Proteolysis and the diurnal decrease of asparaginase activity

The mechanism responsible for the decrease in asparaginase (EC 3.5.1.1) activity in darkened leaves of Pisum sativum L. cv. Little Marvel was investigated. Asparaginase activity, obtained from half-expanded leaves harvested at the end of the dark period, or during the light periods, was inactivated by bromelain (EC 3.4.22.4), ficin (EC 3.4.22.3), both thiol proteases, and trypsin (EC 3.4.21.4), a serine protease. Thrombin (EC 3.4.21.5), pepsin (EC 3.4.23.1), or carboxypeptidase A (EC 3.4.17.1) had no effect on dark- or light-harvested asparaginase preparations. Inactivation of asparaginase activity in crude or purified preparations by ficin was not observed in the presence of leupeptin (an inhibitor of thiol proteases). Supplying leupeptin to detached half-expanded leaves had no effect on the increase of asparaginase observed at the start of the light period, while it maintained asparaginase activity at high levels in leaves excised during or at the end of the light period. These results suggest that decreased asparaginase activity in vivo is brought about by thiol-dependent proteases.     

Taste bud density in circumvallate and fungiform papillae of the bovine tongue

Taste bud quantitation may provide useful parameters for interspecies comparisons of the gustatory system. The present study is a morphometric analysis of bovine taste papillae. Circumvallate and fungiform papillae from six bovine tongues were serially sectioned and, following staining, analyzed. Circumvallate papillae were found to have a mean volume of 3.66 +/- 2.82 mm3, a mean number of taste buds per papilla of 445 +/- 279, and a mean taste bud density of 155 +/- 112 buds/mm3. Values for lateral fungiform papillae for the same three parameters were 0.384 +/- 0.184 mm3, 13.2 +/- 13.4, and 40.8 +/- 46.6 buds/mm3, respectively. Values for dorsal fungiform papillae were 0.438 +/- 0.246 mm3, 4.39 +/- 4.78, and 14.0 +/- 17.1 buds/mm3, respectively. Circumvallate papillae were found to have a significantly greater volume, number of taste buds per papilla, and taste bud density than either type of fungiform papilla. These data should serve as background for biochemical, endocrinological, or neurological studies involving the bovine tongue.     

Identification of estrogen receptors in granulosa cells of immature rats

Nuclear and cytoplasmic binding sites for estradiol (E2-17 beta) in granulosa cells of immature rats were characterized. These binding sites for estrogen were high affinity, low capacity with an affinity constant (Kd) of 1.9 X 10(-10)M (binding capacity, Ro = 80 pM) for nuclear sites and a Kd = 3.5 X 10(-10) M (Ro = 45 pM) for cytosol sites. Binding was specific for biologically active estrogens. The estrogen receptor in granulosa cells is a protein and heat-labile as treatment with protease or pre-incubation at 37 degrees C for 1 h significantly diminished binding. RNase and DNase had no effect on estrogen binding. Sedimentation coefficients for nuclear and cytosol binding components were 5S and 8S respectively, similar to values obtained with uteri. Finally, translocation was demonstrated after a s.c. injection of E2-17 beta. Forty-five minutes post-injection, cytosol binding sites for estradiol were depleted concomitant with accumulation of nuclear binding sites. We concluded that granulosa cells of immature rats have binding sites specific for estradiol which have characteristics similar to the classical estrogen receptor in uteri.     

A low-cost microcomputer-based data acquisition and analysis system for an electron spin resonance spectrometer: data handling of dilute spin labeled nucleic acids

This article describes the construction of an inexpensive and reliable data acquisition system for a Varian E-line Century Series ESR spectrometer utilizing an Apple II Plus microcomputer. All necessary hardware is readily available and used without modification. A BASIC program for routine collection, display, plotting and disk storage of experimental data has been written and subsequently compiled into machine code for high speed operation. The interface offers distinct advantages in spectral resolution as well as instrument control. An example of signal enhancement via computer controlled time averaging is presented for a spin labeled DNA experiment. The technique has recently been applied to studies of relative binding affinities of gene-32 protein for various spin-labeled polynucleotides.     

Two routes for asparagine metabolism in Pisum sativum L.

Asparagine, a major transport compound, is metabolized in Pisum sativum by two enzymes, asparaginase (EC 3.5.1.1) and asparagine-pyruvate aminotransferase. The relative amount of the two enzymes varies between tissues. In developing seeds, there are very high levels of asparaginase but only trace amounts of the aminotransferase. Asparaginase is high in young leaves but falls rapidly during leaf growth; the aminotransferase remains high throughout development. Inhibitor studies with aminooxyacetate and methionine sulfoximine confirm that the aminotransferase is the main enzyme involved in asparagine utilisation in the leaf. Root tissue has low levels of asparaginase and only trace amounts of the aminotransferase. The asparaginase is potassium dependent, but is also partially activated by ammonium ions. The leaf aminotransferase has a lower K _m for asparagine (4.5 mM) than the leaf asparaginase (8 mM). The seed asparaginase has a lower K _m for asparagine (3 mM) than the leaf asparaginase.     

Detection and determination of adenosine diphosphate and related substances in plasma

1. A method is described for detecting and determining the products of metabolism of ADP added to plasma at initial concentrations of about 1mum-ADP. 2. ATP, ADP, AMP, adenosine, inosine and hypoxanthine were detected in human platelet-rich plasma after incubation with ADP and in the presence of either heparin or heparin-citrate. 3. The products of incubation of ADP with human platelet-poor plasma in the presence of heparin were the same as with platelet-rich plasma, except that, when the initial concentration of ADP was 1.5mum, little or no ATP was detected. 4. The ATP detected in platelet-rich plasma when 1.5mum-ADP was initially incubated was present in the platelets and not in the plasma. 5. The time for 50% decay of ADP in either platelet-rich or platelet-poor plasma in the presence of heparin was about 20min. when the initial concentration of ADP was 200mum, but was 6-9min. when the initial ADP concentration was 1.5-2.5mum. The corresponding values in the presence of heparin-citrate were about 45min. and about 9-12min. respectively. 6. Hypoxanthine accumulated to a greater extent in platelet-rich than in platelet-poor plasma after the addition of ADP. 7. After incubation for 15-20min. of either platelet-rich plasma or suspensions of washed platelets in saline with adenosine at an initial concentration of about 3-4mum, ATP, ADP and AMP were detected in the platelets. Similar incubations of washed platelets with inosine also showed the formation of these substances, but to a much less extent. 8. After the addition of adenosine to suspensions of washed platelets in saline, inosine and hypoxanthine were detected in the incubation mixture. After the addition of inosine, hypoxanthine was detected. 9. When ADP at an initial concentration of 1.5mum was added to platelet-rich plasma containing adenosine deaminase, no adenosine was detected in the incubation mixture. There was no difference in the rate of decay of ADP in the presence or absence of the deaminase, but ATP formation was decreased in its presence.     

Development of the small intestine of piglets in response to prenatal elevation of glucocorticoids.

The effects of prenatal adrenal stimulation and synthetic glucocorticoid supplementation on development of the gastro-intestinal tract of the piglet were investigated. Twelve pregnant sows were treated with either ACTH infusion, Isoflupredone injection or Saline between days 105 and 112 of gestation. Neonatal pigs were weighed, bled and sacrificed at 0 or at 6 h. Piglets sacrificed at 6 h were fed bovine colostrum. Transverse sections were prepared from the duodenum, jejunum and ileum for measurement of the villus amplification factor (VAF) and basal membrane circumference. Sows in the ACTH group showed an elevation in cortisol in response to infusion; this decreased after infusion and then rose again at parturition. Piglets from both the ACTH and Saline groups had more villus surface area per unit of body weight (BW) than those born to Isoflupredone-treated animals. The BW of the ACTH piglets was lower (P less than 0.05) than those of piglets in the other groups. When the weight of the stomach and the Small Intestine (SI) was expressed as a function of the body weight, the stomach and SI:BW ratio was larger (p less than 0.05) in pigs born to ACTH-treated sows. The circumference of the ileum was larger at 6 h than at 0 h. Control pigs had a higher concentration of bovine IgG at 4 and 6 h (P less than 0.05). Observations of the light microscopic preparations indicated a less organized epithelium in both ACTH and isoflupredone pigs sacrificed at 0 h. Light and EM preparations of ileum from ACTH pigs sacrificed at 6 h, showed an abundance of dark-stained vacuoles, characteristic of IgG-containing structures. These became less evident in piglets from the Isoflupredone group and even less so in the control groups. The consequences of these phenomena in terms of absorptive capacity are discussed.     

Studies on the limitations to photosynthesis in leaves of the atrazine-resistant mutant ofSenecio vulgaris L.

In leaves of an atrazine-resistant mutant ofSenecio vulgaris the quantum efficiency of CO₂ assimilation was reduced by 21% compared to the atrazine-susceptible wild type, and at a light level twice that required to saturate photosynthesis in the wild type the CO₂ fixation rate in the mutant was decreased by 15%. In leaves at steady-state photosynthesis there was a measurable increase in the reduction state of the photosystem II (PSII) primary quinone acceptor,Q _A. Although this would lead to a decreased rate of PSII electron transport and may thus explain the decrease in quantum efficiency, this cannot account for the fall in the maximum rate of CO₂ fixation. The atrazine-resistant mutant showed an appreciably longer photosynthetic induction time which indicates an effect on carbon metabolism; however, the response of CO₂-fixation rate to intercellular CO₂ concentration revealed no differences in carboxylation efficiency. There were also no differences in the ability to perform a State 1–State 2 transition between the atrazine-resistant and susceptible biotypes and no difference in the profiles of phosphorylated thylakoid polypeptides. It is concluded that the alteration of the redox equilibrium between PSII quinone electron acceptors in the atrazine-resistant biotype limits appreciably the photosynthetic efficiency in non-saturating light. Additionally, there is a further, as yet unidentified, limitation which decreases photosynthesis in the resistant mutant under light-saturating conditions.Abbreviations and symbols DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F _max maximum fluorescence emission - F _o2 minimal fluorescence emission upon exposure to saturating light flash - F _v variable fluorescence emission - F _v2 variable fluorescence emission upon exposure to saturating light flash - kDa kilodalton - PSI, II photosystems I, II - Q _A primary quinone acceptor of PSH - Q _B secondary quinone acceptor of PSII - RuBP ribulose-1,5-bisphosphate