Characterization of the Effects of Divalent Cations on the Coupled Activities of the H-ATPase in Tonoplast Vesicles

The substrate requirement of the H⁺-ATPase in purified corn root tonoplast vesicles was investigated. The coupled activities, ATP hydrolysis and proton pumping, were simultaneously supported only by Mg²⁺ or Mn²⁺. The presence of Ca²⁺ or Ba²⁺ did not significantly affect the coupled activities. The addition of Cd²⁺, Co²⁺, Cu²⁺, and Zn²⁺ inhibited both the hydrolysis of Mg-ATP and the proton transport. However, the inhibition of proton pumping was more pronounced. Based on equilibrium analysis, both ATP-complexed and free forms of these cations were inhibitory. Inhibition of the hydrolysis of Mg-ATP could be correlated to the concentrations of the ATP-complex of Zn. On the other hand, the free Cu²⁺ and Co²⁺ were effective in inhibiting hydrolysis. For proton pumping, the ATP complexes of Co²⁺, Cu²⁺, and Zn²⁺ were effective inhibitors. However, this inhibition could be further modulated by free Co²⁺, Cu²⁺, and Zn²⁺. While the equilibrium concentrations of Cd-ATP and free Cd²⁺ were not estimated, the total concentration of this cation needed to inhibit the coupled activities of the H⁺-ATPase was found to be in the range of 10 to 100 micromolars. The presence of free divalent cations also affected the structure of the lipid phase in tonoplast membrane as demonstrated by the changes of emission intensity and polarization of incorporated 1,6-diphenyl-1,3,5-hexatriene. The differential inhibition caused by these cations could be interpreted by interactions with the protogenic domain of the membrane as previously proposed in “indirect-link” mechanism.     

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H-ATPase: I. Characteristics and Intracellular Localization of the Fusicoccin Receptor in Microsomes from Radish Seedlings

The characteristics of fusicoccin binding were investigated in microsomes from 24-h-old radish (Raphanus sativus L.) seedlings. The time course of fusicoccin binding depended on fusicoccin concentration: equilibrium was reached much faster at 10 nanomolar fusicoccin than at 0.3 nanomolar fusicoccin. Scatchard analysis of equilibrium binding as a function of fusicoccin concentration indicated a single class of receptor sites with a K_d of 1.8 nanomolar and a site density of 6.3 picomoles per milligram protein. Similar values (K_d 1.7 nanomolar and site density 7 picomoles per milligram protein) were obtained from the analysis of the dependence of equilibrium binding on membrane concentration at fixed fusicoccin concentrations. Fusicoccin binding comigrated with the plasma membrane H⁺-ATPase in an equilibrium sucrose density gradient: both activities formed a sharp peak (1.18 grams per milliliter) clearly distinct from that of markers of other membranes which all peaked at lower densities. The saturation profiles of fusicoccin binding and of fusicoccin-induced activation of the plasma membrane H⁺-ATPase, measured under identical conditions, were similar, supporting the view that fusicoccin-induced activation of the plasma membrane H⁺-ATPase is mediated by fusicoccin binding to its plasma membrane receptor.     

Salinity effects on photosynthesis in isolated mesophyll cells of cowpea leaves

Mesophyll cells from leaves of cowpea (Vigna unquiculata [L.] Walp.) plants grown under saline conditions were isolated and used for the determination of photosynthetic CO₂ fixation. Maximal CO₂ fixation rate was obtained when the osmotic potential of both cell isolation and CO₂ fixation assay media were close to leaf osmotic potential, yielding a zero turgor pressure. Hypotonic and hypertonic media decreased the rate of photosynthesis regardless of the salinity level during plant growth. No decrease in photosynthesis was obtained for NaCl concentrations up to 87 moles per cubic meter in the plant growing media and only a 30% decrease was found at 130 moles per cubic meter when the osmotic potential of cell isolation and CO₂ fixation media were optimal. The inhibition was reversible when stress was relieved. At 173 moles per cubic meter NaCl, photosynthesis was severely and irreversibly inhibited. This inhibition was attributed to toxic effects caused by high Cl⁻ and Na⁺ accumulation in the leaves. Uptake of sorbitol by intact cells was insignificant, and therefore not associated with cell volume changes. The light response curve of cells from low salinity grown plants was similar to the controls. Cells from plants grown at 173 moles per cubic meter NaCl were light saturated at a lower radiant flux density than were cells from lower salinity levels.     

Fructose 2,6-bisphosphate hydrolyzing enzymes in higher plants

The phosphatases that hydrolyze fructose 2,6-bisphosphate in a crude spinach (Spinacia oleracea L.) leaf extract were separated by chromatography on blue Sepharose, into three fractions, referred to as phosphatases I, II, and III, which were further purified by various means. Phosphatase I hydrolyzed fructose 2,6-bisphosphate, with a K_m value of 30 micromolar, to a mixture of fructose 2-phosphate (90%) and fructose 6-phosphate (10%). It acted on a wide range of substrates and had a maximal activity at acidic pH. Phosphatase II specifically recognized the osyl-link of phosphoric derivatives and had more affinity for the β-anomeric form. Its apparent K_m for fructose 2,6-bisphosphate was 30 micromolar. It most likely corresponded to the fructose-2,6-bisphosphatase described by F. D. Macdonald, Q. Chou, and B. B. Buchanan ([1987] Plant Physiol 85: 13-16). Phosphatase III copurified with phosphofructokinase 2 and corresponded to the specific, low-K_m (24 nanomolar) fructose-2,6-bisphosphatase purified and characterized by Y. Larondelle, E. Mertens, E. Van Schaftingen, and H. G. Hers ([1986] Eur J Biochem 161: 351-357). Three similar types of phosphatases were present in a crude extract of Jerusalem artichoke (Helianthus tuberosus) tuber. The concentration of fructose 2,6-bisphosphate decreased at a maximal rate of 30 picomoles per minute and per gram of fresh tissue in slices of Jerusalem artichoke tuber, upon incubation in 50 millimolar mannose. This rate could be accounted for by the maximal extractable activity of the low-K_m fructose-2,6-bisphosphatase. A new enzymic method for the synthesis of β-glucose 1,6-bisphosphate from β-glucose 1-phosphate and ATP is described.     

Temporal changes in serum prostaglandin F2alpha and oxytocin in dairy cows with short luteal phases after the first postpartum ovulation

Luteolysis in the cow depends upon an interaction between prostaglandin F(2alpha) (PGF(2alpha)) and oxytocin. The objectives of our study were 1) to determine oxytocin concentrations in postpartum dairy cows and 2) to identify the temporal relationship between oxytocin and PGF(2alpha) release patterns during luteolysis in normal and abbreviated estrous cycles in the postpartum period. Serum oxytocin and PGF(2alpha) metabolite (PGFM) concentrations from nine cows which had short estrous cycles (< 17 d) were compared with those of six cows which had normal estrous cycles. Serum basal oxytocin concentrations in short estrous cycle cows (23.7 to 31.1 pg/ml) were higher (P<0.05) than those of normal estrous cycle cows (14.6 to 19.8 pg/ml). Oxytocin concentrations increased to peak values in both short and normal cycle cows, during luteolysis. Basal PGFM concentrations (112.2 to 137.4 pg/ml) were higher in cows with short cycle (P<0.05) than in cows with normal cycles (62.9 to 87.5 pg/ml). The increase in PGFM concentrations during luteolysis was significant in both normal cycle and short cycle cows (P<0.05). Increases in serum PGFM concentrations were always associated with increases in serum oxytocin concentrations in normal cycle and short cycle cows and the levels decreased simultaneously before the subsequent estrus. Results support the idea of a positive relationship between PGF(2alpha) and oxytocin concentration during the estrous cycle as well as a possible synergistic action of these hormones in the induction of luteolysis in dairy cattle.     

The role of endogenous estrogens in the maturation process of the bovine placenta

The plasma estrogen and progesterone concentrations of 19 pregnant cows (average duration of pregnancy 266.0 +/- 2.3 d at the start of the study) were determined daily from Day 6 pre partum to Day 1 post partum. Parturition was induced in all cows by administration of 10 mg i.m. flumethasone. Values were centered around the delivery date (Day 0) following either induced normal calving (n = 3) or surgical delivery (n = 16). In animals showing spontaneous expulsion of the fetal membranes (Group 1, n = 6) the average total estrogen concentration increased significantly from Day 6 until Day 1 before parturition (1329.2 +/- 317.9 to 3719.8 +/- 951.2 pg/ml in total estrogens). A marked decrease was observed on Day 1 post partum (459.4 +/- 344.2 pg/ml). In comparison with Group 1, animals showing either a delayed or partial expulsion of the fetal membranes, or in which the placenta could be withdrawn 16 h after calving (Group 2, n = 5), had consistently lower total estrogen concentrations between Day 6 (595.4 +/- 174.8 pg/ml) and Day 1 (1884.3 +/- 565.1 pg/ml) before parturition. The estrogen values of the cows with retained placenta (Group 3, n = 8) from Days 6 to 0 pre partum were significantly lower than those of Group 2. Total estrogen concentrations of the three groups 1 d post partum did not differ significantly. It is generally recognized that estrogens play an important role in the maturation process of the placentomes. Our investigation demonstrates that not only is the magnitude of the prepartum rise in estrogens of great influence of the maturation process but the duration of this rise is likewise important. These two factors are vital for a normal expulsion of the fetal membranes.     

Fertility of Bos indicus and Bos indicus x Bos taurus crossbreed cattle after estrus synchronization

Ninety-five cows (79 Boran and 16 Boran-Brahman crossbreeds) and 107 heifers (55 Boran and 52 Boran x Friesian F1 crossbreeds) were used to determine estrus response, estrus response interval and pregnancy rate following synchronization with prostaglandin (PGF(2)alpha), a progesterone-releasing intravaginal device (PRID) and Synchro-mate B (SMB). The proportion of cattle responding to synchronization treatment was 62.5, 43.5 and 57.7% for cows and 85.7, 68.0 and 81.5% for heifers using PGF(2)alpha, PRID and SMB, respectively. The overall mean response was 59 and 81.8% for cows and heifers, respectively. The estrus response of the control animals over a 45-d breeding period was 72.7 and 90% for cows and heifers, respectively. The estrus response interval for cows was 31.8, 22.1 and 18.0 h and it was 51.1, 38.0 and 21.6 h for heifers with PGF(2)alpha, PRID and SMB treatment, respectively. Mean pregnancy rate for cows was 50.0, 34.8, 46.2 and 68.8% and for heifers it was 60.7, 40.0, 55.6 and 77.8% in the PGF(2)alpha, PRID, SMB and control groups, respectively. Based on these findings, it was concluded that both PGF(2)alpha and SMB produce a satisfactory estrus response and pregnancy rate in the cattle studied.     

Ultrasonographic determination of ovarian follicular. Development in superovulated heifers pretreated with FSH-P at the beginning of the estrous cycle

On Day 3 of the estrous cycle (estrus = Day 0), dairy heifers were given either 10 mg i.m. FSH-P (FSH-P primed; n = 9) or a saline vehicle (saline primed; n = 9). On Day 10, all heifers were superovulated with FSH-P (total = 27.7 mg i.m.) in declining doses over 5 d. Heifers were inseminated artificially at estrus. From Day 2 until estrus, the number and size of follicles >2 mm were monitored daily by ultrasonography. The mean (+/- SEM) number of corpora lutea (CL) (6.2 +/- 1.5 vs 10.7 +/- 0.9; P<0.05) and the mean number of recovered embryos and unfertilized ova (3.6 +/- 1.7 vs 8.4 +/- 2.2; P<0.05) were lower in FSH-P-primed than in saline-primed heifers. Prior to initiation of superovulation, follicles >10 mm appeared on Days 6 to 7 in saline-primed heifers but only on Days 8 to 10 in FSH-P-primed heifers (P<0.05). Also, until Day 10, the mean number of follicles 4 to 6 mm and 7 to 10 mm was higher (P<0.05) in FSH-P-primed than in saline-primed heifers. After initiation of the superovulatory treatment (Day 10 to estrus), saline-primed heifers had a greater and faster increase in the mean number of follicles >10 mm (P<0.02) than FSH-P-primed heifers did. Depletion in the number of follicles 2 to 3 mm (P<0.001) between Day 10 and estrus and in the number of follicles 4 to 6 mm (P<0.05) between Day 12 and estrus occurred in both groups of heifers. Decreased superovulatory response and embryo recovery in FSH-P-primed heifers may have been due to the presence of large follicles (>10 mm) prior to the initiation of the superovulatory treatment which reduced the ability of small follicles to grow into larger size classes during superovulatory treatment.     

Effects of cloprostenol, human chorionic gonadotropin and estradiol benzoate treatment on estrus synchronization in dairy cows

Two consecutive experiments were conducted. In Experiment 1, 24 Friesian lactating cows were randomly assigned to two groups. Cows in Group I received intramuscularly (i.m.) 500 mcg of cloprostenol, 1250 IU of human chorionic gonadotropin (hCG) and 5 mg of estradiol benzoate 12 h after cloprostenol treatment. Cows in Group II received 750 IU i.m. of hCG and 3 mg of estradiol benzoate 12 h after cloprostenol treatment. Treatment was given on Day 16 after estrus in both groups. All animals showed estrus within 24 to 48 h after cloprostenol treatment. The average interval from cloprostenol injection to the onset of estrus was not influenced by treatments. Four cows in Group I failed to ovulate and became cystic. In Experiment 2, 71 Friesian lactating cows were randomly assigned to two groups. Cows in Group I received 500 mcg i.m. of cloprostenol after corpus luteum detection by palpation per rectum. Cows in Group II received 500 mcg of cloprostenol plus 750 IU of hCG and 3 mg of estradiol benzoate 12 h after. When estrus ready for service was confirmed by rectal examination, cows were inseminated. The percentage of cows ready for service tended to be lower (P < 0.06) between cows in Group I (88%) and those in Group II (100%). The average interval from cloprostenol treatment to service was longest (P < 0.001) in Group I (78.7 h +/- 14.9, X +/- SD) vs Group II (48 h +/- 2.9). The degree of readiness for service synchrony was lowest (P < 0.001) in Group I (59.3%) vs Group II (94.2%). The pregnancy rates of cows synchronized or treated were not altered by hCG-estradiol benzoate treatment (P > 0.25). These results suggest that in dairy cows treated with cloprostenol following palpation per rectum of a corpus luteum and then with 750 IU of hCG and 3 mg of estradiol benzoate 12 h later, a single fixed-time insemination at 48 h after cloprostenol treatment should be performed.     

Postpartum adrenocortical function in dairy cows with dystocia submitted to cesarean section

The effects of the stress of dystocia on the adrenocortical function post partum in cows (n = 6) requiring a cesarean section were assessed by an adrenocorticotropic hormone (ACTH) stimulation test. Nine cows that calved normally were used as controls. The plasma glucocorticoid levels, before and 60 min after an intramuscular injection of 25 IU ACTH, were 4.4 +/- 0.5 (mean +/- SD) and 21.5 +/- 2.4 ng/ml 1 d post partum, 1.9 +/- 0.3 and 18.4 +/- 2.9 ng/ml 4 d post partum and 2.7 +/- 1.1 and 14.6 +/- 3.3 ng/ml 8 d post partum, respectively. Corresponding values of glucocorticoids in cows with normal calving were 4.5 +/- 3.6 and 18.1 +/- 5.2 ng/ml 1 d post partum, 1.7 +/- 1.3 and 13.2 +/- 5.5 ng/ml 4 d post partum and 1.9 +/- 1.7 and 14.6 +/- 3.3 ng/ml 8 d post partum. There were no statistically significant differences of the values between cows with dystocia requiring a cesarean section and cows with normal calving. The results indicate that dystocia requiring a cesarean section like normal calving does not lead to significant depression of the adrenocortical function post partum.