scholarly journals Expression of acid phosphatase-beta-galactosidase hybrid proteins prevents translocation by depleting a soluble factor.

1990 ◽  
Vol 265 (32) ◽  
pp. 19824-19832
Author(s):  
M R Young ◽  
J Andreadis ◽  
L T Hu ◽  
P B Wolfe
Keyword(s):  
Acid Phosphatase ◽  
Soluble Factor ◽  
Hybrid Proteins ◽  
Beta Galactosidase

scholarly journals Content and thrombin-induced release of acid hydrolases in gel-filtered platelets from patients with storage pool disease

Blood ◽  
1975 ◽  
Vol 46 (1) ◽  
pp. 131-142 ◽  
Author(s):  
H Holmsen ◽  
CA Setkowsky ◽  
B Lages ◽  
HJ Day ◽  
HJ Weiss ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Normal Subjects ◽  
Acid Hydrolases ◽  
Release Reaction ◽  
Dense Granules ◽  
Storage Pool ◽  
Low Concentrations ◽  
Storage Pool Disease ◽  
Beta Galactosidase

Abstract The levels of four acid hydrolases, beta-N-acetyl glucosaminidase, beta- glucuronidase, beta-galactosidase, and acid phosphatase, and the extent of their release (release II) by thrombin was determined in platelets from nine normal subjects, nine patients with storage pool disease, and in normal platelets which had been exposed to aspirin. The levels of all four hydrolases were normal in patients with SPD. However, release of three of these hydrolases (acid phosphatase was an exception) by low concentrations of thrombin (0.015 and 0.04 U/ml) was decreased in the patients as a group, although considerable variation in the extent of release of each enzyme was noted. In contrast, aspirin failed to inhibit release II in normal platelets (except for a slight impairment in the release of beta-N-acetyl glucosaminidase), although release I (serotonin, ATP and ADP) was inhibited. All release defects could be overcome by using higher concentrations of thrombin (0.2 U/ml). The normal levels of acid hydrolases in the platelets of patients with SPD (who are deficient in the platelet dense granules) suggest that these enzymes are not normally stored in the dense granules, but rather in alpha-granules. The findings also support the conclusions of previous studies that the release reaction is impaired in SPD. This release defect appears to be different from that seen in normal platelets after exposure to aspirin.

scholarly journals Effect of a transitory ischaemia on the structure-linked latency of rat liver acid phosphatase and β-galactosidase

1981 ◽  
Vol 196 (3) ◽  
pp. 861-866 ◽  
Author(s):  
R Wattiaux ◽  
S Wattianx-De Coninck
Keyword(s):  
Blood Flow ◽  
Acid Phosphatase ◽  
Distribution Pattern ◽  
Rat Liver ◽  
Differential Centrifugation ◽  
Liver Lysosomes ◽  
Secondary Rise ◽  
Beta Galactosidase ◽  
Free Activity ◽  
Normal Latency

The structure-linked latency of acid phosphatase and beta-galactosidase was studied in rat liver lobes made ischaemic for 1 or 2 h and then recirculated with blood for increasing periods. Free activity of acid phosphatase and unsedimentable activity of beta-galactosidase are increased in homogenates of ischaemic livers. When ischaemia had been maintained for 1 h, the recovery of normal latency for both enzymes was observed 1 h after re-establishment of the blood flow. After a 2 h period of ischaemia, unmasked activity markedly decreases during the first 1 h after restoration of blood flow; after that, a large and irreversible secondary rise takes place. Chlorpromazine, injected 30 min before or just after induction of ischaemia, extensively prevents the latency decrease occurring during restoration of blood flow. Modifications of the hydrolase distribution pattern obtained after differential centrifugation are in agreement with the latency changes. These results suggest that a 2 h ischaemia causes an alteration of the liver lysosomes that is largely reversible and that restoration of blood flow induces an irreversible alteration of these organelles. Chlorpromazine treatment prevents the irreversible lesion from taking place.

scholarly journals Absence of step changes in activity of certain enzymes during the cell cycle of budding and fission yeasts in synchronous cultures

1983 ◽  
Vol 61 (1) ◽  
pp. 339-349
Author(s):  
J. Creanor ◽  
S.G. Elliott ◽  
Y.C. Bisset ◽  
J.M. Mitchison
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Acid Phosphatase ◽  
Kluyveromyces Lactis ◽  
The Other ◽  
Linear Pattern ◽  
Synchronous Cultures ◽  
Asynchronous Control ◽  
Alpha Glucosidase ◽  
Beta Galactosidase

Synchronous cultures prepared by selection from an elutriating rotor were used to measure activity changes during the cell cycle of the following enzymes: acid phosphatase in Schizosaccharomyces pombe and Saccharomyces cerevisiae, alpha-glucosidase in S. cerevisiae and beta-galactosidase in Kluyveromyces lactis. There was no sign of step rises in activity in acid phosphatase but there were indications in S. cerevisiae of the linear pattern with rate doublings once per cycle that had been found previously in S. pombe. There was also no sign of step rises in the other two enzymes, in contrast to earlier results using different techniques. Asynchronous control cultures showed little or no perturbations after the first hour.

scholarly journals Content and thrombin-induced release of acid hydrolases in gel-filtered platelets from patients with storage pool disease

Blood ◽  
1975 ◽  
Vol 46 (1) ◽  
pp. 131-142 ◽  
Author(s):  
H Holmsen ◽  
CA Setkowsky ◽  
B Lages ◽  
HJ Day ◽  
HJ Weiss ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Normal Subjects ◽  
Acid Hydrolases ◽  
Release Reaction ◽  
Dense Granules ◽  
Storage Pool ◽  
Low Concentrations ◽  
Storage Pool Disease ◽  
Beta Galactosidase

The levels of four acid hydrolases, beta-N-acetyl glucosaminidase, beta- glucuronidase, beta-galactosidase, and acid phosphatase, and the extent of their release (release II) by thrombin was determined in platelets from nine normal subjects, nine patients with storage pool disease, and in normal platelets which had been exposed to aspirin. The levels of all four hydrolases were normal in patients with SPD. However, release of three of these hydrolases (acid phosphatase was an exception) by low concentrations of thrombin (0.015 and 0.04 U/ml) was decreased in the patients as a group, although considerable variation in the extent of release of each enzyme was noted. In contrast, aspirin failed to inhibit release II in normal platelets (except for a slight impairment in the release of beta-N-acetyl glucosaminidase), although release I (serotonin, ATP and ADP) was inhibited. All release defects could be overcome by using higher concentrations of thrombin (0.2 U/ml). The normal levels of acid hydrolases in the platelets of patients with SPD (who are deficient in the platelet dense granules) suggest that these enzymes are not normally stored in the dense granules, but rather in alpha-granules. The findings also support the conclusions of previous studies that the release reaction is impaired in SPD. This release defect appears to be different from that seen in normal platelets after exposure to aspirin.

Identification and regulation of a gene required for cell fusion during mating of the yeast Saccharomyces cerevisiae

1987 ◽  
Vol 7 (8) ◽  
pp. 2680-2690
Author(s):  
G McCaffrey ◽  
F J Clay ◽  
K Kelsay ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Fusion ◽  
Subcellular Fractionation ◽  
Alpha Cells ◽  
Cell Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Hybrid Proteins ◽  
Regulatory Strategies ◽  
Absolute Requirement ◽  
Beta Galactosidase

We have devised a screen for genes from the yeast Saccharomyces cerevisiae whose expression is affected by cell type or by the mating pheromones. From this screen we identified a gene, FUS1, whose pattern of expression revealed interesting regulatory strategies and whose product was required for efficient cell fusion during mating. Transcription of FUS1 occurred only in a and alpha cells, not in a/alpha cells, where it was repressed by a1 X alpha 2, a regulatory activity present uniquely in a/alpha cells. Transcription of FUS1 showed an absolute requirement for the products of five STE genes, STE4, STE5, STE7, STE11, and STE12. Since the activators STE4, STE5, and STE12 are themselves repressed by a1 X alpha 2, the failure to express FUS1 in a/alpha cells is probably the result of a cascade of regulatory activities; repression of the activators by a1 X alpha 2 in turn precludes transcription of FUS1. In addition to regulation of FUS1 by cell type, transcription from the locus increased 10-fold or more when a or alpha cells were exposed to the opposing mating pheromone. To investigate the function of the Fus1 protein, we created fus1 null mutants. In fus1 X fus1 matings, the cells of a mating pair adhered tightly and appeared to form zygotes. However, the zygotes were abnormal. Within the conjugation bridge the contained a partition that prevented nuclear fusion and mixing of organelles. The predicted sequence of the Fus1 protein (deduced from the FUS1 DNA sequence) and subcellular fractionation studies with Fus1-beta-galactosidase hybrid proteins suggest that Fus1 is a membrane or secreted protein. Thus, Fus1 may be located at a position within the cell where it is poised to catalyze cell wall or plasma membrane fusion.

scholarly journals Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (11) ◽  
pp. 2347-2355 ◽  
Author(s):  
S D Emr ◽  
I Schauer ◽  
W Hansen ◽  
P Esmon ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Lacz Gene ◽  
Gene Fusions ◽  
Glucose Medium ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Hybrid Proteins ◽  
Suc2 Gene ◽  
Beta Galactosidase

The yeast SUC2 gene codes for the secreted enzyme invertase. A series of 16 different-sized gene fusions have been constructed between this yeast gene and the Escherichia coli lacZ gene, which codes for the cytoplasmic enzyme beta-galactosidase. Various amounts of SUC2 NH2-terminal coding sequence have been fused in frame to a constant COOH-terminal coding segment of the lacZ gene, resulting in the synthesis of hybrid invertase-beta-galactosidase proteins in Saccharomyces cerevisiae. The hybrid proteins exhibit beta-galactosidase activity, and they are recognized specifically by antisera directed against either invertase or beta-galactosidase. Expression of beta-galactosidase activity is regulated in a manner similar to that observed for invertase activity expressed from a wild-type SUC2 gene: repressed in high-glucose medium and derepressed in low-glucose medium. Unlike wild-type invertase, however, the invertase-beta-galactosidase hybrid proteins are not secreted. Rather, they appear to remain trapped at a very early stage of secretory protein transit: insertion into the endoplasmic reticulum (ER). The hybrid proteins appear only to have undergone core glycosylation, an ER process, and do not receive the additional glycosyl modifications that take place in the Golgi complex. Even those hybrid proteins containing only a short segment of invertase sequences at the NH2 terminus are glycosylated, suggesting that no extensive folding of the invertase polypeptide is required before initiation of transmembrane transfer. beta-Galactosidase activity expressed by the SUC2-lacZ gene fusions cofractionates on Percoll density gradients with ER marker enzymes and not with other organelles. In addition, the hybrid proteins are not accessible to cell-surface labeling by 125I. Accumulation of the invertase-beta-galactosidase hybrid proteins within the ER does not appear to confer a growth-defective phenotype to yeast cells. In this location, however, the hybrid proteins and the beta-galactosidase activity they exhibit could provide a useful biochemical tag for yeast ER membranes.

scholarly journals Identification and regulation of a gene required for cell fusion during mating of the yeast Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (8) ◽  
pp. 2680-2690 ◽  
Author(s):  
G McCaffrey ◽  
F J Clay ◽  
K Kelsay ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Fusion ◽  
Subcellular Fractionation ◽  
Alpha Cells ◽  
Cell Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Hybrid Proteins ◽  
Regulatory Strategies ◽  
Absolute Requirement ◽  
Beta Galactosidase

We have devised a screen for genes from the yeast Saccharomyces cerevisiae whose expression is affected by cell type or by the mating pheromones. From this screen we identified a gene, FUS1, whose pattern of expression revealed interesting regulatory strategies and whose product was required for efficient cell fusion during mating. Transcription of FUS1 occurred only in a and alpha cells, not in a/alpha cells, where it was repressed by a1 X alpha 2, a regulatory activity present uniquely in a/alpha cells. Transcription of FUS1 showed an absolute requirement for the products of five STE genes, STE4, STE5, STE7, STE11, and STE12. Since the activators STE4, STE5, and STE12 are themselves repressed by a1 X alpha 2, the failure to express FUS1 in a/alpha cells is probably the result of a cascade of regulatory activities; repression of the activators by a1 X alpha 2 in turn precludes transcription of FUS1. In addition to regulation of FUS1 by cell type, transcription from the locus increased 10-fold or more when a or alpha cells were exposed to the opposing mating pheromone. To investigate the function of the Fus1 protein, we created fus1 null mutants. In fus1 X fus1 matings, the cells of a mating pair adhered tightly and appeared to form zygotes. However, the zygotes were abnormal. Within the conjugation bridge the contained a partition that prevented nuclear fusion and mixing of organelles. The predicted sequence of the Fus1 protein (deduced from the FUS1 DNA sequence) and subcellular fractionation studies with Fus1-beta-galactosidase hybrid proteins suggest that Fus1 is a membrane or secreted protein. Thus, Fus1 may be located at a position within the cell where it is poised to catalyze cell wall or plasma membrane fusion.

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