Effect of thyrotropin on glycosaminoglycans synthesized by primocultured thyroid cells

The synthesis of glycosaminoglycans (GAGs) was investigated in porcine thyroid cells under the influence or not of thyrotropin. After labelling with [³H] glucosamine and [³⁵S] $\text{SO}{}_4{}^{\mathrm{2?}}$, enriched GAG-fractions prepared from culture media, cells, and eventually substrate adhering materials, were analyzed by cellulose acetate electrophoresis combined with specific degradations. They comprised heparan sulfate and hyaluronic acid together with an unknown sulfated component labile to endo-β-galactosidase. Whereas global labellings of newly made GAGs were not significantly modified by thyrotropin, we reproducibly observed with the hormone a substantial increase in the proportion of hyaluronic acid [³H] label and, when cells organized into follicles, of the proportion of cell-associated [³H] GAGs. This system thus offers an interesting model to study how the responsiveness to an hormone and the reorganization that follows might implicate specific glycoconjugates.     

Regulation of calcium accumulation and efflux from platelet vesicles: Possible role for cyclic-AMP-dependent phosphorylation and calmodulin

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ activity of about 10 nmol (min·mg)^?1 and an ATP-dependent Ca²⁺ uptake of about 10 nmol (min·mg)^?1. When incubated in the presence of Mg[γ-³²P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [³²P]phosphate-labeled Ca²⁺ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca²⁺ or Ca²⁺ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of ¹²⁵I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol ($\text{M}{}_{\mathrm{r}}\text{= 94 000, 87 000, 60 000 and 43 000}$). Some minor calmodulin-binding proteins were enriched in the membrane fractions ($\text{M}{}_{\mathrm{r}}\text{= 69 000, 57 000, 39 000 and 37 000}$). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca²⁺ uptake is essentially unaltered, while the Ca²⁺ capacity is diminished as a consequence of a doubling in the rate of Ca²⁺ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca²⁺ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation.     

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.     

Fluorography--limitations on its use for the quantitative detection of 3H- and 14C-labeled proteins in polyacrylamide gels

The suitability of fluorography for the detection of 3H- and 14C-labeled proteins on polyacrylamide gradient gels has been investigated. It was found that the absorbance of the fluorographic film image produced by a given level of radioactivity decreased as the acrylamide concentration in the gel increased. The use of Coomassie brilliant blue protein dyes to stain the gel prior to fluorography reduced the absorbance of the fluorographic image. It is concluded that quantitative fluorography can only be applied to unstained gels of a uniform acrylamide concentration.     

Ammoniacal silver staining of proteins: mechanism of glutaraldehyde enhancement

When the conditions for detecting proteins by ammoniacal silver staining (B. R. Oakley, D. R. Kirsch, and N. R. Morris (1980) Anal. Biochem. 105, 361-363.) following gel electrophoresis were varied, it was noted that glutaraldehyde pretreatment was necessary for maximal staining, which could not be explained simply as the result of "fixation." Further studies indicated that glutaraldehyde enhancement of protein staining with this silver reagent was probably due to oxidation of the aldehyde groups by silver ions, resulting in metallic silver depositions within the gel which act as nucleation sites for additional metallic silver localization in the protein bands upon the addition of formaldehyde developer. This proposed mechanism is consistent with the Tollen's reaction, as well as some aspects of the photographic process. Consistent with this notion, silver-staining intensities are directly related to mole percentage lysine of various standard proteins.     

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.     

Soybean leaf urease: Comparison with seed urease

Soybeans, Glycine max (L.) Merr., from ureides for transport of nitrogen from the root nodule to the shoot. The most direct routes for ureide utilization include the degradation of ureide-derived urea to NH₃ and CO₂. Ureolytic activity was found in leaf disks of soybean and exhbited optimal activity at pH 7 in the presence of a high concentration of urea (250 m M ). In vitro studies showed neither urea amidolyase nor urea dehydrogenase activity in soybean leaves and the ureolytic activity was characterized as urease. Several biochemical properties of soybean leaf urease were determined and compared to seed urease properties. Soybean leaf urease differed from that of seed in five ways: pH optima (5.25 and 8.75), apparent K_m (0.8 m M ), no inhibition by hydroxyurea, faster electrophoretic mobility and no cross-reactivity with soybean seed urease antibodies. The data suggest that urease is the primary urea metabolizing enzyme present in soybean leaves. The properties of soybean leaf urease support the conclusion that a unique isozyme of urease is present in leaf tissue.     

Sulfate influx across the rabbit ileal brush border membrane: Sodium and proton dependence,and substrate specificities

Summary In intact ileal mucosa, uptake of SO₄ across the brush border membrane requires the presence of Na and is saturable, withK1/2=1.3mm at 140mm Na (P.L. Smith, S.A. Orellana & M. Field, 1981.J. Membrane Biol. 63:199–206). The present study examines the substrate specificities and transport stoichiometry of the Na-dependent SO₄ uptake process. The effects of variations in medium anion and cation composition on lumen-to-epithelium influx of SO₄ (J _me ^SO4 ) were determined under short-circuit conditions.J _me ^SO4 is inhibited by thiosulfate, but not by phosphate, methylsulfate, vanadate or taurocholate. Cl is weakly inhibitory. Uptake of SO₄ is poorly supported by Li, and is unaffected by K, indicating a specific dependence on Na. At low SO₄ concentration (0.22mm),J _me ^SO4 is a hyperbolic function of medium Na concentration; the corresponding Hill plot is linear with a slope of 1.0, suggesting a transport stoichiometry of 1 Na: 1 SO₄. At high SO₄ concentration (6.7mm), the Na-dependent SO₄ velocity curve is sigmoidal and yields a Hill plot which is again linear but has a slope of 1.56, suggesting transport of more than 1 Na per SO₄. SO₄ uptake in presence of Na exhibits a dependence on medium pH. At 0.22mm SO₄ and 140mm Na,J _me ^SO4 was doubled by lowering pH from 7.4 to 6.8. However, at 6.7mm SO₄ and 140mm Na, changing pH had no effect onJ _me ^SO4 over the range 6.8 to 8.5. The pH dependence ofJ _me ^SO4 at 6.7mm SO₄ was restored when medium Na was lowered to 3mm, suggesting that pH sensitivity is a function of the concentration of preformed NaSO ₄ ^– ion pair. The results suggest that SO₄ influx across the ileal brush border occurs by electroneutral Na⁺/NaSO ₄ ^– or Na⁺/H⁺/SO ₄ ^2– cotransport, the former being favored by high concentrations of Na and SO₄.     

Application of sodium dodecyl sulfate electrophoresis of hemolysate to the biochemical systematics of the rockfish Sebastes

Sodium dodecyl sulfate electrophoresis of rockfish hemolysate was carried out to investigate problems in the systematics of the genus Sebastes. Results generally revealed species-specific electropherograms that were generally intra-specifically invariant. Experimental findings supported the distinction of S. chrysomelas and S. carnatus as valid species which hitherto had not been accomplished on a morphometric or meristic basis. Moreover, common protein subunits occurred within all specimens studied, and additional interspecific differences suggested several biochemical subgroupings (caurinus, nebulosus; paucispinis, goodei; carnatus, chrysomelas, atrovirens, melanops, mystinus and serranoides). These proposed subgroupings generally differed from previous classifications based on morphometric and meristic data. Some of these differences may ultimately be attributed to pressure adaptive differences in the hemoglobin molecule.