scholarly journals The effect of intralysosomal sucrose storage on the turnover of hamster fibroblast lysosomal and Golgi-apparatus enzymes

1976 ◽  
Vol 158 (2) ◽  
pp. 401-407 ◽  
Author(s):  
M J Warburton ◽  
C H Wynn
Keyword(s):  
Golgi Apparatus ◽  
Half Life ◽  
Cellular Protein ◽  
Enzyme Synthesis ◽  
Pulse Labelling ◽  
Sucrose Uptake ◽  
Total Cellular Protein ◽  
Sucrose Storage ◽  
Alkaline Ribonuclease ◽  
Synthesis And Degradation

1. The effect of overloading of hamster fibroblast lysosomes with sucrose on the turnover of lysosomal and Golgi-apparatus enzymes was studied. Arylsulphatase B and UDP-galactose-N-acetylglucosamine galactosyltransferase were chosen as appropriate marker enzymes. The relative contributions of changes in the rates of synthesis and degradation to the increased activities of these enzymes after uptake of sucrose were examined by isotopic-labelling experiments. The effects of sucrose uptake on the degradation of total cellular protein and of the cytoplasmic enzyme, alkaline ribonuclease, were determined for comparative purposes. 2. The rates of enzyme synthesis in the presence and absence of sucrose were compared by pulse-labelling the cells with 14C-labelled amino acids, followed by isolation of purified enzymes and determination of their radioactivity. Sucrose uptake produced increases of 270% and 90% respectively in the rates of synthesis of arylsulphatase B and galactosyltransferase, whereas the rate of synthesis of alkaline ribonuclease was not affected. 3. The rates of degration of the enzymes were estimated by measuring the decay with time of the radioactivity of purified enzymes from prelabelled cells. In the absence of sucrose, the apparent half-lives of arylsulphatase B and galactosyltransferase were about 30 days and 40h respectively. After uptake of sucrose, the half-life of arylsulphatase B decreased to about 10 days and that of galactosyltransferase to 10h. Neither the half-life of alkaline ribonuclease (4 days) nor the rate of degradation of total cellular protein was affected by the uptake of sucrose. 4. These results indicate that the hyperactivity of lysosmal and Golgi-apparatus enzymes after the uptake of sucrose is accompanied by increases in the rates of both synthesis and degradation, and that the increased rates of degradation are insufficient to prevent accumulation of the excess of enzymes synthesized; also that the effects of sucrose uptake are restricted to the vacuolar apparatus.

scholarly journals Constant expression and activity of protein phosphatase 2A in synchronized cells.

1991 ◽  
Vol 11 (8) ◽  
pp. 4282-4285 ◽  
Author(s):  
R Ruediger ◽  
J E Van Wart Hood ◽  
M Mumby ◽  
G Walter
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Cellular Protein ◽  
Quantitative Comparison ◽  
Resting Cells ◽  
Constant Amount ◽  
Total Cellular Protein ◽  
B Subunit ◽  
Phosphatase 2A ◽  
Constant Expression

The levels of the A, B, and C subunits of protein phosphatase 2A in extracts from synchronized embryonic bovine tracheal cells were determined by immunoblotting with subunit-specific antibodies. A constant amount of each subunit was found in resting cells as well as in growing cells from all stages of the cell cycle. The phosphatase activity of protein phosphatase 2A was also constant. A quantitative comparison showed that the A and C subunits were present in similar amounts, whereas the B subunit was present at a significantly lower level. Together, the A, B, and C subunits represented approximately 0.2% of the total cellular protein.

scholarly journals Stoffwechsel aromatischer Pflanzeninhaltsstoffe I

1969 ◽  
Vol 24 (2) ◽  
pp. 234-239 ◽  
Author(s):  
Wolfgang Barz
Keyword(s):  
Cicer Arietinum ◽  
Half Life ◽  
Biochanin A ◽  
Pulse Labelling ◽  
Phaseolus Aureus ◽  
Cicer Arietinum L ◽  
Plant Products ◽  
Biological Half Life ◽  
Total Turnover ◽  
Secondary Plant

The turnover of the isoflavones, formononetin and biochanin A, in Cicer arietinum L. and of the isoflavone, daidzein, and the coumestan, coumestrol, in Phaseolus aureus Roxb. has been determined by pulse labelling. The biological half-life of formononetin, daidzein and coumestrol has been found to be approximately 50 hours, while the turnover of biochanin A appeared to be very slow. The excretion of all four plant products through the roots into the medium has been measured. Approximately 3 percent of the total turnover of isoflavones in the plants can be accounted for by excretion into the medium.The results are discussed in view of the concept that secondary plant products may well be subject to further metabolism.

Quantitation of a 55K cellular protein: similar amount and instability in normal and malignant mouse cells

1982 ◽  
Vol 2 (7) ◽  
pp. 763-771
Author(s):  
P T Mora ◽  
K Chandrasekaran ◽  
J C Hoffman ◽  
V W McFarland
Keyword(s):  
Half Life ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cellular Protein ◽  
T Antigen ◽  
Similar Amount ◽  
Parent Cell ◽  
Primary Cells ◽  
Quantitative Expression ◽  
Tumorigenic Cells

Quantitative expression of a specific 55,000 (55K)-molecular-weight cellular protein was studied in two groups of mouse embryo fibroblast (clonal) cells originating from two parent clones, one of which possessed high tumorigenicity and the other of which possessed very low tumorigenicity. From the clone with low tumorigenicity, tumor lines and clones were obtained by selecting rare spontaneously transformed highly tumorigenic (mutant) cells. Cells were labeled during exponential growth for 3 h at 37 degrees C, with [35S]methionine, and the cellular 55K protein was immunoprecipitated with a monoclonal antibody and quantitated. There were low and approximately equal amounts of 55K protein in cells (clones) with both low and high tumorigenicity from both groups of cells, and there was no correlation at all between quantitative expression of 55K protein and of cellular tumorigenicity. There was approximately 10- to 20-fold more 55K protein in all simian virus 40-transformed T antigen-positive derivative clones, as shown previously. The T antigen-negative revertant tumor lines and clones obtained by an immunological in vivo selection method had low amounts of 55K protein, similar to the parent cell before simian virus 40 transformation. In all of the T antigen-negative cells, including the highly tumorigenic cells, degradation (turnover?) of the 55K protein was rapid, and a half-life of 15 to 60 min was estimated from pulse-chase experiments. In all of the T antigen-positive cells the 55K protein was stable (half-life greater than 10 h). In primary cells established from the tumors induced by highly tumorigenic cells there was a very low or no detectable amount of the 55K protein. This is in contrast to the primary cells obtained from early murine embryos in which we have reported high amounts of (stable) 55K proteins.

Expression of the ornithine decarboxylase gene in response to asparagine in intestinal epithelial cells

1996 ◽  
Vol 271 (1) ◽  
pp. G164-G171 ◽  
Author(s):  
J. Y. Wang ◽  
M. J. Viar ◽  
P. M. Blanner ◽  
L. R. Johnson
Keyword(s):  
Ornithine Decarboxylase ◽  
Half Life ◽  
Cellular Protein ◽  
Mrna Levels ◽  
Intestinal Epithelial ◽  
Normal Rat ◽  
Fetal Bovine ◽  
Mucosal Growth ◽  
Rate Limiting ◽  
In Cells

Refeeding fasted rats significantly stimulates mucosal growth and ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, but the exact mechanism responsible for induction of ODC at the molecular level is unknown. Of normal dietary constituents, the amino acid asparagine markedly increases ODC activity and mucosal growth when administered intragastrically. The current study examined the expression of the ODC gene in IEC-6 cells (a line of normal rat small intestinal crypt cells) after exposure to asparagine. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum. They were deprived of serum for 24 h before experiments. Exposure to asparagine at the dose of 10 mM resulted in the rapid increase in ODC mRNA levels. The increased expression of the ODC gene began 1 h after and peaked between 3 and 5 h after treatment with asparagine. Maximum increases in ODC mRNA levels were approximately fivefold the normal value. Increased levels of ODC mRNA in cells exposed to asparagine were paralleled by increases in ODC protein and enzyme activity and cellular polyamine levels. The half-life of mRNA for ODC in unstimulated IEC-6 cells was approximately 30 min and increased to > 2 h in cells exposed to 10 mM asparagine. The half-life of ODC activity also was increased in asparagine-treated cells. When cellular protein synthesis was inhibited by cycloheximide, asparagine superinduced ODC mRNA levels. Furthermore, asparagine also significantly stimulated DNA synthesis in IEC-6 cells. These results indicate that 1) asparagine stimulates ODC in IEC-6 cells through multiple pathways and 2) increased ODC mRNA levels result partly from a delay in the rate of degradation. These findings suggest that luminal amino acids stimulate gut mucosal growth in association with their ability to regulate ODC gene expression.

scholarly journals Developmental regulation of rat lung Cu,Zn-superoxide dismutase

1987 ◽  
Vol 246 (3) ◽  
pp. 697-703 ◽  
Author(s):  
M A Hass ◽  
D Massaro
Keyword(s):  
Superoxide Dismutase ◽  
Half Life ◽  
Specific Activity ◽  
Developmental Regulation ◽  
Progressive Increase ◽  
Enzyme Synthesis ◽  
Enzyme Activation ◽  
Sod Activity ◽  
Adult Rats ◽  
Copper Chelation

In the present investigation we found that lung Cu, Zn-superoxide dismutase (SOD) activity (units/mg of DNA) increases steadily in the rat from birth to adulthood. The specific activity (units/micrograms of enzyme) of Cu, Zn-SOD was unchanged from birth to adulthood, excluding enzyme activation as a mechanism responsible for the increase in enzyme activity. Lung synthesis of Cu, Zn-SOD peaked at 1 day before birth and decreased thereafter to adult values. Calculations, based on rates of Cu, Zn-SOD synthesis and the tissue content of the enzyme, indicated that lung Cu, Zn-SOD activity increased during development owing to the rate of enzyme synthesis exceeding its rate of degradation by 5-10%. These calculations were supported by measurements of enzyme degradation in the neonatal (half-life, t1/2, = 12 h) and adult lung (t1/2 = greater than 100 h); the difference in half-life did not reflect the rates of overall protein degradation in the lung, since these rates were not different in lungs from neonatal and adult rats. We did not detect differences in the Mr or pI of Cu, Zn-SOD during development, but the susceptibility of the enzyme to inactivation by heat or copper chelation decreased with increasing age of the rats. We conclude that the progressive increase in activity of Cu, Zn-SOD is due to a rate of synthesis that exceeds degradation of the enzyme. The data also suggest that increased stabilization of enzyme conformation accounts for the greater half-life of the enzyme in lungs of adult compared with neonatal rats.

scholarly journals Effect of heterologous expression of acyl-CoA-binding protein on acyl-CoA level and composition in yeast

1993 ◽  
Vol 290 (2) ◽  
pp. 369-374 ◽  
Author(s):  
S Mandrup ◽  
R Jepsen ◽  
H Skøtt ◽  
J Rosendal ◽  
P Højrup ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Heterologous Expression ◽  
Binding Protein ◽  
Cellular Protein ◽  
Serine Residue ◽  
Yeast Saccharomyces Cerevisiae ◽  
Total Cellular Protein ◽  
Gal1 Promoter

We have expressed a bovine synthetic acyl-CoA-binding protein (ACBP) gene in yeast (Saccharomyces cerevisiae) under the control of the GAL1 promoter. The heterologously expressed bovine ACBP constituted up to 6.4% of total cellular protein and the processing was identical with that of native bovine ACBP, i.e. the initiating methionine was removed and the following serine residue was N-acetylated. The expression of this protein did not affect the growth rate of the cells. Determination of the yeast acyl-CoA pool size showed a close positive correlation between the ACBP content of the cells and the size of the acyl-CoA pool. Thus ACBP can act as an intracellular acyl-CoA pool former. Possible physiological functions of ACBP in cells are discussed.

scholarly journals Decreased actin content of lymphocytes from patients with chronic lymphocytic leukemia

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 536-541
Author(s):  
R Stark ◽  
LF Liebes ◽  
D Nevrla ◽  
M Conklyn ◽  
R Silber
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Lymphocytes ◽  
Protein A ◽  
Cellular Protein ◽  
Lymphocytic Leukemia ◽  
T And B Cells ◽  
Total Cellular Protein ◽  
Normal Human ◽  
Cell Chronic Lymphocytic Leukemia ◽  
Normal Human Blood

Actin, a major cytoskeletal protein, was quantitated in normal and chronic lymphocytic leukemia lymphocytes. The actin content of normal human blood lymphocytes was 2.2 +/- 0.4 mg/10(9) cells and represented 6.6% +/- 1.8% of the total cellular protein. A significant decrease (p less than 0.001) was noted in chronic lymphocytic leukemia lymphocytes that contained 1.4 +/- 0.3 mg actin/10(9) cells, constituting 4.3% +/- 1.1% of the total protein. Normal T and B cells did not differ in actin content. Reduced actin levels were found in the T as well as in the B lymphocytes of “B-cell” chronic lymphocytic leukemia. The possible importance of the decreased actin level in the anomalous capping response and motility of chronic lymphocytic leukemia lymphocytes is discussed.

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