The presence of a high-
K
m hexokinase activity was tested in both dog and boar spermatozoa. Hexokinase kinetics from dog extracts showed the presence of a specific activity (dog-sperm ...glucokinase-like protein, DSGLP), in the range of glucose concentrations of 4–10 mM, whereas boar sperm did not show any DSGLP activity. Furthermore, dog-sperm cells, but not those of boar, showed the presence of a protein which specifically reacted against a rat-liver anti-glucokinase antibody. This protein also had a molecular weight equal to that observed in rat-liver extracts, suggesting a close similarity between both the proteins. This glucokinase-like protein was distributed in the peri- and post-acrosomal zones of the head, and the midpiece and principal piece of tail of dog spermatozoa. These results indicate that dog spermatozoa have functional high-
K
m hexokinase activity, which could contribute to a very fine regulation of their hexose metabolism. This strict regulation could ultimately be very important in optimizing dog-sperm function along its life-time.
Incubation of hepatocytes isolated from fasted rats with
14Cglucose for short periods of time showed that the initial stages of glycogen synthesis occur near the plasma membrane. Incubation with
...14Cglucose followed by cold glucose demonstrated that glycogen synthesis is always active at the hepatocyte periphery and that previously synthesised glycogen moves towards the centre of the cell, while its place is filled by newly synthesised molecules. However, the reverse experiment, incubation with cold glucose before addition of
14Cglucose, showed that, as glycogen synthesis progresses, it also becomes gradually active in more internal sites of the hepatocyte. These results indicate a spatial order in the synthesis of hepatic glycogen.
Changes in the intracellular distribution of liver glycogen synthase (GS) might constitute a new regulatory mechanism for the activity of this enzyme at cellular level. Our previous studies indicated ...that incubation of isolated hepatocytes with glucose activated GS and resulted in its translocation from a homogeneous cytosolic distribution to the cell periphery. These studies also suggested a relationship with insoluble elements of the cytoskeleton, in particular actin. Here we show the translocation of GS in a different experimental model that allows the analysis of this phenomenon in long-term studies. We describe the reversibility of translocation of GS and its effect on glycogen distribution. Incubation of cultured rat hepatocytes with glucose activated GS and triggered its translocation to the hepatocyte periphery. The relative amount of the enzyme concentrated near the plasma membrane increased with time up to 8 h of incubation with glucose, when the glycogen stores reached their maximal value. The lithium-induced covalent activation of GS was not sufficient to cause its translocation to the cell periphery. The intracellular distribution of GS closely resembled that of glycogen. Our results showed an interaction between GS and an insoluble element of the hepatocyte matrix. Although no co-localization between actin filaments and GS was observed in any condition, disruption of actin cytoskeleton resulted in a significantly lower percentage of cells in which the enzyme translocated to the cell periphery in response to glucose. This observation suggests that the microfilament network has a role in the translocation of GS.
This manuscript describes a new strategy for introducing secondary school students to biochemistry. To bridge the gap between secondary education and the university, the Department of Biochemistry ...and Molecular Biology of the University of Barcelona, in collaboration with the SEBBM (The Spanish Society for Biochemistry and Molecular Biology), designed a course with lectures and practical classes for students in their last year of secondary school. The impact of this course on society has been considerable, and it is now a reference model for other disciplines.
Intra-testicular inoculation of an adenoviral vector carrying the fusion gene
Aequorea victoria green fluorescence protein/rat-liver glycogen synthase (GFP/LGS) resulted in the presence of GFP/GLS in ...spermatozoa from 7
days to, at least, 16
days after inoculation. The GFP/LGS was detected in the sperm heads after an “in vitro” fertilization procedure, either before or after the oocyte penetration. Our results indicate that spermatozoa carrying GFP/LGS protein conserved their fertilizing ability and were also detectable after oocyte penetration. This technique will allow to develop an easy system to follow the fate of mature sperm proteins.
Changes in the intracellular distribution of liver glycogen synthase (GS) might constitute a new regulatory mechanism for the activity of this enzyme at cellular level. Our previous studies indicated ...that incubation of isolated hepatocytes with glucose activated GS and resulted in its translocation from a homogeneous cytosolic distribution to the cell periphery. These studies also suggested a relationship with insoluble elements of the cytoskeleton, in particular actin. Here we show the translocation of GS in a different experimental model that allows the analysis of this phenomenon in long-term studies. We describe the reversibility of translocation of GS and its effect on glycogen distribution. Incubation of cultured rat hepatocytes with glucose activated GS and triggered its translocation to the hepatocyte periphery. The relative amount of the enzyme concentrated near the plasma membrane increased with time up to 8h of incubation with glucose, when the glycogen stores reached their maximal value. The lithium-induced covalent activation of GS was not sufficient to cause its translocation to the cell periphery. The intracellular distribution of GS closely resembled that of glycogen. Our results showed an interaction between GS and an insoluble element of the hepatocyte matrix. Although no co-localization between actin filaments and GS was observed in any condition, disruption of actin cytoskeleton resulted in a significantly lower percentage of cells in which the enzyme translocated to the cell periphery in response to glucose. This observation suggests that the microfilament network has a role in the translocation of GS.
The presence of a high‐K
m hexokinase activity was tested in both dog and boar spermatozoa. Hexokinase kinetics from dog extracts showed the presence of a specific activity (dog‐sperm ...glucokinase‐like protein, DSGLP), in the range of glucose concentrations of 4–10 mM, whereas boar sperm did not show any DSGLP activity. Furthermore, dog‐sperm cells, but not those of boar, showed the presence of a protein which specifically reacted against a rat‐liver anti‐glucokinase antibody. This protein also had a molecular weight equal to that observed in rat‐liver extracts, suggesting a close similarity between both the proteins. This glucokinase‐like protein was distributed in the peri‐ and post‐acrosomal zones of the head, and the midpiece and principal piece of tail of dog spermatozoa. These results indicate that dog spermatozoa have functional high‐K
m hexokinase activity, which could contribute to a very fine regulation of their hexose metabolism. This strict regulation could ultimately be very important in optimizing dog‐sperm function along its life‐time.
We have studied the intracellular distribution in vivo of glucokinase (GK) and glucokinase regulatory protein (GKRP) in livers of fasted and refed rats, using specific antibodies against both ...proteins and laser confocal fluorescence microscopy. GK was found predominantly in the nucleus of hepatocytes from starved rats. GK was translocated to the cytoplasm in livers of 1- and 2-h refed animals, but returned to the nucleus after 4 h. GKRP concentrated in the hepatocyte nuclei and its distribution did not change upon refeeding. These results show that, in physiological conditions, GKRP is present predominantly in the nuclei of hepatocytes and that the translocation of hepatic GK from and to the nucleus is operative in vivo.
PDF
