Effects of phosphorylation, MgATP, and ionic strength on the rates of papain degradation of heavy and light chains of smooth muscle heavy meromyosin at the S1-S2 junction

The effects of ionic strength, MgATP, and phosphorylation on the degradation rates of heavy meromyosin (HMM) by papain have been compared to their effects on the sedimentation coefficient (s20,w) to determine the relationship of the degradation rate to the equilibrium between the flexed and the extended forms (Suzuki, H., Stafford, W. F., Slayter, H. S., and Seidel, J. C. (1985) J. Biol. Chem. 260, 14810-14817). At 0.025 M NaCl, where HMM is predominantly in the flexed form, MgATP, Mg-adenylyl imidodiphosphate or MgADP reduce kH by 80-90%. MgATP exerts its optimal effect at this ionic strength, where at least 70% of HMM is flexed in the presence or absence of MgATP, suggesting that nucleotides reduce kH by decreasing the proteolytic susceptibility of the flexed form. At 0.5 M NaCl, where HMM is in the extended form, MgATP has no effect on kH. At low ionic strengths phosphorylation decreases kH but increases it in the presence of MgATP. Plots of kH against s20,w determined at various ionic strengths are linear, the data for phosphorylated and dephosphorylated HMM falling on the same line. Thus, raising the ionic strength or phosphorylating the 20-kDa light chain appears to alter kH by increasing the fraction of HMM in the extended form. The degradation rate of the 20-kDa light chain (kL) of dephosphorylated HMM responds to changes in ionic strength in essentially the same way as does kH, suggesting that the response of kL to changes in ionic strength can also be attributed to conversion of HMM to the extended form. However, kL for phosphorylated HMM measured in the presence of MgATP exhibits very little dependence on ionic strength.     

J chain in Rana catesbeiana high molecular weight Ig

A polypeptide homologous to human and mouse J chain has been identified in the high molecular weight (HMW) Ig of the bullfrog, Rana catesbeiana. In previous studies, we had detected a component that was similar in size to mammalian J chains and that, relative to L chains, migrated rapidly to the anode in alkaline-urea PAGE; however, its mobility was less than that of mammalian J chains. We now demonstrate that this component is covalently linked to the H chain of R. catesbeiana HMW Ig. All of the disulfide bridges of this polypeptide, like those of human and mouse J chain, can be cleaved by reducing agents even in the absence of denaturing solvents. The putative frog J chain was isolated by a procedure that did not require preliminary purification of the HMW Ig. The chain differed in amino acid composition from L chains but resembled J chains from several other species. Tryptic peptides were isolated and sequenced. Except for a single heptapeptide, the peptides could be aligned by virtue of their similarity to segments of human and mouse J chain. Of the 116 residues that were placed, 55 were identical with residues in human J chain and 60 with residues in mouse J chain. The six cysteine residues identified in the frog J chain are at the same positions as six of the eight cysteines in the human and mouse J chains. The results indicate significant conservation in structure between amphibian and mammalian Ig J chains.     

Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes

Summary A systematic study was made of the action of 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) on active Ca²⁺ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca²⁺-selective electrodes or use of tracer⁴⁵Ca²⁺. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS. Ca²⁺ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K⁺ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca²⁺ pump, exchanging one Ca²⁺ ion for one proton.     

Phosphate sequestration by glycerol and its effects on photosynthetic carbon assimilation by leaves

Glycerol induced a limitation on photosynthetic carbon assimilation by phosphate when supplied to leaves of barley (Hordeum vulgare L.) and spinach (Spinacia oleracea L.). This limitation by phosphate was evidenced by (i) reversibility of the inhibition of photosynthesis by glycerol by feeding orthophosphate (ii) a decrease in light-saturated rates of photosynthesis and saturation at a lower irradiance, (iii) the promotion of oscillations in photosynthetic CO₂ assimilation and in chlorophyll fluorescence, (iv) decreases in the pools of hexose monophosphates and triose phosphates and increases in the ratio of glycerate-3-phosphate to triose phosphate, (v) decreased photochemical quenching of chlorophyll fluorescence, and increased non-photochemical quenching, specifically of the component which relaxed rapidly, indicating that thylakoid energisation had increased. In barley there was a massive accumulation of glycerol-3-phosphate and an increase in the period of the oscillations, but in spinach the accumulation of glycerol-3-phosphate was comparatively slight. The mechanism(s) by which glycerol feeding affects photosynthetic carbon assimilation are discussed in the light of these results.Abbreviations Chl chlorophyll - C _i intercellular concentration of CO₂ - P phosphate - PGA glycerate-3-phosphate - Pi orthophosphate - triose-P sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate     

Limitation of photosynthesis by changes in temperature

The aim of this work was to examine the effect of abrupt changes in temperature in the range 5 to 30°C upon the rate of photosynthetic carbon assimilation in leaves of barley (Hordeum vulgare L.). Measurement of the CO₂-assimilation rate in relation to the intercellular partial pressure of CO₂ at different temperatures and O₂ concentrations and at saturating irradiance showed that as the temperature was decreased photosynthesis was saturated at progressively lower CO₂ partial pressures and that the transition between the CO₂-limited and ribulose-1,5-bisphosphate-regeneration-limited rate became more abrupt. Feeding of orthophosphate to leaves resulted in an increased rate of CO₂ assimilation at lower temperatures at around ambient or higher CO₂ partial pressures both in 20% O₂ and in 2% O₂ and it removed the abruptness in the transition between the CO₂-limited and ribulose-1,5-bisphosphate-regeneration-limited rates. Phosphate feeding tended to inhibit carbon assimilation at higher temperatures. The response of carbon assimilation to temperature was altered by feeding orthophosphate, by changing the concentrations of CO₂ or of O₂ or by leaving plants in the dark at 4°C for several hours. Similarly, the response of carbon assimilation to phosphate feeding or to changes in 2% O₂ was altered by leaving the plants in the dark at 4°C. The mechanism of limitation of photosynthesis by an abrupt lowering of temperature is discussed in the light of the results.Abbreviations A rate of CO₂ assimilation - P _i intercellular partial pressure of CO₂ - RuBP ribulose-1,5-bisphosphate     

Alarm reaction and alert state inGambusia Affinis (Pisces,Poeciliidae) in response to chemical stimuli from injured conspecifics

We studied the behavior of the Poeciliid fishGambusia affinis after the introduction of 3 substances into their tank: a homogenization ofGambusia affinis, a homogenization of the Anabantid fishBetta splendens, and a blank made of distilled water. The response of the fish was measured as a change in their spatial distribution in the tank after the introduction of the substance. Two sizes of fish were used, and theGambusia homogenization produced clear alarm reactions in both, the fish fleeing to the bottom of the tank. This is one of a few examples available of recognition of alarm substances in non-ostariophysian fish. In addition, we found that the small fishes that had recently been exposed to the alarm substance stayed in an ‘alert state’, in which they had an increased sensitivity to mechanical and visual fright stimuli.     

Behavioral analysis of the choice of oviposition site by single females ofDrosophila Melanogaster (Diptera: Drosophilidae)

The oviposition behavior of single females of Drosophila melanogasterwas studied in population cages over 24 h. Each female shows a different behavior, but they can be arbitrarily separated into those which concentrate their egg laying in only one tube and those which spread it over more than two tubes. A comparison is made between females extracted from the Valdivian population and flies from lines selected for high and low aggregation. When one-tube females were grouped and compared with more-than-one-tube females, the aggregation indices between these groups were significantly different.     

Organisation and functions of the actV A region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor

Summary Sequence analysis of the actVA region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor revealed a succession of six open reading frames (ORFs), all running in the same direction and extending over 5.32 kb. The protein product of actVA-ORF1 strongly resembles that of another gene, elsewhere in the act cluster (actII-ORF2), which codes for a trans-membrane protein previously implicated in actinorhodin export from the mycelium. This suggests that the two gene products may co-operate in actinorhodin export, perhaps being sufficient for self-protection of the organism against suicide. At least four of the other five ORFs are implicated in the control of the C-6 and C-8 ring-hydroxylation reactions, lacking in actVA mutants, that occur at middle to late stages in the actinorhodin biosynthetic pathway. This conclusion was reached by genetic mapping of actVA mutants to actVA-ORF3 and-ORF5 (and perhaps -ORF4), and by the finding of strong resemblances between the protein products of actVA-ORF2 and -ORF6 and the products of genes of the oxytetracycline or tetracenomycin gene clusters that have been implicated in ring-hydroxylation reactions in the biosynthesis of these other aromatic polyketide antibiotics.