Biochemical and autoradiographic studies of TRH receptors in sections of rabbit spinal cord

Receptors for thyrotropin-releasing hormone (pGlu-His-Pro-NH2, TRH) on thaw-mounted sections of rabbit spinal cord have been identified biochemically and visualized by light microscopic autoradiography. Binding of [3H] [3-Me-His2]TRH to 20 microns sections exhibited high apparent affinity and a pharmacological specificity almost identical to that previously demonstrated for spinal TRH receptors in membranes. In autoradiograms, the highest density of TRH receptors appeared in the substantia gelatinosa of the dorsal gray and around the central canal, with intermediate levels in the ventral gray.     

Studies on the complex formed between glucagon and dicaprylphosphatidylcholine

The interaction between glucagon and dicaprylphosphatidylcholine (DCPC) was studied by fluorescence, circular dichroism and calorimetry, as well as by ¹H- and ³¹P-nuclear magnetic resonance. The water-soluble lipid-protein complex was also characterized by gel filtration and ultracentrifugation. The complex appeared to be monodisperse by sedimentation equilibrium measurements, with a molecular weight of (4.55 ± 0.57)·10⁴. This complex contained approximately 7 molecules of glucagon and 35 molecules of phospholipid. Proton-decoupled ³¹P-NMR spectra of the phospholipid in the lipid-protein complex display narrower resonances than those of sonicated vesicles of DCPC, and ¹H-³¹P coupling could be detected in proton coupled spectra. These NMR results, together with gel-filtration results, suggest that glucagon ‘solubilizes’ phospholipid aggregates, forming a lipid-protein complex which is smaller than sonicated preparations of DCPC. ¹H-NMR resonance of both the methionine methyl group (met-27) and the aromatic envelope of glucagon are broadened by the phospolipid, indicating that the C-terminal region and the aromatic residues are involved in the interaction with the phospholipid. Nuclear magnetic resonance titrations of the imidazole ring C(2) and C(4) protons of the histidine residue of glucagon show that DCPC lowers the pK of the imidazole. The alterations caused by the phospholipid in the far and near ultraviolet CD spectra of glucagon reflect, respectively, the increased helix content of the hormone and the fact that the aromatic residues are located in a more structured environment. The phospholipid also alters the fluorescence properties of glucagon, shifting the fluorescence emission maximum of the hormone to shorter wavelength, and enhancing its relative intensity. This suggests that the fluorophore is experiencing a more hydrophobic environment in the presence of the lipid. Binding of glucagon to the phospholipid was analysed by Scatchard plots of the enhancement of fluorescence caused by the phospholipid and showed that the equilibrium binding constants of glucagon to DCPC are (4.4 ± 0.5)·10⁴M^?1 and (7.5±0.5)·10⁴M^?1, at 15°C and 25°C, respectively. The average number of moles of phospholipid bound per mole of glucagon is 4.4±0.6. The isothermal enthalpy of reaction of glucagon with DCPC is ?20.5 kcal/mol of glucagon at 25°C and ?32.5 kcal/mol of glucagon at 15°C. The observed enthalpies can arise from glucagon-induced cyrstallization of the phospholipid, from the non-covalent interactions between the peptide and lipid as well as from the lipid-induced conformational change in the protein. These results demonstrate that, unlike the complexes formed between glucagon and phospholipids which form more stable bilayers, the complex formed between glucagon and DCPC is stable over a wide range of temperatures, including temperatures well above the phase transition.     

A high recovery method for concentrating microgram quantities of protein from large volumes of solution

A method is presented for the recovery of 40-80% of the protein from a 1 microgram/ml solution. The final protein pellet is free of detergent and other ionic compounds and is thus compatible with any denaturing solution. The primary structure of the protein is unaffected by the procedure, making the final pellet an ideal sample for any analytical procedure to determine protein structure.     

Spectroscopic characterization of extracts from humic and fulvic fractions: IR and1H NMR spectra

Summary Infrared and proton resonance spectra have been used to characterize fraction extracted sequentially from humic and fulvic acids by diethylether, acetone, dioxane, tetrahydrofuran, pyridine and dimethylformamide. The results showed that the same solvents extracted structurally similar components from both humic and fulvic acids. On the other hand, the spectra showed solvent-dependent differences, some being characteristic for a preponderance of aliphatic structures, others for aromatic structures.     

Specific photoperiodic stimulation of dry matter production in a high-latitude cultivar of Poa pratensis

Vegetative plants of Poa pratensis L. cv. Holt (origin 69°N) raised in short days gave large and significant increases in plant dry weight, plant height and leaf area upon exposure to continuous light, compared with 8-h short days, at essentially identical daily inputs of radiant energy (8-h summer daylight ± low intensity extension). For example, by the fourth harvest (after 26, 34 and 46 days at 21, 15 and 9°C, respectively), the dry weights of plants in long days were 81, 163 and 195% greater than those of the corresponding short-day controls at the respective temperatures. Plant leaf areas in long days were between two and four times as large as control values by the end of the experiment. This was mainly due to increased leaf length caused by long-day stimulation of cell extension and division. However, the photoperiod did not affect the partitioning of assimilates amongst leaves, culms and stolons. Most of these effects could also be brought about by exogenous gibberellin application to plants in short days. However, in contrast to the effect of long days, gibberellin treatment also induced stem internode elongation even in these vegetative plants. Examination by standard growth analysis procedures revealed that the observed increases in relative growth rate were due primarily to increased net assimilation rate followed, several days later, by increases in leaf area ratio when newly-emerged leaves began to constitute a significant proportion of the leaf area. It is concluded that these reactions are of great adaptive significance for growth at the marginal temperatures prevailing at high latitudes.     

The effect of soil water status on critical phosphorus concentrations inStylosanthes hamata cv. Verano

Summary The effect of soil water status on the critical phosphorus concentration (CPC) determined in apices and whole tops ofStylosanthes hamata cv. Verano was investigated in a glasshouse trial. The species was grown with six rates of P and three ranges of soil water potential and was harvested at 10 and 14 weeks after germination. The CPC of both whole tops and apices decliced between the two harvests. At the first harvest the CPC of both whole tops and apices increased as the soil water potential decreased but at the second harvest there was no effect of soil water potential on CPC. It is suggested that at the earlier harvest water stress was delaying physiological development, resulting in a CPC characteristic of chronologically younger tissue, but that by the second harvest the decline in CPC with age had ceased for all water treatments.     

Requirements for antiribosomal activity of pokeweed antiviral protein

It has been known for some time that pokeweed antiviral protein acts by enzymatically inhibiting protein synthesis on eucaryotic ribosome systems. The site of this action is known to be the ribosome itself. In this paper we show that the pokeweed antiviral protein reaction against ribosomes is a strong function of salt concentrations, where 160 mM K⁺ and 3 mM Mg²⁺ retards the reaction, while 20 mM K⁺ and 2 mM Mg²⁺ allows maximum reaction rate. It is also shown, however, that an unidentified protein in the postribosomal supernatant solution, together with ATP, allows the ribosome to be attacked even in the presence of high salt. Kinetic analysis of the antiviral protein reaction has been carried out under both sets of conditions, and reveals that the turnover number for the enzyme is about 300–400 mol/mol per min. in each case. The K_m for ribosomes is 1 μM in the presence of low salt and 0.2 μM at higher salt in the presence of postribosomal supernatant factors plus ATP. The antiviral protein reaction is also shown to be pH dependent and is controlled by a residue with pK_a value of approx. 7.0, apparently a histidine. Stoichiometric reaction of the enzyme with iodoacetamide results in a significant loss of antiribosomal activity.     

Decomposition of cellulose,soil organic matter and plant litter in a temperate grassland soil

The formation of mor humus in an experimental grassland plot, which has been acidified by long-term fertiliser treatment, has been studied by comparing the rates of cellulose, soil organic matter and plant litter decay with those in an adjacent plot with near-neutral pH and mull humus. The decomposition of cellulose filter paper in litter bags of 5 mm, 1-mm and 45-μm mesh size buried at 3 to 4 cm depth the plots was followed by measuring the weight loss and changes in glucose content over a 6 month period. Soil pH was either 5.3 or 4.3. Decomposition of native soil organic matter and plant litter in soil from the same plots were followed using CO₂ evolution in laboratory microcosms. Cellulose weight loss at pH 5.3 was greatest from the 5-mm mesh bags and least from the 45-um mesh bags. At pH 4.3 there was little weight loss from bags and no significant differences in weight loss between bags with different sized mesh. There was, however, a reduction in the glucose content of the hydrolysed and derivatised filter paper with time. The decomposition rate of native soil organic matter in the low pH soil was increased to that observed in the less acid soil when the pH of the former was increased from 4.3 to 5.3. The increase in decomposition rate of added plant litter in the more acid soil as a result of CA(OH)₂ addition was only 60% of that observed in the soil with pH 5.3. These data support the hypothesis that the absence of soil animals and the restricted microbial decomposition in the acidic soil was responsible for mor humus formation.     

Seasonal nitrogen and carbon concentrations in white,brown and woody fine roots of sugar maple (Acer saccharum Marsh)

Small diameter (<1.0-mm) Acer saccharum Marsh roots were separated into white, brown and woody development state classes and analyzed for total N and C concentrations in April, July and October of 1988. White roots had greater concentrations of N and C than either brown or woody roots at each sampling date, and the N concentration of brown roots was consistently greater than that of woody roots. There were no temporal changes in N concentrations in any of the roots. C was slightly elevated in mid-summer in all three classes of roots. The data suggest the possible existence of an N translocation mechanism in ageing and developing fine roots. More research should be undertaken to establish the mechanisms of N loss in developing fine roots.     

Aluminum geochemistry in peatland waters

The chemical speciation of aluminum was examined in surface water samples from Sphagnum peatlands in north-central Minnesota, from peatlands along the Canadian east coast, and from bogs in the Pennine Mountain area of England. In highly organic ([DOC] 50 mg L^–1 ), low pH waters, 80–90% of total dissolved Al was complexed with organic matter (OM), while in waters with low DOC ([DOC] 5 mg L^–1) 54–86% of total dissolved Al existed as Al⁺³ or other inorganic Al species. Batch titrations of OM with Al revealed a high Al binding capacity, 1.4–2.8 mol (mg DOC)^–1, that generally was unsaturated with Al. Titrations of OM with Al in conjunction with a continuous distribution model were used to determine Al-OM conditional stability constants. Binding capacity (mol Al (mg DOC)^–1) and strength (formation constant) increased from pH 3 to 5 but decreased above pH 5 due to formation of AI-hydroxy species including A1(OH)₃ (s). The high binding capacity of OM in bog waters facilitates metal mobility, especially in low pH (< 5) wetlands where metal solubility is high and OM concentrations are highest. Results showed that the relative degree of organic matter saturation with metal ions was important in modeling AI speciation in bog waters.