content of GRCs. Taken altogether, tGPGG expression in GRCs is stimulated by hyperosmotic challenge, and this may catalyze initial glycogen degradation to provide the adjacent ionocytes with energy to carry out iono- and osmoregulatory functions.

High Levels of Thymidine Phosphorylase as an Independent Prognostic Factor in Renal Cell Carcinoma

We investigated whether thymidine phosphorylase (TP) protein level in renal cell carcinoma (RCC) correlates with clinicopathological characteristics and clinical outcomes. TP protein level was measured in 116 RCC specimens and in 90 non-neoplastic kidney tissues using a sandwich-type enzyme-linked immunosolvent assay. The median TP protein level in RCC tissues was 9.76-fold (range, 3.2-933.9) higher than those in non-neoplastic kidney tissues (P < 0.0001). TP protein level was correlated with T classification, histological grade and mode of infiltration. TP as a prognostic variable was studied using a logistic regression model. TP at higher levels (128 U/mg protein or greater) would play a role as an independent prognostic factor (odds ratio, 13.73; 95% confidence interval, 2.09-90.41; P = 0.0064). TP at high levels can be regarded as an unfavorable independent prognostic factor. These results may pave a way for a novel approach to effective treatment of RCC.

Human Polynucleotide Phosphorylase Protein in Response to Oxidative Stress

8-Oxo-7,8-dihydroguanine (8-oxoGua) is generated in nucleic acids as well as in their precursors due to the actions of oxygen radicals produced through a normal cellular metabolism. Since oxidized guanine can pair with both cytosine and adenine, it causes alterations in the phenotypic expression when it is present in RNA. To prevent such an outcome, organisms must have some mechanism for eliminating such oxidized guanine nucleotides from RNA and its precursors. In mammalian cells, MTH1 and NUDT5 proteins degrade 8-oxoGTP and 8-oxoGDP to 8-oxoGMP, which is an unusable form for RNA synthesis. In a search for proteins functioning at the RNA level, polynucleotide phosphorylase (PNP) protein has been suggested to be a good candidate for such a role. The human PNP protein has an ability to bind specifically to RNA containing 8-oxoGua. When human cells are exposed to agents that induce oxidative stress, such as hydrogen peroxide and menadion, the amounts of PNP protein decrease rapidly while amounts of other proteins in the cells do not change after such treatments. No specific decrease in the PNP protein level is observed when cells are treated with ACNU and cycloheximide at doses sufficient to provide the same degree of growth suppression. These results imply that the PNP protein might thus play a role in excluding oxidized forms of RNA from the translation mechanism.

Tissue Microarray Analysis of Methylthioadenosine Phosphorylase Protein Expression in Melanocytic Skin Tumors

Using tissue microarrays, we investigated whether methylthioadenosine phosphorylase (MTAP) protein expression is associated with clinicopathologic variables in benign and malignant melanocytic skin tumors. Cytoplasmic MTAP expression was detected in 227 (72.1%) of 315 informative cases. Expression was significantly reduced in primary malignant melanomas and in melanoma metastases compared with benign nevi (P<.001 for both). No difference was noted in MTAP expression between primary malignant melanomas and melanoma metastases. In primary malignant melanomas, a Ki67-labeling index less than 5% was associated with MTAP expression (P = .04), suggesting that loss of MTAP expression is associated with proliferation. No other variables had significant associations with MTAP expression. Lymph node metastases demonstrated significantly higher MTAP expression compared with skin metastases (P = .01). In the overall cohort, MTAP expression was not associated with prognosis. Among 26 patients with MTAP-positive melanomas and tumor recurrence, 18 patients who received interferon therapy had a significant benefit compared with 8 patients who did not receive interferon therapy (P = .009). This was not seen in the patients with MTAP-negative tumors. Conclusion Methylthioadenosine phosphorylase protein expression may be a predictive marker of interferon therapy resistance in patients with melanoma and disease progression.

Glucose 6-Phosphate Produced by Gluconeogenesis and by Glucokinase Is Equally Effective in Activating Hepatic Glycogen Synthase

Glucose 6-phosphate (Glc-6-P) produced in cultured hepatocytes by direct phosphorylation of glucose or by gluconeogenesis from dihydroxyacetone (DHA) was equally effective in activating glycogen synthase (GS). However, glycogen accumulation was higher in hepatocytes incubated with glucose than in those treated with DHA. This difference was attributed to decreased futile cycling through GS and glycogen phosphorylase (GP) in the glucose-treated hepatocytes, owing to the partial inactivation of GP induced by glucose. Our results indicate that the gluconeogenic pathway and the glucokinase-mediated phosphorylation of glucose deliver their common product to the same Glc-6-P pool, which is accessible to liver GS. As observed in the treatment with glucose, incubation of cultured hepatocytes with DHA caused the translocation of GS from a uniform cytoplasmic distribution to the hepatocyte periphery and a similar pattern of glycogen deposition. We hypothesize that Glc-6-P has a major role in glycogen metabolism not only by determining the activation state of GS but also by controlling its subcellular distribution in the hepatocyte.

Specific binding of 8-oxoguanine-containing RNA to polynucleotide phosphorylase protein.

8-Oxoguanine, an oxidized form of guanine, has the potential to pair with both cytosine and adenine, and thus, the persistence of this base in messenger RNA would cause translational errors. To prevent such an outcome, organisms probably have a mechanism for recognizing RNA molecules carrying 8-oxoguanine and prevent them from entering into the cellular translational machinery. We now report that the Escherichia coli cell possesses proteins that bind specifically to RNA carrying 8-oxoguanine. On incubation with a cell-free extract, 8-oxoguanine-containing RNA is stable while normal RNA is degraded by cellular nucleases. The RNase protection assay and gel shift assay revealed that some proteins bind specifically to 8-oxoguanine-containing RNA, hence preventing nuclease attacks. Among the complexes that were detected, one with a 77 kDa protein exhibits tight binding between RNA and protein components. This protein was identified as polynucleotide phosphorylase, encoded by the pnp gene. pnp(-)() mutants are hyperresistant to paraquat, a drug that induces oxidative stress in the cell. Binding of Pnp protein to 8-oxoguanine-containing RNA would inhibit cell growth, probably due to withdrawal of such RNA from the translational machinery. The Pnp protein may, therefore, discriminate between an oxidized RNA molecule and a normal one, thus contributing a high fidelity of translation.

Insulin Replacement Therapy in Diabetic Rats Using an Osmotic Pump Normalizes Expression of Enzymes Key to Hepatic Carbohydrate Metabolism

Intensively treating type I diabetics with continuous subcutaneous insulin infusions or multiple daily insulin injections to normalize mean blood glucose concentrations significantly reduces the onset of secondary diabetic complications when compared to conventionally treated diabetics. Our studies focused on characterizing hepatic enzyme expression in intensively and conventionally treated diabetic rats. Alloxan-induced diabetic rats were conventionally treated with insulin injected twice daily or intensively treated with similar daily dosages of insulin administered via a surgically implanted osmotic pump. Our results demonstrate a significant difference in hepatic enzyme expression when these treatment regimes are compared. In conventionally treated diabetic rats, phosphoenolpyruvate carboxykinase (PEPCK) protein and mRNA levels remained slightly elevated when compared to normal animals, glycogen phosphorylase (GP) protein levels were still slightly decreased, and glycogen synthase (GS) protein and mRNA levels remained at the elevated levels observed in untreated diabetics. In contrast, the protein and mRNA levels of all three enzymes were normalized in the insulin pump-treated animals. These results suggest that intensive insulin therapy improves glycemia directly by normalizing hepatic gene expression while conventional insulin therapy normalizes plasma glucose concentrations indirectly.

Structural and Functional Characterization of theDrosophilaGlycogen Phosphorylase Gene

We identified a P element insertional mutant of theDrosophilaglycogen phosphorylase (DGPH) gene. Glycogen phosphorylase protein concentration and enzyme activity are decreased while glycogen content is increased in flies homozygous for the mutant allele. TheDGPHgene has been cloned and sequenced; its open reading frame codes for a protein of 844 amino acids with a predicted molecular mass of 97 kDa. Comparison of the conceptual amino acid sequence of theDrosophilaglycogen phosphorylase with glycogen phosphorylase sequences from other organisms shows a high degree of homology to mammalian enzymes. All the residues of the allosteric effector binding sites, the active site, and the site of phosphorylation are exactly conserved, but some of the residues of the glycogen storage site are not.

Role of E and CArG boxes in developmental regulation of muscle glycogen phosphorylase promoter during myogenesis.

Muscle glycogen phosphorylase (MGP) transcript and protein levels increase during skeletal muscle development in tandem with the products of other muscle genes responsible for glucose and glycogen metabolism. Previous studies demonstrated that a 269 bp region 5' to exon 1 of MGP is sufficient for developmental regulation in the C2C12 myogenic cell line (Froman et al., 1994). This genomic region (-209 to +60) contains four consensus E box motifs, a CArG-like sequence, and a GC-rich domain. Native MGP transcripts were not detected in pluripotent CH310T1/2 fibroblasts, but low levels of MGP mRNA were measured in CH310T1/2 cells that were stably transfected with MyoD. Three of the E box motifs in the MGP proximal promoter interacted with C2C12 nuclear proteins. However, cotransfection of the MGP promoter with myogenic regulatory factors, including MyoD and myogenin, produced less than 2-fold activation compared with 20-fold activation of the desmin promoter. Mutational analyses of the MGP promoter demonstrated that increased expression in C2C12 myotubes did not require any of the E box motifs or the CArG-like element. A small region (-76 to -68) upstream of GC-rich domain (-64 to -51) significantly reduced promoter activities in both myoblasts and myotubes. The functional studies suggest that MGP is developmentally regulated during myogenesis by alternative pathways that utilize unidentified regulatory elements or ancillary factors.

Co-determination of the angiogenic factors thymidine phosphorylase and vascular endothelial growth factor in node-negative breast cancer: prognostic implications.

Experimental and clinical studies have shown that human breast cancer is an angiogenesis-dependent neoplasm. In fact, several authors have demonstrated that the determination in primary tumors of the degree of vascularization (microvessel counts) as well as of some angiogenic peptides is of prognostic value. However, which are the most important mediators of angiogenesis and their relationship with other relevant biological markers needs further investigation. In the series of 260 women with node-negative breast cancer (NNBC) on which we previously assessed vascular endothelial growth factor (VEGF), we have now also determined thymidine phosphorylase (TP) protein as well as p53 protein and Cathepsin-D cytosolic levels using immunometric methods. The median concentrations of TP, p53 and Cathepsin-D were 105.4U/mg (range 1.2-843.1), 0.22 ng/mg (range 0.0-41.65) and 33.80nmol/mg (range 4.20-216.0), respectively. We found that TP concentrations were associated with Cathepsin-D and p53, but not with VEGF. VEGF (p<0.0001) and p53 (p = 0.03 and p = 0.012, respectively) were found to be statistically significant prognostic variables for both relapse-free survival (RFS) and overall survival in univariate analysis. Conversely, TP and Cathepsin-D levels did not correlate with prognosis. In multivariate analysis for RFS, VEGF levels (p<0.0001), TP levels (p = 0.050) and their first-order interaction terms (p = 0.027) were statistically significant prognostic indicators. Cathepsin-D and p53 protein levels did not retain significance in the model inclusive of all the above variables. The predictive capability of the complete model was satisfactory (Harrell c statistic = 0.72). Moreover, these results suggest a possible potentiation of the capability of predicting the likelihood of recurrence by the co-determination of TP and VEGF. The probability of recurrence was particularly high in the patients with primary tumors characterized by elevated levels of both angiogenic factors. This is the first study showing in vivo that two different angiogenic peptides concur in the progression of human breast cancer. The biology and possible therapeutic implications of this observation are discussed.

The Effects of Streptozotocin-induced Diabetes and Insulin Supplementation on Expression of the Glycogen Phosphorylase Gene in Rat Liver

We have previously observed that the chronic effects of streptozotocin-induced diabetes cause a decrease in the total hepatic glycogen phosphorylase activity with a corresponding reduction in the phosphorylase protein levels. These effects were normalized by insulin administration to diabetic rats. There was no change in the total glycogen synthase activity as a result of diabetes or insulin supplementation. These results are extended to examine the effects of diabetes and insulin administration to diabetic animals on the expression of phosphorylase and glycogen synthase enzymes. The expression (i.e. mRNA levels) of phosphorylase was down-regulated (45% of normal levels) in diabetic livers, and this was normalized by insulin supplementation to diabetic animals. Diabetes or insulin supplementation to diabetic rats showed no effect on the transcription rate of phosphorylase. As expected, diabetes (or insulin administration to diabetic animals) did not cause any alteration in the mRNA levels or in the transcription rate of hepatic glycogen synthase. The stability of phosphorylase mRNA was then examined using hepatocytes prepared from normal and diabetic rats. Diabetes caused a decrease in the half-life of phosphorylase mRNA from 14 h in normal hepatocytes to 6.5 h in diabetic hepatocytes. Insulin supplementation to the medium of diabetic hepatocytes increased the half-life of phosphorylase mRNA to a level comparable with normal values. This study indicates that the chronic effect of insulin on the activation of the total hepatic phosphorylase activity (and protein) is mediated through the stabilization of its mRNA levels.

Induction of the plastidic starch-phosphorylase gene in potato storage sink tissue

The expression of the gene encoding the plastidic enzyme starch phosphorylase (EC 2.4.1.1) varies according to tissue carbohydrate status. Incubation of excised potato (Solanum tuberosum L. cv. Desiree) leaves carrying a portion of the stem under a short photoperiod resulted in a drastic accumulation of starch, accompanied by a rapid increase in the level of phosphorylase mRNA and by a similar change in phosphorylase protein level. However, under the same incubation conditions, the transcriptional activity of the phosphorylase promoter in transgenic plants did not change markedly. Therefore, the increased expression of the phosphorylase gene in petioles of stem cuttings is not controlled by the level of initiation of transcription. Phosphorylase mRNA accumulated to a high level in petioles of detached leaves kept under constant light for 24 h, but not in petioles kept in the dark. The effect of light on the accumulation of the mRNA was appreciably reduced if the petioles were incubated in ethylendiaminetetraacetic acid (EDTA), a treatment known to increase phloem exudation in detached leaves. The inhibition by EDTA could be partially counteracted by the addition of sucrose to the incubation solution. Furthermore, incubation of petioles in darkness in solutions with high levels of sucrose resulted in enhanced expression of the gene. These results suggest that sucrose, the main compound transported by phloem in potato, is involved in the regulation of the starch phosphorylase gene. This also indicates that conditions favouring starch synthesis lead to increased expression of the phosphorylase gene.

Amylolytic Activity in Germinating Sweetpotato (Ipomoea batatas L.) Roots

In vitro activity measurements indicate that storage sweetpotato roots contain high amounts of extractable amylolytic enzymes. These storage roots also have a very high starch content, a characteristic indicating that the in vitro measurements estimate potential amylolytic activity rather than actual physiological activity. We are interested in optimizing the use of endogenous amylases when processing sweetpotato roots and have undertaken a study to identify physiological parameters that control in vivo starch breakdown. Sweetpotato roots were allowed to germinate for 35 days in controlled conditions. Using a combination of in vitro activity measurements and immunochemical detection, the spatial distribution and changes in activity levels for the three major amylolytic enzymes in storage sweetpotato roots—α-amylase, β-amylase, and starch phosphorylase—have been followed. After 6 days, α-amylase protein increased in the outer starchy parenchymatous tissues surrounding the cambium layers, a result suggesting a de novo synthesis of the enzyme in cambium or laticifers layers. β-Amylase was abundant throughout the root at all times, and its high levels did not directly affect starch degradation rates. Starch phosphorylase protein level remained constant, while its extractable activity increased. Starch content decreased during sweetpotato seed root germination. However, the amount of starch that disappeared during germination was low compared with the calculated starch hydrolysis potential estimated by amylolytic activity measurements.

Identification of an S100 target protein: glycogen phosphorylase

An S100 binding protein from skeletal muscle, R95 000, has been purified, identified as glycogen phosphorylase, and shown to be regulated in vitro by the S100α isoform. When a soluble skeletal muscle fraction was subjected to a standard purification procedure for glycogen phosphorylase, R95 000 copurified with the 95 000 molecular weight glycogen phosphorylase protein standard on SDS-polyacrylamide gels, as well as having glycogen phosphorylase activity. In addition, purified glycogen phosphorylase a and b interacted with both S100 isoforms, S100α and S100β, by gel overlay and affinity chromatography. While S100β had no effect on the enzymatic activity of glycogen phosphorylase a, S100α inhibited the enzymatic activity of glycogen phosphorylase a in a calcium-independent manner. Altogether, these data suggest that glycogen phosphorylase may be an intracellular S100α target in skeletal muscle fibers. Furthermore, these results suggest that the inhibition of glycogen phosphorylase a activity may be responsible for the lack of fatigability of slow-twitch fibers, which express S100α, when compared to fast-twitch fibers, which do not express S100 proteins.