Analytical polyphosphate extraction from Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, inorganic polyphosphate is analyzed by polyphosphate extraction and subsequent quantification. Recently, we developed a method for polyphosphate quantification, and length determination of short chain polyphosphate. However, the lack of a simple, optimized and validated method for analytical polyphosphate extraction has both hindered the advance in this research field, and prevented comparability of results between laboratories. Hence, the goal of this study was to develop an analytical method for polyphosphate extraction from S. cerevisiae. Several literature methods were compared with special attention to omission of polyphosphate precipitation steps, because these work neither at low polyphosphate concentrations nor quantitatively. The best literature protocol, which takes 5.5 h and requires five reaction tubes per sample, was optimized here in regards to the amount of extracted polyphosphate and simplification of the work flow. The final protocol extracts 40 % more polyphosphate than the best literature method, takes only 30 min, requires just one reaction tube per sample, and is, therefore, proposed as the new gold standard for analytical polyphosphate extraction from S. cerevisiae. In combination with our recently published polyphosphate quantification method, total polyphosphate in S. cerevisiae can now be analyzed within 2 h.

A continuous enzyme-coupled assay for triphosphohydrolase activity of HIV-1 restriction factor SAMHD1.

The development of deoxynucleoside triphosphate (dNTP)-based drugs requires a quantitative understanding of any inhibition, activation, or hydrolysis by off-target cellular enzymes. SAMHD1 is a regulatory dNTP-triphosphohydrolase that inhibits HIV-1 replication in human myeloid cells. We describe here an enzyme-coupled assay for quantifying the activation, inhibition, and hydrolysis of dNTPs, nucleotide analogues, and nucleotide analogue inhibitors by triphosphohydrolase enzymes. The assay facilitates mechanistic studies of triphosphohydrolase enzymes and the quantification of off-target effects of nucleotide-based antiviral and chemotherapeutic agents.

Expression of FHIT, p16, p53 and EGFR as prognostic markers in thyroid tumors of uncertain malignant potential.

PURPOSE: Thyroid tumors of uncertain malignant potential (TT-UMP) constitute a relatively new diagnosis. The purpose of this study was to analyze the relationship between immunohistochemical panels, prognostic parameters and TT-UMP. METHODS: Group I was composed of patients diagnosed as differentiated thyroid carcinoma (DTC) and Group II of patients diagnosed as TT-UMP. The prognostic scores of patients were calculated using data according to the well-known prognostic scoring systems MACIS, AMES, AGES. Evaluations of antibodies were based on the presence of nuclear staining for p16 and p53, membranous and cytoplasmic staining for epidermal growth factor receptor (EGFR) and cytoplasmic staining for fragile histidine triad (FHIT). RESULTS: Statistically significant difference was noted (p< 0.05) between Group I and Group II according to MACIS and AMES. No statistical difference was found in terms of immunostaining between groups when stained with p16, p53 and FHIT. On the other hand, in Group II a moderate positive correlation was detected between MACIS and EGFR. CONCLUSION: According to our findings p53 was not important in tumor genesis at early stages in well-differentiated thyroid carcinomas and p16 loss of expression could be used as a finding to help in difficult microscopic diagnosis. TT-UMP is a gray zone of lesions requiring specific therapeutic procedures and postoperative follow-up. A positive correlation was detected between EGFR and TT-UMP, leading to assume that this situation could be used as a new tool in the follow-up of these patients in the future.

Multilevel correlations in the biological phosphorus removal process: From bacterial enrichment to conductivity-based metabolic batch tests and polyphosphatase assays.

Enhanced biological phosphorus removal (EBPR) from wastewater relies on the preferential selection of active polyphosphate-accumulating organisms (PAO) in the underlying bacterial community continuum. Efficient management of the bacterial resource requires understanding of population dynamics as well as availability of bioanalytical methods for rapid and regular assessment of relative abundances of active PAOs and their glycogen-accumulating competitors (GAO). A systems approach was adopted here toward the investigation of multilevel correlations from the EBPR bioprocess to the bacterial community, metabolic, and enzymatic levels. Two anaerobic-aerobic sequencing-batch reactors were operated to enrich activated sludge in PAOs and GAOs affiliating with "Candidati Accumulibacter and Competibacter phosphates", respectively. Bacterial selection was optimized by dynamic control of the organic loading rate and the anaerobic contact time. The distinct core bacteriomes mainly comprised populations related to the classes Betaproteobacteria, Cytophagia, and Chloroflexi in the PAO enrichment and of Gammaproteobacteria, Alphaproteobacteria, Acidobacteria, and Sphingobacteria in the GAO enrichment. An anaerobic metabolic batch test based on electrical conductivity evolution and a polyphosphatase enzymatic assay were developed for rapid and low-cost assessment of the active PAO fraction and dephosphatation potential of activated sludge. Linear correlations were obtained between the PAO fraction, biomass specific rate of conductivity increase under anaerobic conditions, and polyphosphate-hydrolyzing activity of PAO/GAO mixtures. The correlations between PAO/GAO ratios, metabolic activities, and conductivity profiles were confirmed by simulations with a mathematical model developed in the aqueous geochemistry software PHREEQC.

Selective monitoring of the enzymatic activity of the tumor suppressor Fhit.

Cancer is a leading cause of death worldwide. Functional inactivation of tumor suppressor proteins, mainly by mutations in the corresponding genes, is a key event in cancer development. The fragile histidine triade protein (Fhit) is a tumor suppressor that is frequently affected in different cancer types. Fhit possesses diadenosine triphosphate hydrolase activity, but although reduction of its enzymatic activity appears to be important for exerting its tumor suppressor function, the regulation of Fhit activity is poorly understood. Here, we introduce a novel fluorogenic probe that is suited to selectively analyze the enzymatic activity of Fhit in extracts derived from human cells. This novel method will allow in-depth insight into the mechanisms involved in Fhit regulation in biologically relevant setups and, thus, into its role in the development of cancer.

Intramitochondrial calcium regulation by the FHIT gene product sensitizes to apoptosis.

Despite the growing interest in the Fhit tumor suppressor protein, frequently deleted in human cancers, the mechanism of its powerful proapoptotic activity has remained elusive. We here demonstrate that Fhit sensitizes the low-affinity Ca(2+) transporters of mitochondria, enhancing Ca(2+) uptake into the organelle both in intact and in permabilized cells, and potentiating the effect of apoptotic agents. This effect can be attributed to the fraction of Fhit sorted to mitochondria, as a fully mitochondrial Fhit (a chimeric protein including a mitochondrial targeting sequence) retains the Ca(2+) signaling properties of Fhit and the proapoptotic activity of the native protein (whereas the effects on the cell cycle are lost). Thus, the partial sorting of Fhit to mitochondria allows to finely tune the sensitivity of the organelle to the highly pleiomorphic Ca(2+) signals, synergizing with apoptotic challenges. This concept, and the identification of the molecular machinery, may provide ways to act on apoptotic cell death and its derangement in cancer.

Aberrant expression of DNA damage response proteins is associated with breast cancer subtype and clinical features.

Landmark studies of the status of DNA damage checkpoints and associated repair functions in preneoplastic and neoplastic cells has focused attention on importance of these pathways in cancer development, and inhibitors of repair pathways are in clinical trials for treatment of triple negative breast cancer. Cancer heterogeneity suggests that specific cancer subtypes will have distinct mechanisms of DNA damage survival, dependent on biological context. In this study, status of DNA damage response (DDR)-associated proteins was examined in breast cancer subtypes in association with clinical features; 479 breast cancers were examined for expression of DDR proteins γH2AX, BRCA1, pChk2, and p53, DNA damage-sensitive tumor suppressors Fhit and Wwox, and Wwox-interacting proteins Ap2α, Ap2γ, ErbB4, and correlations among proteins, tumor subtypes, and clinical features were assessed. In a multivariable model, triple negative cancers showed significantly reduced Fhit and Wwox, increased p53 and Ap2γ protein expression, and were significantly more likely than other subtype tumors to exhibit aberrant expression of two or more DDR-associated proteins. Disease-free survival was associated with subtype, Fhit and membrane ErbB4 expression level and aberrant expression of multiple DDR-associated proteins. These results suggest that definition of specific DNA repair and checkpoint defects in subgroups of triple negative cancer might identify new treatment targets. Expression of Wwox and its interactor, ErbB4, was highly significantly reduced in metastatic tissues vs. matched primary tissues, suggesting that Wwox signal pathway loss contributes to lymph node metastasis, perhaps by allowing survival of tumor cells that have detached from basement membranes, as proposed for the role of Wwox in ovarian cancer spread.

Correlation between LRIG1 and LRIG2 expressions and expression of 11 tumor markers, with special reference to tumor suppressors, in CIN and normal cervical epithelium.

OBJECTIVE: Novel biological markers LRIG1 and LRIG2 have been associated with favorable as well as poor prognosis, respectively, in different cancer types, including cervical cancer. The aim of this study was to investigate possible interactions between these proteins and other tumor markers, and as diagnostic adjuncts in CIN. METHODS: Cervical biopsies from 171 women, with normal epithelium, and low-grade and high-grade CIN were stained for LRIG1 and LRIG2, and 11 additional tumor markers. The tumor markers were chosen to be relevant in cervical neoplasms. Staining was evaluated semiquantitatively. RESULTS: Expression of LRIG1 and LRIG2 was found to correlate with increasing CIN grade, as well as with expression of tumor suppressor FHIT, independent of histological grade. In addition, tumor promoter LRIG2 expression correlated negatively with expression of tumor suppressor retinoblastoma protein and positively with IL-10. The latter correlation did not however remain after adjustment for CIN grade. p53 and p16 expressions correlated positively with LRIG1 expression in univariate analyses, but significance did not hold after adjustment for CIN grade. CONCLUSION: LRIG1 and LRIG2 expressions were seen in precancerous cervical epithelium and found to increase with increasing grade. There was an association between expression of these glycoproteins and FHIT tumor suppressor protein, independently of histological grade.

High-risk HPV infection and CIN grade correlates to the expression of c-myc, CD4+, FHIT, E-cadherin, Ki-67, and p16INK4a.

OBJECTIVE: : This study aimed to investigate correlations between a panel of biomarkers/tumor markers and high-risk (HR) human papillomavirus (HPV)-positive versus HR-HPV-negative cervical lesions. MATERIALS AND METHODS: : The study included 188 women who consecutively attended a colposcopy clinic because of PAP smears suggesting cervical intraepithelial neoplasia (CIN), and 40 women with normal vaginal cytology. Tissue microarray blocks were prepared from representative cervical cone or punch biopsies. Sections were stained for 12 biological markers, previously shown to be relevant in cervical neoplasms, and expression was correlated to the presence or absence of HR-HPV in cervical lesions. RESULTS: : No correlations between expression of biomarkers and HPV status were found in normal epithelium. Expression of c-myc, CD4, Ki-67, and p16 correlated significantly to HR-HPV-infected epithelium compared with HR-HPV-negative epithelium. When adjustment was made for CIN grade, only the expression of Ki-67 correlated significantly with HPV status and CIN grade. Human papillomavirus status was stratified to normal epithelium, low-grade CIN, and high-grade CIN. Fragile histidine triad (FHIT), E-cadherin, Rb, Ki-67, and p16 expression was significantly increased in HPV-positive tissue by increasing CIN grade. No correlation to tumor marker expression was observed in the HPV-negative tissue. CONCLUSIONS: : This study described correlations, previously not investigated, between HPV status and tumor marker expression, that is, E-cadherin, Rb, and fragile histidine triad. Surprisingly, p16 was not, although Ki-67 expression was, independently correlated to HPV positivity. The results of this study suggest that p16 instead correlates independently with increasing CIN grade.

Clinicopathological significance and prognostic value of leukemia-related protein 16 expression in invasive ductal breast carcinoma.

To explore the expression of leukemia-related protein 16 (LRP16) in invasive ductal breast carcinoma and analyze its correlation with clinicopathological feature and prognosis, immunohistochemistry was performed on 100 cases of invasive ductal breast carcinoma. Medical records were reviewed and clinicopathological analysis was performed. Leukemia-related protein 16 expression was detected in 33 of 100 cases (33%) of the invasive ductal breast carcinoma. Expression of LRP16 in carcinoma was obviously higher than that in normal breast tissue. LRP16 protein expression was found in 27.6% (21/76) of carcinoma at stage I and II, and 50.0% (12/24) of carcinoma at stage III and IV. LRP16 expression was found correlative with metastasis in the axillary lymph node (P = 0.001), stage (P = 0.042), estrogen receptor (ER) expression (P = 0.001), fragile histidine triad (FHIT) expression (P = 0.015) and CD133 expression (P = 0.038), but not with grade (P = 0.543), tumor size (P = 0.263), age (P = 0.840), menopause (P = 0.701) and HER-2 gene amplification (P = 0.463). The difference of the mean disease free survival (DFS) time between cancer patients with LRP16 expression (43.7 months) and those without (77.7 months) was statistically significant (Log rank = 9.989, P = 0.002). The difference of the mean overall survival (OS) time between cancer patients with LRP16 expression (50.0 months) and those without (120.0 months) was statistically significant (Log rank = 9.977, P = 0.002). Our finding suggests that expression of LRP16 protein is correlated with the stage, metastasis, prognosis and expression of ER, progesterone receptor, Ki-67, CD133 and FHIT in invasive ductal breast carcinoma.

Clinicopathological significance of the fragile histidine triad transcription protein expression in laryngeal carcinogenesis.

The fragile histidine triad (FHIT), frequently lost in many cancers, was identified as a candidate tumor suppressor gene at chromosome 3p locus 14.2. Loss of the FHIT protein because of the alteration or loss of heterozygosity by genetic deletion occurs in a variety of epithelial tumors including head and neck cancer. However, the biological function of the FHIT protein is still unknown and its role in intrinsic cellular proliferation remains particularly controversial in preinvasive lesions and invasive tumors of the head and neck. To clarify the role of the FHIT protein in laryngeal squamous cell carcinoma (LSCC) and to examine whether the expression of FHIT could be a prognostic parameter for laryngeal carcinogenesis, we investigated the relationship between the expression of the FHIT protein, other tumor suppressor gene products (p53 and p16), the cellular proliferation marker (Ki-67) and the survival time of patients with LSCC. In our study, there were significant differences (p<0.05) in the expression of FHIT between low grade dysplasia and LSCC. Additionally, survival time analysis showed a significant correlation between the reduction of FHIT expression and the length of disease-free survival (p<0.05) in patients with T1-T2 N0 laryngeal carcinoma. However, we did not confirm a relationship between the expression of FHIT, the other tumor suppressor gene products (p53 and p16) or the cellular proliferation marker (Ki-67). In conclusion, we provided evidence that the reduction of FHIT levels may be a useful prognostic indicator for the clinical outcome of laryngeal SCC. Our findings indicated that FHIT utilizes a pathway independent of p53 and is involved in abnormal cell proliferation via the breakdown of G0-G1 arrest in the larynx and apoptosis during multistep carcinogenesis of the larynx.

Mononuclear phagocytes in the blood of turtles characterized by ultrastructural and cytochemical analyses and by phagocytic activity.

Ultrastructural and cytochemical characteristics of mononuclear phagocyte cells in turtles are not well described in the literature, especially in Phrynops hilarii. Thus, the aim of this study was to evaluate these characteristics in the mononuclear phagocyte cells and their phagocytic activity "in vitro" using the turtle P. hilarii as an experimental animal model. The six turtles used in the study were observed in two seasons, spring and summer. Results showed that mononuclear phagocytes incubated only in diluted solution or with colloidal charcoal have cytoplasm phagolysosomes. The cells incubated with colloidal charcoal and further exposed to the cytochemical reaction for acid beta-glycerophosphatase, showed cytoplasm phagolysosomes filled by charcoal particles being digested and some positively stained lysosomes. Acid beta-glycerophosphatase positive reaction was present in lysosomes and inside the phagolysosomes, while acid cytidine 5-monophosphatase staining occurred in lysosome surroundings. A positive reaction for trimetaphosphatase was also found inside phagolysosomes. In conclusion, the presence of lysosomal enzymes like trimetaphosphatase and cytidine-5'-sodium monophosphate, in the circulating blood of P. hilarii indicate that mononuclear phagocytes participate in the phagocytic process by gathering many phagocytic cells and forming multinucleated giant cells, which probably have a role in the blood "clearance" process.

Loss expression of active fragile sites genes associated with the severity of breast epithelial abnormalities.

BACKGROUND: WWOX and FHIT are two candidate tumor suppressor genes located in active fragile sites, the damage of which has been associated with the development of breast cancer. The association of the expression of these genes and the development of breast cancer has not been fully explored. We evaluated mRNA and protein expression of WWOX and FHIT in breast tissue with normal histological appearances, atypical ductal hyperplasia, ductal carcinoma in situ, and invasive cancer to see if a progressive decline in expression was present. METHODS: Reverse transcription-polymerase chain reaction and Western blotting were used to evaluate the specimens for mRNA and protein expression, including 28 specimens with normal tissue, 28 specimens with atypical ductal hyperplasia, 33 specimens with ductal carcinoma in situ, and 51 specimens with invasive ductal carcinoma. RESULTS: Compared with in situ and invasive cancer specimens, both normal and atypical hyperplasia specimens had greater rates of detectable mRNA (WWOX rate ratio = 2.95, 95% CI 1.24 - 7.08; FHIT rate ratio = 4.58, 95% CI 1.82 - 11.81) and Western blotting detectable protein (WWOX rate ratio = 4.12, 95% CI 1.63 - 10.73; FHIT rate ratio = 3.76, 95% CI 1.44 - 10.06). For both proteins, differences between normal and atypical hyperplasia specimens and between in situ and invasive carcinoma specimens were explainable by chance (P > 0.05 for each analysis). Within each histological category, differences among fractions of specimens showed that FHIT and WWOX mRNA and protein expression were explainable by chance (P > 0.05 for each analysis). CONCLUSION: Expression of FHIT and WWOX decreases along with breast tissue progress from a normal histological appearance to atypical ductal hyperplasia, in situ cancer, and the final invasive cancer.

Low expression of FHIT and PTEN correlates with malignancy of gastric carcinomas: tissue-array findings.

To clarify the roles of FHIT (fragile histidine triad) and PTEN (phosphatase and tensin homology deleted from human chromosome 10) expression in the genesis and progression of gastric cancers, we examined expression of FHIT and PTEN on tissue microarray containing gastric normal mucosa (n=49), adenoma (n=49), noncancerous mucosa adjacent to carcinoma (n=84) and carcinoma (n=249) by immunohistochemistry. Their expression was compared with clinicopathologic parameters of tumors, including expression of p53 and cysteine protease protein 32 as well as survival time of patients with carcinoma. The results showed expression of FHIT and PTEN were lower in gastric carcinoma than those in normal mucosa, noncancerous mucosa adjacent to carcinoma and adenoma of the stomach (P<0.05). FHIT and PTEN expression showed a significantly negative association with depth of invasion, lymphatic invasion, and lymph node metastasis, liver metastasis, and Union Internationale Contre le Cancer staging of gastric carcinoma (P<0.05). Intestinal-type gastric carcinomas highly expressed FHIT and PTEN protein, compared with diffuse-type ones (P<0.05). Expression of FHIT and PTEN were positively related with expression of p53 and cysteine protease protein 32 in gastric carcinoma (P<0.05), as well as favorable prognosis of the patients with the tumors (P<0.05). There was positive relationship between FHIT and PTEN expression in gastric carcinoma (P<0.05). It was suggested that down-regulated expression of FHIT and PTEN contributed to gastric carcinogenesis possibly by involving in the imbalance between apoptosis and proliferation of cells. Their altered expression underlay the molecular basis of invasion, metastasis, differentiation of gastric carcinoma.

Prevalence of fragile histidine triad expression in tumors from saudi arabia: a tissue microarray analysis.

AIM: The fragile histidine triad (FHIT) gene was discovered and proposed as a tumor suppressor gene for most human cancers. It encodes the most active common human chromosomal fragile region, FRA3B. We studied the prevalence of loss of FHIT expression in various tumors and correlated its loss with various clinicopathologic features. METHODS: To determine whether the absence of FHIT expression correlates with clinical variables such as grade, stage, and survival time, we assessed FHIT expression using immunohistochemistry. More than 1,800 tumors from more than 75 tumor categories were analyzed by immunohistochemistry in a tissue microarray format. RESULTS: Loss of FHIT expression ranged from 19% in ovarian tumors to 67% in lung cancers. Clinical and pathologic features like grade, stage, tumor size, and lymph node metastasis showed correlation with loss of FHIT expression in some tumors. No difference was seen in the survival patterns and loss of FHIT expression in any of the tumor groups studied. CONCLUSIONS: Loss of FHIT expression is an ubiquitous event in the multistep, multifactorial carcinogenesis process. FHIT may be altered at different stages in different types of cancers. Most of the tumors with a wider prevalence of loss of FHIT expression as an early event show a correlation with clinicopathologic features. However, in some of the tumors, FHIT expression is lost as a late event and is only seen in a fraction of the tumors.

Loss of Fhit expression is associated with poorer survival in gastric cancer but is not an independent prognostic marker.

Several studies have reported conflicting results regarding correlations of the loss of Fhit expression with clinicopathological parameters in gastric cancer. We investigated the immunohistochemical expression of Fhit in 362 cases of sporadic advanced gastric adenocarcinoma. The series included 64 cases with microsatellite instability associated with defective mismatch repair genes. Fhit expression resulted absent in 72% of the tumors analyzed. Absence of Fhit expression was more frequent in cases with diffuse and mixed histotype compared to the intestinal histotype (P=0.009). Absence of Fhit expression also correlated with tumor stage (P<0.001), lymph node involvement (P<0.001), presence of distant metastasis (P=0.033), and increasing histological grade (P=0.005). Retained Fhit expression also correlated with microsatellite instability as 61% of instable tumors had lost Fhit expression compared to 74% of microsatellite stable cancers (P=0.050). While loss of Fhit correlates with poorer survival in univariate analysis, it is not an independent prognostic factor in multivariate analysis and is thus not of clinical utility.

The fragile histidine triad gene: a molecular link between cigarette smoking and cervical cancer.

PURPOSE: Smoking is an epidemiologic risk factor for cervical cancer. The fragile histidine triad (FHIT) gene is a tumor suppressor gene that is altered in 80% of tobacco-associated lung cancers. We hypothesized that reduced FHIT protein expression, homozygous deletions (HD) or hemizygous deletions (HemiD) and microsatellite alterations (MA) at the FHIT/FRA3B locus occur more commonly in cervical cancers of smokers than nonsmokers. EXPERIMENTAL DESIGN: Archival tissues of 58 patients with stage IA1 to IB2 squamous cell carcinoma of the cervix were identified. FHIT protein expression was studied with immunohistochemistry. Laser capture microdissection was used to isolate tumor and normal DNA. HD/HemiD of FHIT exons 4 and 5 were analyzed by monoplex real-time PCR. MA at FHIT/FRA3B were studied with multiplex nested PCR with three fluorescently labeled microsatellite markers (D3S1300, D3S1312, and D3S1480). RESULTS: Eighteen of 26 tumors from smokers (69%) and 13 of 32 nonsmokers (41%; P < 0.05) showed loss of FHIT protein expression. Thirty-seven stage IB tumors yielded sufficient DNA for analyses. HD or HemiD of both exons tested occurred in 8 of 17 smokers (47%) and 2 of 20 nonsmokers (10%; P < 0.05). MA at more than two sites were found in 11 of 17 tumors of smokers (65%) and 6 of 20 nonsmokers (30%; P < 0.05). Mean composite genomic FHIT alteration scores were significantly higher for tumors of smokers versus nonsmokers (0.67 versus 0.40; P < 0.02). CONCLUSION: Loss of FHIT expression, HD, HemiD, and MA at the FHIT/FRA3B locus occur significantly more commonly in cervical cancers of smokers. These findings suggest that the tumor suppressor gene FHIT may represent a molecular target in cigarette smoking-associated cervical carcinogenesis.

A 46 kDa NTPase common to rat liver nuclear envelope, mitochondria, plasma membrane, and endoplasmic reticulum.

A 46 kDa ATP binding polypeptide of the nuclear envelope, virtually identical to the nuclear envelope NTPase putatively involved in mRNA efflux [6], is present in all rat liver cell membranes. Its presence in nuclear envelope is not the result of cross contamination during isolation.

Subcellular distribution of "intersecting' beta-N-acetylglucosaminyltransferase in Dictyostelium discoideum. A likely marker for the Golgi apparatus.

Glycoprotein processing in Dictyostelium discoideum is characterized by enzyme catalyzed steps not reported in other organisms. One of these is the formation of a beta 1 --> 4 linkage between GlcNAc and the mannose linked to the core mannose in the alpha 1 --> 6 position of N-glycosides. A simple and sensitive assay for this GlcNAc transferase activity, using a tri-mannose acceptor and a low concentration of UDP-GlcNAc, was developed. Homogenates of the organism were subjected to sub-cellular fractionation by centrifugation in discontinuous sucrose gradients. The specific activity was enriched 4-5-fold in a crude membrane fraction. The transferase was purified 10-12-fold in a membrane fraction that bands on top of 1.1 M sucrose. This fraction was also enriched in nucleotidyldiphosphatase. The enriched fraction was deficient in glucose-6-phosphatase, an endoplasmic reticulum marker. Approx. 80% of the transferase activity was latent, and unavailable to protease. Purified membranes were either subjected to phase separation in Triton X-114, or sodium carbonate extraction or sonication. In each case, the transferase behaved as an intrinsic membrane protein. Several secreted and lysosomal proteins are modified by the enzyme. These data support the idea that the GlcNAc transferase is present as an integral Golgi membrane protein and that at least the catalytic center of the transferase is on the lumenal side of the vesicles.

Membrane-bound and soluble polyphosphatases of mitochondria of Saccharomyces cerevisiae: identification and comparative characterization.

Isolated mitochondria of Saccharomyces cerevisiae possess polyphosphatases insensitive to a number of inhibitors of ATPase and pyrophosphatase of the same organelles and differing from the last two by neutral pH optima and molecular masses. After subfractionation of mitochondria, the polyphosphatase activity is distributed among the membrane and soluble preparations. The membrane-bound and soluble polyphosphatase activities are represented by different enzymes distinguished by molecular masses, substrate specificity, Km values, and relation to mono- and divalent cations. The membrane-bound polyphosphatases have molecular masses of 120 and 76 kDa, and the soluble one of about 36 kDa. All three enzymes appear to have a monomeric structure. The soluble polyphosphatase activity is stimulated by divalent cations in contrast to the membrane-bound one which is inhibited by the same cations, including Mg2+. Monovalent cations do not actually change the activity of the soluble enzyme, but stimulate it in the membrane preparation. Specific activities for the hydrolysis of polyphosphates with average chain lengths of 9-188 phosphate residues increase under increasing degree of substrate polymerization in the membrane preparation and are actually unchanged in the soluble one. The affinity of the soluble enzyme to polyphosphates is 5-10 times higher than that of the membrane-bound polyphosphatases.