Search PubMed⌕ Search

Biomedical subjects

V Hansson

Publications and source records attributed to V Hansson.

At least 163 records · Page 9Linked to original sources
PubMed1981

Purification and kinetic properties of the soluble Mn2+-dependent adenylyl cyclase of the rat testis.

The soluble Mn2+-dependent adenylyl cyclase (AC) of the rat testis was purified 1500-fold with some 19% yield of the initial activity. These results were accomplished by conventional separation techniques including (NH4)2SO4 precipitation of testis cytosol (106 000 x g), gel filtration (Sephadex G-200) and ion-exchange chromatography (Sephadex DEAE-A50) followed by Sephadex G-100 gel filtration and isoelectric focusing. Analysis by polyacrylamide-gel electrophoresis (PAGE) of aliquots from each purification step revealed the following. (a) The Mn2+-dependent AC migrated with a Rf value of 0.40 irrespective of the degree of purification. (2) The AC peak from the isoelectric focusing column separated into 2 major protein bands; however, only one band (Rf 0.40) had AC activity. The molecular weight emerging from the position of migration on the Sephadex G-200 and G-100 columns appeared to be consistent at 47 000-48 000 D, as estimated from the relationship log MW versus elution volume. The purified enzyme fulfilled the requirements for a simple Michaelis-Menten kinetics with an apparent Km for Mn2+ and MnATP2- of 6.7 and 2.5 mM, respectively. Varying the concentrations of ATP or Mn2+ separately did not alter the apparent affinity (Kmapp) for the other parameter. These and previous data from our laboratory show that the physico-chemical and kinetic properties (molecular weight and Kmapp for Mn2+ and MnATP2-) do not alter during purification. Furthermore, the additional step of affinity chromatography seems obligatory if a homogeneous AC preparation is to be obtained.

Adenylyl Cyclases↗
PubMed1981

Age dependent changes in FSH responsive adenylyl cyclase and FSH binding in rat testes.

FSH responsive adenylyl cyclase (AC) activities were examined in both homogenates and membrane particles from 9-71 days old rat testes, and FSH binding was determined in membrane particles from the same animals. FSH responsive AC was measured in the presence of either GTP (0.04 mM) or the non-metabolizable GTP analogue, GMP-P(NH)P (0.04 mM). From days 9 and 11 there was a gradual decrease in both basal and FSH stimulated AC activities and a very similar decrease in FSH binding. The relative stimulation by FSH, however, was fairly constant (2-3 fold stimulation). The excellent correlation between specific FSH binding and FSH responsive AC activities during sexual maturation does not support the notion that uncoupling of the FSH receptors from the AC is a primary reason for the attenuated FSH response during pubertal development. The age dependent decrease in both basal and FSH stimulated AC activities during sexual maturation is a reflection of the relative increase in germ cells (containing no FSH receptors and FSH responsive AC). The LH/hCG responsive AC in membrane particles from the same ages showed an age dependent increase in both absolute activities and relative responses, probably indicating the increased number of Leydig cells in the developing testis.

Adenylyl Cyclases↗
PubMed1981

Cellular localization of the Mn2+-dependent adenylyl cyclase (AC) in rat testis.

In the present study we have examined the exact cellular localization of the Mn2+-dependent adenylyl cyclase (AC) in the rat testis. Several pieces of evidence indicate that this testis specific enzyme is exclusively localized to haploid cells in the testis: firstly, the appearance of Mn2+-dependent adenylyl cyclase coincides in time with the occurrence of haploid cells (IC) in the rat testis. Secondly, tests of tfm rats and mice, in which little or no haploid cells are found, exhibit negligible Mn2+-dependent AC activity. Finally, separation of various testicular cells by BSA gradient sedimentation (Staput fractionation), revealed that this enzyme activity follows the location of haploid cells; the highest specific AC activities being found in the fractions containing elongated spermatids. Some activity found in the primary spermatocyte fraction (4C cells) can to a large extent be explained by the contamination of haploid cells in this fraction.

Adenylyl Cyclases↗
PubMed1981

Preliminary physico-chemical characterization of the soluble Mn2+-dependent adenylate cyclase in the rat testis.

This paper reports some physico-chemical properties of the Mn+-dependent adenylate cyclase (AC) of the rat testis. The AC activity in the crude cytosol (106 000 x g) migrates as a single peak in a linear sucrose gradient (5--20%) with a sedimentation coefficient (S20,w) of 4,1. The enzyme exhibits an Einstein-Stokes radius (rs) of 30.6 A as assessed by gel filtration (Kav = 0.50) on a calibrated Sephadex G-200 column. The molecular shape is close to spherical (f/f0 = 1.25). Isoelectric focussing of the AC peak (Kav = 0.50) from the Sephadex G-200 column revealed a pI of 5.7. Ion exchange chromatography of the crude cytosol on a Whatman DE-52 column gave a single peak eluting between 0.13--0.17 M NaCl. Electrophoresis on polyacrylamide gels resulted in a migrating AC peak with an apparent Rf of 0.40 relative to bromophenol blue. The Mn2+-dependent AC is not retained on a Con-A Sepharose 4B column indicating that the enzyme molecule does not contain sugars possessing alpha-D-manno- or alpha-D-glucopyranosyl terminal groups. (NH4)2SO4 precipitates the AC activity (4 degrees C) between 30--60% saturation. The yield approximates 85% of total AC activity with the specific activity reaching a plateau (110 pmoles cAMP/mg protein/min.) at 50% saturated (NH4)2SO4. The soluble Mn+-dependent AC of the rat testis thus appears to be relatively symmetrical with a size of approximately 52 000 D and a negative charge at physiological pH. Apparently it does not contain sugar moieties and it exhibits little molecular heterogeneity.

Adenylyl Cyclases↗
PubMed1981

Heterogeneity in end-terminal sugars of rabbit and rat androgen binding protein (ABP).

The interaction between rabbit and rat androgen binding protein (ABP) and rabbit serum testosterone binding globulin (TeBG) with concanavalin A (Con A) was studied using affinity chromatography on Con A-Sepharose 4 B columns. When partly purified rat ABP, equilibrated with [3H]5 alpha-dihydrotesterone [3H]DHT was applied to Con A-Sepharose columns, approximately 50% of the ABP was retained by the column, whereas the remaining was eluted with the break-through protein fraction. A similar picture was found using partly purified rabbit ABP, or crude rabbit rete testis fluid. These studies indicate that both rat and rabbit ABP are glycoproteins, showing heterogeneity in their end-terminal sugars. When partly purified rabbit TeBG was examined by Con A-Sepharose affinity chromatography, the TeBG was completely retained by the column. The different elution patterns between rabbit ABP and rabbit TeBG indicate that these proteins, although showing identical physico-chemical and immunological properties (Weddington et al. 1975a,b), possess differences in their carbohydrate content.

Androgen-Binding Protein↗
PubMed1981

Fine structure and FSH binding of Sertoli cells in the blue fox (Alopex lagopus) in different stages of reproductive activity.

A clearly different Sertoli cell morphology was found in the immature blue fox and in the adults in and out of season. Immature cells had small, basally, located nuclei rich in peripheral heterochromatin, and few cytoplasmic organelles. Sertoli cells from adults in season had basally located, large, convoluted nuclei with homogenous nucleoplasma and prominent nucleoli. The basal and intermediate parts of the cytoplasm contained an extensively developed ER, numerous mitochondria, free ribosomes, lipid droplets and residual bodies, while the apical cytoplasm showed few distinct structures. In Sertoli cells from adults out of season the nuclei were dislocated towards the lumen, and apart from numerous dilated cisternae of ER, there were generally fewer organelles, but more glycogen particles. A marked rise in FSH binding was found towards the breeding season.

Aging↗
PubMed1981

Effects of castration, sex steroids, LHRH and glucocorticoids on LHRH binding in the anterior pituitary of male rats.

In the present study we have examined the effects of gonadotrophin releasing hormone (LHRH), sex steroids and glucocorticoids on the binding of LHRH to receptors in the pituitary of male intact and castrated rats. In intact rats, LHRH (10 microgram/day) treatment for 11 days caused a significant increase in LHRH binding, whereas testosterone (500 microgram/day) or oestradiol (50 microgram/day) were inhibitory. 17-hydroxyprogesterone and dexamethasone were without effects. In castrated rats, LHRH caused a marginal decrease in LHRH binding. Much greater inhibition was observed with testosterone and oestradiol. 17-hydroxyprogesterone reduced binding to that of intact controls, whereas dexamethasone was ineffective. When different doses of sex steroids were tested, both oestradiol, testosterone and 5 alpha-dihydrotestosterone inhibited LHRH in a dose-dependent manner. The lowest doses of steroids causing significant inhibition of LHRH binding in castrated animals were 0.5, 50 and 500 microgram/day for oestradiol, 5 alpha-dihydrotestosterone and testosterone, respectively. The present study shows that pituitary receptors for LHRH are regulated both by sex steroids and LHRH itself.

Animals↗
PubMed1981

Age-related changes in [125I]hLH specific binding to rat interstitial cells.

Enriched Leydig cell suspensions were prepared from rats ranging from 5 to 80 days of age. The cells were incubated with [125I]hLH in vitro, and the proportion of LH binding cells determined by means of autoradiography. The proportion of 3 beta-hydroxy-steroid-dehydrogenase (3 beta-HSD) positive cells was also determined. By means of Scatchard analysis and determination of grain counts of LH positive cells, an increase in LH receptors per LH binding cells with increasing age was found. LH binding cells from animals 5 and 10 days of age had few LH receptors, more than a doubling occurred between day 10 and day 20, a significant increase was seen between day 20 and 40 whereas only a slight increase in LH receptors per LH binding cell was seen thereafter. Scatchard analysis and grain count analysis of labelled cells gave essentially the same results. The grain count distribution showed cells distributed around low grain values in animals of all ages, whereas a subpopulation of cells with high grain counts appeared in maturing and mature animals. Only a small fraction of LH positive cells had detectable levels of 3 beta-HSD activity in animals 5 and 10 days of age. With increasing age the proportion of 3 beta-HSD positive cells approached that of LH binding cells, indicating that rat interstitial cells acquire LH receptors well before any 3 beta-HSD activity can be traced.

3-Hydroxysteroid Dehydrogenases↗
PubMed1981

Changes in rat testicular adenylate cyclase activities and gonadotrophin binding during unilateral experimental cryptorchidism.

Unilaterally cryptorchid rats were examined at 3, 8, 15, 22 and 28 days after operation. There was a selective decrease in the adenylate cyclase (ATP pyrophosphate--lyase (cyclizing), EC 4.6.1.1) responses to gonadotrophin stimulation in the abdominal testis. This was associated with a parallel decrease in specific FSH and LH binding. There was no reduction in the response of testicular adenylate cyclases to prostaglandin (PG) E-1 or fluoride stimulation, indicating that both the GTP binding protein (N-component) and the catalytic subunit of the adenylate cyclase complexes were intact. The reduction in FSH-responsive adenylate cyclase activity in the abdominal testis was not due to a change in the Km for adenylate cyclase activation, but was due to a reduction in maximal velocities. Unilateral cryptorchidism was also associated with a rapid decline in soluble Mn2+-dependent adenylate cyclase activity in germ cells (spermatids). By 3 days after operation there was an 82% decrease in germ cell adenylate cyclase activity. The loss of soluble Mn2+-dependent adenylate cyclase activity was associated with a parallel decrease in Sertoli cell secretion of androgen binding protein, indicating that Sertoli cell factors may be important for the maintenance of germ cell adenylate cyclase activity. The desensitization of the gonadotrophin--responsive adenylate cyclases and the loss of gonadotrophin receptors in Leydig and Sertoli cells were not due to changes in plasma gonadotrophin values because LH concentrations were within normal limits and plasma FSH was only marginally elevated in the cryptorchid rats. No significant alterations of any of these parameters were seen in the scrotal testis of unilaterally cryptorchid rats when compared to values for intact controls.

Adenylyl Cyclases↗
PubMed1981

Specific prolactin binding in the rat adrenal gland: its characterization and hormonal regulation.

Rat adrenal prolactin receptors possess the same hormonal specificity as those in the prostate gland and liver, but are less stable during storage and after freezing. There is a gradual decrease in specific prolactin binding to the adrenal during sexual maturation in male rats; maximum binding capacity of 980 fmol/mg protein is at 25 days of age decreasing to approximately 100 fmol/mg protein at day 90. Prolactin receptors in the prostate are high at 25 days of age (700 fmol/mg protein), decrease sharply by day 30 (180 fmol/mg protein) and then gradually increase. Ovariectomy resulted in a significant rise in total prolactin binding in the adrenal gland, while the administration of oestradiol or testosterone reduced the binding, the reverse of changes in prolactin binding in the liver. Only oestrogen increased serum levels of prolactin in female rats. Ovine prolactin (500 micrograms) given to female rats resulted in a rapid increase over a period of 2-8 h total prolactin receptors in the adrenal, and these then decreased to normal levels, indicating a possible positive regulation of prolactin receptors by homologous hormone.

Adrenal Glands↗
PubMed1981

Temporal relationship between hCG induced desensitization of LH/hCG responsive adenylyl cyclase and downregulation of LH/hCG receptors in the rat testis.

A single s.c. injection of 75 IU of human chorionic gonadotropin (hCG) into adult male rats caused a transient desensitization of the LH/hCG responsive adenylyl cyclase (AC) in membrane particles from rat testis. Two hours after the injection of hCG, LH/hCG responsive AC was reduced by 40%, whereas LH binding was still normal. After 24 hr LH/HCG responsive AC was lost, whereas LH binding only showed a marginal decrease. During the next 2-3 days there was a gradual loss of LH/HCG receptors, which reached a nadir at day 4 (14% of control levels). At day y and 9 following the hCG injection LH/hCG receptors and LH/hCG responsive AC gradually returned towards normal. Fluoride (F-) stimulated AC activity showed only minor changes throughout the period investigated. The initial loss of AC responsiveness to hCG was associated with maximal levels of circulating hCG. However, desensitization persisted for several days after hCG was cleared form the circulation. AC desensitization preceded LH receptor downregulation and may support the notion that uncoupling of the catalytic subunit of the AC from Leydig cell LH/hCG receptors is a requirement for subsequent receptor internalization.

Adenylyl Cyclases↗
PubMed1981

Testicular cyclic nucleotide phosphodiesterase in the rat. Kinetic properties and changes with age.

The assay for cyclic nucleotide phosphodiesterase has been applied, with certain modifications, to the measurement of the soluble forms of these enzymes in the rat testis. The homogenization and incubation conditions were adjusted to achieve linear product formation as a function of time and protein concentrations and the resulting products were isolated by ion exchange chromatography using 5 mM HCl as the eluting agent. Phosphodiesterase activities were present in the testicular cytosol (105,000 g supernatant) of adult rats which were capable of hydrolyzing cAMP with a high (Km2 microM) and low Km20 microM) affinity and GMP with a relatively high affinity (Km3 microM). The low affinity cAMP enzyme activity could be stimulated with divalent ions such as calcium, magnesium, and manganese. At 18 days of age, all three enzyme activities were present in the testis, although both the high and low affinity cAMP phosphodiesterase displayed maximal rates (Vmax) that were only one third of the adult testis (when expressed per mg protein).

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Characterization of protein carboxyl methylase activities in rat testis: presence of testis specific charge isomers.

The physicochemical and molecular properties of testicular Protein Carboxyl Methylase (PCM) have been studied. The testicular enzyme was stable at high ionic strength, independent of metal ions and resistant to reducing or alkylating agents, and displayed a narrow pH optimum of pH 5.8-6.0. The enzyme had a molecular weight of approximately 25,000 daltons and a Km for S-adenosyl-L-methionine (SAM) of 1.3 microM. Varying concentrations of gelatine (exogenous methyl acceptor protein) did not change the affinity of the enzyme for SAM. The molecular properties of the enzyme were characterized using gel filtration, hydroxyapatite and DEAE-cellulose chromatography, and isoelectric focusing. Three charge isomers exhibiting PCM activity were isolated with pI values of 6.1, 6.7, and 7.35 while comparative data from the pituitary gland revealed primarily the presence of the most acid isozyme (pI 6.1). These results suggest that the different testicular charge isozymes may be involved in the selective methylation of specific testicular proteins.

Animals↗