Rise In Specific Activity Research Articles

High specific activity is not optimal: 18 F-fluoroestradio positron emission tomography-computed tomography results in a breast cancer xenograft.

The purpose of the preliminary study was to investigate whether high specific activity (SA) of 18 F-fluoroestradiol was optimal in breast cancer diagnosis. Imaging at variable SA was conducted in a ZR-75-1 xenograft model of estrogen-receptor positive human breast cancer in 6 mice. The region of interest was manually drawn, and the percent of injected dose per gram of the tumor and muscle in the regions of interest were recorded. Tumor-to-muscle ratio (T/M) was calculated and compared in each group with different SAs. In addition, the correlation between blood estradiol and sex hormone-binding globulin and the value of T/M were also analyzed. The value of T/M increased initially with the rise of SA and it reached the peak at SA of 1.6Ci/μmol. After that, the value fell down sharply and remained stable from SA of 3.1Ci/μmol. The value of T/M was highest at SA of 1.6Ci/μmol (P<.001). Additionally, the blood levels of estradiol and sex hormone-binding globulin showed no correlation with the value of T/M (P>.05). High SA of 18 F-fluoroestradiol leads to low T/M results in breast cancer xenograft models. We should control SA in a reasonable range to obtain high-quality images.

Cell-wall composition and polysaccharide synthase activity changes following photoinduction in Trichoderma viride.

The differentiation and metabolism in the soil-borne saprophytic deuteromycete Trichoderma viride are subject to control by light. We have investigated the effect of illumination of mycelia on the activities of cell-wall synthesizing enzymes beta-1,3-glucan synthase and chitin synthase and on the composition of cell alls. After 10 min illumination of dark-grown mycelia with white fluorescent light at 600 1x, a gradual rise in specific activity of membrane-bound enzymes beta-1,3-glucan synthase by about 130% and a decrease in specific activity of chitin synthase by about 50% in relation to the dark control were observed. The changes in enzyme activities were caused by de novo synthesis of corresponding polysaccharide synthases(s) and/or their regulatory components since they were not observed when protein synthesis was blocked with 50 microg/ml cycloheximide. The content of beta-1,3-glucan in the cell walls of illuminated mycelia has increased by 35-50% in comparison to the dark control while the content of chitin remained practically unchanged.

Tissue lipids and intestinal ATPases in rabbits fed cassava ( Manihotuttilisima) and plantain ( Musaparadisica) diets

The effect of two vegetable sources of dietary fiber (DF) from Africa, plantain and cassava, has been studied on lipid metabolism and intestinal ATPases in cholesterol-fed rabbits. In an eight-week experiment, it was shown that both sources of DF are capable of reducing serum cholesterol levels in rabbits by diverting cholesterol from the blood to the liver. The DF from plantain had more effect. When the rabbits were switched from test to control and back to the test diets only those on plantain diet maintained a steady state level of serum cholesterol. The other diets caused a drop in serum cholesterol levels in the absence of dietary cholesterol and a sharp rebound on the introduction of cholesterol-based diets. Cassava diet was hypertriglyceridimic at all times. The ATPases were highly elevated in the ileum of rabbits fed the cassava diet and this was associated with the higher content of insoluble and particulate components of cassava which may cause cell sloughing and alteration in the morphology of the intestine. Also, because the available minerals will be distributed between those that will physically interact with DF and those to be transported, a rise in specific activity of the vectorial enzyme was stimulated to meet the challenge of competition for minerals.

Phosphoinositide interconversion in thrombin-stimulated human platelets.

Stimulation of platelets and other secretory cells by agonists results in the degradation of phosphoinositides by phospholipase C. Kinetic studies suggest that hydrolysis of phosphatidylinositol 4,5-diphosphate (PI-4,5-P2) is an initial event in this process. Platelets contain much larger amounts of phosphatidylinositol (PI) than PI-4,5-P2, and approximately 50% of total phosphoinositides are degraded upon stimulation. We have investigated whether degradation of PI occurs by direct phospholipase C hydrolysis or by phosphorylation to PI-4,5-P2 followed by phospholipase C action on the latter compound. When platelets are incubated for 3 min with 32Pi prior to stimulation, the phosphoinositides are labeled to different specific activities. Under these nonequilibrium conditions, the time course of change in specific activity reflects turnover. The rise in specific activity of phosphatidylinositol 4-phosphate (PI-4-P) is similar in stimulated and unstimulated cells, indicating that there is little increase in the conversion of PI to PI-4-P during thrombin stimulation. In addition, the specific activity of the 4-phosphate in PI-4-P during thrombin stimulation is less than both the 5-phosphate of PI-4,5-P2 and the phosphate group of phosphatidic acid, indicating that the 4-phosphate moiety is not labeled to equilibrium with ATP. This finding is inconsistent with a rapid flux of PI via PI-4-P to PI-4,5-P2 during thrombin stimulation, in which case the 4-phosphate would be at maximum specific activity. We, therefore, conclude that the bulk of PI breakdown that occurs in thrombin-stimulated platelets occurs via direct phospholipase C hydrolysis of PI.

Turnover of succinyl-CoA:3-oxoacid CoA-transferase in glioma and neuroblastoma cells. Specific influence of acetoacetate in neuroblastoma cells.

The specific activity of succinyl-CoA:3-oxo-acid CoA-transferase (3-oxoacid CoA-transferase, EC 2.8.3.5) increases significantly during growth in culture in both mouse neuroblastoma N2a and rat glioma C6 cells. To investigate the mechanism(s) responsible for this, antibody specific for rat brain 3-oxoacid CoA-transferase was raised in rabbits. Immunotitrations of 3-oxoacid CoA-transferase from neuroblastoma and glioma cells on days 3 and 7 of growth after subculture showed that the ratio of 3-oxoacid CoA-transferase activity to immunoprecipitable enzyme protein remained constant, indicating that differences in specific activity of the enzyme at these times in both cell types reflect differences in concentration of enzyme protein. In glioma cells, the relative rate of 3-oxoacid CoA-transferase synthesis was about 0.04-0.05% throughout 9 days in culture. In contrast, the relative rate of synthesis of 3-oxo-acid CoA-transferase in neuroblastoma cells was about 0.07-0.08% on days 3, 5 and 7 after subculture, but fell to 0.052% on day 9. The degradation rates of total cellular protein (t1/2 = 28 h) and 3-oxoacid CoA-transferase (t1/2 = 46-50 h) were similar in both cell lines. The rise in specific activity of the enzyme in both cell lines from days 3 to 7 without a significant increase in the relative rate of synthesis reflects a slow approach to steady-state conditions for the enzyme secondary to its slow degradation. Differences in 3-oxoacid CoA-transferase specific activity between the two cell lines are apparently due to a difference of about 60% in relative rates of enzyme synthesis. The presence of 0.5 mM-acetoacetate in the medium significantly increased the specific activity of 3-oxoacid CoA-transferase in neuroblastoma cells during the early exponential growth phase. This treatment increased the relative rate of synthesis of 3-oxoacid CoA-transferase by 23% (P less than 0.025) in these cells on day 3, suggesting that substrate-mediated induction of enzyme synthesis is a mechanism of regulation of 3-oxoacid CoA-transferase.

Choline Acetyltransferase and Cholinesterases in the Developing Xenopus Retina

To understand the developmental regulation of acetylcholine (ACh) synthesis in the Xenopus retina, the properties of choline acetyltransferase (CAT) and cholinesterase (ChE), as well as histochemical localization of ChE in the retina, were studied during development. CAT activity first became detectable in the developing eyecup at stages 35/36. This was followed by a rapid, 50-fold rise in specific activity between stages 35/36 and 44. Since this rapid rise coincided with an almost identical increase in total ACh synthesis in whole retinae found in previous studies, it is suggested that this increase was sufficient to account for the rapid increase in total ACh synthesis. Moreover, it also correlated with increased rates of synaptogenesis in both the inner and the outer plexiform layers. Total ChE was resolved into specific and nonspecific ChE by the use of tetraisopropylpyrophosphoramide. Total ChE activities first became detectable at stages 35/36. Specific ChE [acetylcholinesterase (AChE)] increased from 50% at stage 39 to 95% of total ChE activities at stage 66. Again, the most rapid increase in both total ChE and AChE activities occurred between stages 35/36 and 44. Histochemical studies showed that AChE was localized predominantly in the two plexiform layers, with the inner plexiform layer more heavily stained at all stages. Moreover, a stratified staining pattern, clearly discerned in the inner plexiform layer, also correlated with synaptogenesis during this early period of retinal development.

Protein kinase activity associated with Epstein-Barr virus-determined nuclear antigen

A cyclic AMP-independent protein kinase activity was found associated with Epstein-Barr virus-determined nuclear antigen (EBNA). The specific activity of protein kinase (casein as substrate) rose concurrently with the rise in specific activity of CF activity for EBNA during a series of chromatographic purifications on (1) Sephadex G-200, (2) DNA-cellulose, (3) DEAE-Sephadex A-50, and (4) heparin-Sepharose (or blue sepharose instead of 3 and 4). The kinase activity contained in EBNA thus obtained (about 50% pure as determined by gel electrophoresis) could be immunoprecipitated almost quantitatively with anti-EBNA-positive IgG but not with negative IgG. This kinase immunoprecipitable with antibody to EBNA was detected only in Raji cells and not in EBNA-negative lymphoblastoids, RAMOS cells, or BJAB cells. The EBNA-associated kinase in the immunoprecipitate was unable to phosphorylate exogenous casein efficiently. The analysis for in vitro phosphorylated products in the immunoprecipitate by gel electrophoresis showed that EBNA itself was phosphorylated at serine and threonine residues.

Hypertrophic and hyperplastic effects of thyroxine on the submandibular gland of the mouse.

The nature of the trophic response of the mouse submandibular gland to thyroxine (T4) was examined. Adult female Swiss-Webster mice were given daily subcutaneous injections of T4 (1 microgram/gm body weight) for two or four days; two injections of tritiated thymidine (3H-TdR) were given 24 and 29 hours after the last injection of hormone, and the mice were killed one hour after the last injection of 3H-TdR. One gland was analyzed chemically for DNA content and for incorporation of 3H-TdR, while the other was used to prepare autoradiograms. The cellular composition of each gland was analyzed by counting 1000 nuclei, and the frequency and labelling index (LI) of six cell types were established. A rise in specific activity of DNA and a fall in its concentration were seen in response to T4. The LI for the entire gland more than doubled. The LIs and frequencies of granular convoluted tubule and granular intercalated duct cells were increased more than those of acinar and nongranular intercalated duct cells; striated and excretory duct cells were not affected. It is concluded that the enlargement of the submandibular gland of the mouse caused by T4 is due to both hyperplastic and hypertrophic effects.

Axonal transport of [35S]methionine-labeled proteins in two intra-brain tracts of the rat.

Abstract[35S]Methionine was stereotaxically injected into the dorsalateral geniculate body (DLGB) of adult male rats, and 1 h to 10 days post‐injection the DLGB and projection site (striate cortex) were dissected out and solubilized in 1% sodium dodecyl sulfate. Samples were analyzed for acid‐precipitable radioactivity, and radioactivity in different molecular weight classes was determined following discontinuous gel electrophoresis on both tube and slab gels. Acid‐precipitable radioactivity in the DLGB peaked by 4 h and then declined over the time period studied. The molecular weight distribution pattern was complex and did not change appreciably with time. Radioactivity in the striate cortex arrived in at least three waves: rapidly transported proteins arrived between 2 and 4 h; a second wave of transport began to arrive at about 7 h post‐injection and there was a slight rise in specific activity for 2 days; finally, at 3 days post‐injection, there was a steep increase with the arrival of the bulk of the transported material. The electrophoretic distribution pattern of proteins arriving in the first wave included 40–50 identifiable bands ranging in molecular weight from 13,000 to 200,000. Of particular interest was a radioactive band of apparent molecular weight of 110,000, which was prominent at 4 h, but by 12 h showed very little labeling. The second wave of radioactivity contained primarily proteins of molecular weight classes already present, although there were quantitative differences. Several proteins in the molecular weight range of 43,000 to 78,000 were identifiable as characteristic of the third wave of transported material. Results from a study following injection of a hippocampus were similar: the electrophoretic distribution pattern of radioactive proteins extracted from the injected hippocampus resembled that of the DLGB, and also did not vary appreciably with time, while radioactive proteins in the contralateral hippocampus had an electrophoretic distribution pattern similar to that of the striate cortex and changed with time in a similar manner.

Selective changes in tRNA methyltransferase activity in confluent monolayers of WI‐38 cells stimulated to proliferate

In quiescent confluent monolayers of WI-38 cells, the specific activity of the tRNA methyltransferases falls to 20% of the level found in log phase cells. When the resting cells are stimulated to proliferate by a change to fresh medium, the enzyme show a rapid rise in specific activity which correlates with early increases in the rate of tRNA synthesis. The specific activity of the enzymes continues to rise throughout the period of DNA synthesis, at the end of which it is somewhat higher than that of log phase cells. The increases in enzyme activity could be blocked by exposure of the stimulated cells to Actinomycin D (2 microgram/ml). The increases in activity were not equivalent for the different base-specific enzymes. The contribution of the N2-methylguanine specific enzyme remained relatively constant, while that of the N2,N2-dimethyl-guanine specific and 1-methyladenine specific enzymes doubled and tripled, respectively, by late S phase. The contributions of the 1-methylguanine and the 7-methylguanine specific enzymes fell to a few percent of the total by late S phase. This indicates non-coordinate variations in the expression of the different base-specific enzymes after stimulation of resting cells and may be related to altered isoaccepting tRNA profiles observed in resting and growing cells.

Effect of pulmonary lavage on lung lecithin synthesis in the Syrian hamster

Bronchopulmonary lavage with 0.15 m saline stimulated the uptake of [1,2- 14C]choline into both the surface-active dipalmitoyl lecithin (DPL) and the unsaturated lecithins (UPC) of Syrian hamster lung. Labeling occurred in the tissue lecithins prior to label appearance in the lecithins in the alveolar space. In control lung tissue the rise in specific activity of saturated lecithin lagged behind that of the unsaturated lecithin fraction. The data were consistent with the view that alveolar lecithins are secreted by the tissue into the alveoli and that unsaturated lecithins are the precursors of dipalmitoyl lecithin. Labeled DPL and UPC were transferred from the tissue to the alveoli at approximately the same rate. Bronchopulmonary lavage did not greatly affect the rate of loss of label from alveolar lecithins. Type II cell proliferation, as measured by [ 3H]thymidine uptake, did not occur during the 18-hr period in which stimulated lecithin synthesis was observed.

Alkaline protease in the larvae of the army worm, Spodoptera litura

The alkaline protease activity in the gut of Spodoptera litura was found to increase with the development of larvae and decreased with the onset of pupation. Fasting of the 5th instar larvae caused a slight increase in protease activity at 4 hr, which declined consistently on further starvation. The optimum pH for the gut protease was 11.0, with a shoulder between pH 8.0 and 9.0. The protease was inactivated upon dialysis of the crude enzyme solution at room temperature but not at 4°C. Incubation of the crude enzyme solution at pH 11.0 and 37°C for 22 hr resulted in a three-fold rise in specific activity of the alkaline protease which declined on further incubation. The three-fold purified preparation, obtained by incubation of the crude enzyme solution, was passed through Sephadex G-75 to give a seven-fold purification and 70% yield. The preparation was not completely homogeneous and showed three clearly separable protein bands by polyacrylamide gel electrophoresis. The partially purified protease exhibited no shoulder between pH 8 to 9, like the crude preparation.

Protein and RNA Synthesis in Endometrium of the Rat Uterus During Early Progestation1

To assess the rate and timing of nucleic acid and protein synthesis in the endometrium of the rat during early progestation, two series of experiments were performed. Measures were made of a) the incorporation of labeled glycine into endometrial subcellular fractions and b) the uptake of labeled leucine and uridine into TCA-insoluble material of endometrium. Of the glycine incorporated during the cycle, 50-70 percent was distributed into the nuclear fraction, with 30-50 percent in the other two fractions. Following the induction of pseudopregnancy, about 97 percent of the label was associated with the nuclear fraction. By Day 4 of progestation, a shift in the distribution occurred, such that 68 percent of the glycine was associated with the soluble and mitochondrial fractions. Calculated as specific activity, different patterns of activity were evident in each fraction: 1) no changes occurred during the estrous cycle in the nuclear and mitochondrial fractions and 2) the soluble fraction showed peak activity during proestrus. Following cervical stimulation, a significant, transient rise in specific activity was measured on Day 1 in all three fractions. Subsequently, a second peak in activity occurred on Day 3 in the soluble and mitochondrial fractions. The data suggest that at the onset of progestation, a change in the ability of the tissue to utilize exogenous substrate may Occur: glycine may be compartmentalized, and thus withheld from the general metabolic machinery of the cell, or it may be utilized preferentially in the biosynthesis of nucleic acid. Following this initial period, a greater proportion of label may be utilized in various other metabolic pathways. Total incorporation of leucine and uridine showed similar patterns: peak activity, measured during proestrus, declined to minimal activity during estrus and metestrus. After stimulation of the uterine cervix, incorporation declined to a low level by Day 1 of pseudopregnancy. After Day 2, a significant increase occurred, which was maintained through Day 6. Calculated as specific activity, the incorporation of these substrates showed no significant day-to-day changes during the cycle. Following induction of pseudopregnancy, a more gradual decline in the specific activity of uridine was followed by a transiently increased leucine activity on Day 1, but the responses were marginal (and not significant). Between Days 2-5 a dramatic rise in the specific activity of both substrates was measured. These experiments suggest that at the beginning of progestation the uterine endometrium generally reflects the effect of postovuiatory hormone deprivation, though local changes in biosynthetic activity, induced by early progestogenic action may be evident. Following Day 2, however, a second, dramatic increase in the rate of biosynthetic activity occurs which can be measured as an increased rate of synthesis of RNA and protein, and increased protein content. These changes in endometrium, evident by Day 3, reach their peak before Day 4, the time of maximal sensitivity of the uterus to decidualizing stimuli.

Studies on protein multimers: VII. Stabilization of E.coli β-galactosidase by a 260-nm absorbing compound in crude preparations

The properties of crude β-galactosidase preparations from E. coli ML308 have been compared with those of purified samples. With purification the 280 260 - nm absorbance ratio rises and the enzyme becomes less stable to heat denaturation. A small molecular-weight ligand, responsible for the nonprotein 260-nm absorbance, has been isolated. When added to purified enzyme there occurs a rise in specific activity and an increase in heat stability.

Development of intestinal adenyl cyclase and its response to cholera enterotoxin.

Adenyl cyclase activity in intestinal membranes has been studied during development in the rabbit fetus from fetal day 17 to 10 days postnatally and in the human fetus from the 10th to the 17th wk of gestation. In the rabbit, the enzyme was already present by fetal day 17 and showed a fourfold peak rise in specific activity by 22 days. By 28 days, the specific activity had fallen toward adult levels and remained constant throughout gestation and the 1st wk of life. Fluoridestimulated activity showed a similar curve, and was 2.5-5 times the basal values. Activities in jejunum and ileum were comparable at all time points studied. Phosphodiesterase activity did not change during gestation. When fetal intestinal segments were incubated in vitro with purified cholera enterotoxin, adenyl cyclase activity in subsequently prepared membranes was increased two- to threefold. This level was not regularly further elevated by fluoride ion. Lithium ion inhibited both the basal and fluoride-stimulated enzyme activity in membranes prepared from rabbit fetuses at term. Lactase activity (reflecting the development of the microvilli) in either whole intestinal homogenates or in the membrane fractions showed a differnet pattern of development, with a rise beginning on fetal day 24 and a plateau just after birth. In intestinal membranes prepared from human fetuses, the activity of both basal and fluoride-stimulated adenyl cyclase tripled from the 10th to the 17th wk of gestation. The data both in the rabbit and in man show that intestinal adenyl cyclase is capable of responding to cholera enterotoxin quite early in gestation. In the rabbit, this occurs before the time of appearance or ville or of an enzyme marker (lactase) for microville. The results support the concept that adenyl cyclase is present in plasma membrane other than the brush border.

DNA-dependent RNA polymerase from trophozoites and cysts of Acanthamoeba castellanii

DNA-dependent RNA polymerase was solubilized and partially purified from nuclei of Acanthamoeba castallanii trophozoites. Two distinct peaks having enzymatic activity were recovered after DEAE-Sephadex chromatography, and some of the properties of each were examined. Maximal activity for each peak required added DNA, Mg 2+, Mn 2+ and the nucleoside triphosphates. The pH optima were in the range 8.0–8.4. The products were acid-insoluble and ribonuclease sensitive. Rifampin and α-amanitin inhibited the activity of Peak II, while cycloheximide inhibited the Peak I polymerase. Since A. castellanii encysts, the pattern of RNA polymerase specific activity during encystment was examined. The greatest enzymatic activity was observed from 0–20 h after induction, with an apparent rise in specific activity at 10–12 h. This pattern of polymerase specific activity was consistent with the pattern of RNA turnover in encysting cells.

Purification of a human placental cholesteryl ester hydrolase

A low but significant activity of cholesteryl ester hydrolase (EC 3.1.1.13) activity has been demonstrated in human placenta. A 352-fold purification of this enzyme has been achieved that gives only one band on polyacrylamide gel electrophoresis. The rise in specific activity with purification does not appear to be attributable to a decrease in pool size of cholesteryl ester.

Developmental regulation of alkaline phosphatase in Dictyostelium discoideum.

The kinetics of accumulation of alkaline phosphatase activity were determined in cells of wild-type and morphologically aberrant mutant strains of Dictyostelium discoideum induced to develop synchronously on membrane supports. The enzyme specific activity increased slowly in wild-type cells until culmination when a dramatic rise in specific activity occurred. The patterns of accumulation in the mutant strains, as well as previous electrophoretic analysis, suggest that the two phases of accumulation may result from the synthesis of distinct isozymes. The rapid accumulation of alkaline phosphatase was found to require concomitant protein synthesis. Ribonucleic acid synthesis, on the other hand, could be inhibited during the 8 hr immediately preceding culmination without affecting the amount of enzyme accumulated. When ribonucleic acid synthesis was inhibited earlier in development, the accumulation of alkaline phosphatase was reduced. Comparison of these results with work on other developmentally controlled enzymes suggests that both transcriptional and translational control occurs during development of D. discoideum. The accumulation of alkaline phosphatase was shown to require specific cellular topography during culmination, suggesting that intercellular interactions which allow synthesis of alkaline phosphatase occur at that stage.

Synchrony of enzyme accumulation in a population of differentiating slime mold cells

1. 1. Studies of the cellular slime molds have previously revealed the accumulation of a number of enzymes at different stages during the development of multicellular fruiting structures. 2. 2. A study of the spatial distribution of one of the enenzymes (UDPGlc pyrophosphorylase) during the period of its accumulation revealed a uniform rise in specific activity throughout the developing fruiting body. This suggests that all cells accumulate the enzyme simultaneously rather than as a wave of accumulation starting at a particular region. 3. 3. The distribution of the enzyme UDPGal: polysaccharide transferase served as a control for the techniques employed. The mucopolysaccharide product is known to be restricted to spores, and the enzyme was found to be accumulated only in regions containing incipient spore cells.

Purification and properties of lysozyme produced by Staphylococcus aureus.

A method based on cold ethyl alcohol fractionation at different pH levels and ionic strengths and on gel filtration on a Sephadex G-200 column was used to concentrate and purify lysozyme from the culture supernatant fluid of Staphylococcus aureus strain 524. The final, nondialyzable product exhibited a 163-fold rise in specific activity over that of the starting material. Staphylococcal lysozyme is a glycosidase which splits N-acetylamino sugars from the susceptible substrate. Staphylococcal lysozyme was shown to be similar to egg white lysozyme in its optimal temperature for reaction, optimal pH, activation by NaCl and Ca(++) ions, inhibition by sodium citrate and ethylenediaminetetraacetate, and inactivation by Cu(++) ions and sodium dodecyl sulfate. It differs from the egg white lysozyme in its temperature susceptibility range (staphylococcal lysozyme is inactivated at 56 C). It acts on whole cells and cell walls of Micrococcus lysodeikticus, murein from S. aureus 524, and cell walls of S. epidermidis Zak. The last substrate was not susceptible to the action of egg white lysozyme in the test system used. The mechanism of action of staphylococcal lysozyme seems to be analogous to that of egg white lysozyme; however, the biological specificity of the two enzymes may be different.