Posttranslational Processing of Carboxypeptidase E, a Neuropeptide-Processing Enzyme, in AtT-20 Cells and Bovine Pituitary Secretory Granules

Abstract: Carboxypeptidase E (CPE) functions in the posttranslational processing of peptide hormones and neurotransmitters. Like other peptide processing enzymes, CPE is present in secretory granules in soluble and membrane-associated forms that arise from posttranslational processing of a single precursor, “proCPE.” To identify the intracellular site of proCPE processing, the biosynthesis and posttranslational processing were investigated in the mouse anterior pituitary-derived cell line, AtT-20. Following a 15-min pulse with [³⁵S]Met, both soluble and membrane-bound forms of CPE were identified, indicating that the posttranslational processing event that generates these forms of CPE occurs in the endoplasmic reticulum or early Golgi apparatus. The relative proportion of soluble and membrane-bound forms of CPE changed when cells were chased for 2 h at 37°C but was unaffected when cells were chased at either 20 or 15°C, suggesting that further processing of membrane forms to the soluble form occurs in a post-Golgi compartment. Treatment of the cells with chloroquine did not alter the relative distribution of soluble and membrane forms, suggesting that an acidic compartment is not required for this processing event. Overexpression of CPE did not influence the distribution of soluble and membrane forms of CPE, indicating that the CPE-processing enzymes are not rate-limiting. To examine directly CPE-processing enzymes, bovine anterior pituitary secretory vesicles were isolated. An enzyme activity that releases the membrane-bound form of CPE was detected in the purified secretory vesicle membranes. This enzyme, which removes the C-terminal region of CPE, is partially inhibited by EDTA and phenylmethylsulfonyl fluoride and is activated by CaCI₂. Together, the data indicate that posttranslational processing of CPE occurs in secretory granules and that this activity may be mediated by a prohormone convertase-like enzyme.     

Two subpopulations of α1-adrenergic receptors with high and low affinity for agonists: Short-term exposure to agonists reduced the high-affinity sites

Short-term receptor regulation by agonists is a well-known phenomenon for a number of receptors, including β-adrenergic receptors, and has been associated with receptor changes revealed by radioligand binding. In the present study, we investigated the rapid changes in α₁-adrenergic receptors induced by agonists. α₁-receptors were studied on DDT₁ MF-2 smooth muscle cells (DDT₁-MF-2 cells) by specific [³H]prazosin binding. In competition binding on membranes and on intact cells at 4°C or at 37°C in 1-min assays, agonists competed for a single class of sites with relatively high affinity. By contrast, in equilibrium binding at 37°C on intact cells agonists competed with two receptor forms (high- and low-affinity). We quantified the receptors in the high-affinity form by measuring the [³H]prazosin binding inhibited by 20 μM norepinephrine (this concentration selectively saturated the high-affinity sites). The low-affinity sites were measured by subtracting the binding of [³H]prazosin to the high-affinity sites from the total specific binding. High-affinity receptors were 85% of the total sites in binding experiments at 4°C, but only 30% at 37°C. On DDT₁-MF-2 cells preequilibrated with [³H]prazosin at 4°C, and then shifted to 37°C for a few minutes, norepinephrine selectively reduced the high-affinity sites by 30%. We suggest that at 4°C it is the native form of α₁-receptors that is measured, with most of the sites in the high-affinity form, while during incubation at 37°C the norepinephrine present in the binding assay converts most of the receptors to an apparent low-affinity form, so that they are no longer recognized by 20 μM norepinephrine. The nature of this low-affinity form was further investigated. On DDT₁-MF-2 cells preincubated with the agonist and then extensively washed at 4°C (to maintain the receptor changes induced by the agonist) the number of receptors recognized by [³H]prazosin at 4°C was reduced by 38%. After fragmentation of the cells, the number of receptors measured at 4°C was the same in control and norepinephrine-treated cells, suggesting that the disruption of cellular integrity might expose the receptors which are probably sequestered after agonist treatment. In conclusion, the appearance of the low affinity for agonists at 37°C may be due to the agonist-induced sequestration of α₁-adrenergic receptors, resulting in a limited accessibility to hydrophilic ligands.     

In vitro culture of Plasmodium yoelii blood stages

Lewis-Hughes P. H. and Howell M. J. 1984. In vitro culture of Plasmodium yoelii blood stages. International Journal for Parasitology14: 447–451. Plasmodium yoelii infected reticulocytes were cultured for 72 h at either 37 or 20°C in MEM (Eagle's modification) medium containing, in addition, glucose, para-aminobenzoic acid and 5% foetal calf serum, buffered at pH 7.3 with sodium bicarbonate/ HEPES and maintained under 10% CO₂ in air. Red blood cell numbers were more stable at 20°C than at 37°C. Culture at both temperatures resulted in an increase in parasitaemia of the reticulocyte population over the initial 36 h at 37°C and for at least 72 h at 20°C. The effects of different temperatures appeared to be related to the continued presence of target cells. Parasites were not detected after 72 h culture at 37°C, but persisted for up to 120 h at 20°C. Increasing parasitaemia at both temperatures was associated with changes in the numbers of some parasite development types. Early falls in schizont numbers were associated with an increase in the numbers of ring forms. Trophozoite numbers tended to remain constant throughout the culture period. Viability of parasites cultured for 36 h was confirmed by their infectivity to CBA mice. In addition, parasites progressively incorporated H³-leucine into TCA-precipitable material over the initial 36 h of culture.     

The division protein FtsZ interacts with the small heat shock protein IbpA in Acholeplasma laidlawii

Small heat shock proteins (sHSPs) control the proteins stability in the cell preventing their irreversible denaturation. While many mycoplasmas possess the sHSP gene in the genome, Acholeplasma laidlawii is the only mycoplasma capable of surviving in the environment. Here we report that the sHSP IbpA directly interacts with the key division protein FtsZ in A. laidlawii, representing the first example of such interaction in prokaryotes. FtsZ co-immunoprecipitates with IbpA from A. laidlawii crude extract and in vitro binds IbpA with K_D ~ 1 μM. Proteins co-localize in the soluble fraction of the cell at 30–37 °C and in the non-soluble fraction after 1 h exposition to cold stress (4 °C). Under heat shock conditions (42 °C) the amount of FtsZ decreases and the protein remains in both soluble and non-soluble fractions. Furthermore, in vitro, FtsZ co-elutes with IbpA_His6 from A. laidlawii crude extract at any temperatures from 4 to 42 °C, with highest yield at 42 °C. Moreover, in vitro FtsZ retains its GTPase activity in presence of IbpA, and the filaments and bundles formation seems to be even improved by sHSP at 30–37 °C. At extreme temperatures, either 4 or 42 °C, IbpA facilitates FtsZ polymerization, although filaments under 4 °C appears shorter and with lower density, while at 42 °C IbpA sticks around the bundles, preventing their destruction by heat. Taken together, these data suggest that sHSP IbpA in A. laidlawii contributes to the FtsZ stability control and may be assisting appropriate cell division under unfavorable conditions.     

The characterization of a membrane-bound protein carboxylmethylation system in brain

The membrane-bound component of the cerebral protein carboxylmethylation system, consisting of the membrane-bound enzyme protein carboxylmethyltransferase II (PCMT) and of selected membrane-bound methyl accepting proteins (MAP), is described. The cellular localization of this membrane-bound protein carboxylmethylation system is shown to include, in addition to nerve cell bodies and purified synaptosomes, astrocytes and oligodendroglia. The membrane-bound nature of the protein carboxylmethylation system was investigated and these studies revealed a tight association which exposure to several detergents could only partially solubilize. The membrane-bound PCMT could be shown to undergo activation after treatment with Na-deoxycholate and CHAPs, while after its detergent-induced solubilization PCMT activation was observed after Na-deoxycholate, Nonidet P-40 and Lubrol-P_X. Solubilization of the carboxylmethylation system in CHAPS appeared to be more effective at 0°C than at 25°C or 37°C. Detergent treatment was shown to be deleterious to the MAPs as PCMT substrates, particularly when the exposure was extended to more than 1 h. These observations prompted exposure of the brain membranes and of their Lubrol-P_X and Nonidet P-40 extracts to NH₄OH, treatment which promotes the conversion of protein asparagine residues to atypical l-isoaspartate residues, recently shown (in synthetic peptides) to be the single most effective residue recognized for carboxylmethylation by PCMT. We found up to a 400% enhancement of the carboxylmethylation of solubilized membrane MAPs by the equally solubilized PCMT (which resisted the alkaline treatment virtually unscathed) after 90 min at 37°C in 0.05 M NH₄OH. However, when brain membrane Lubrol-P_x extracts were first subjected to bis(I,I-trifluoroacetoxy)-iodobenzene, a reagent which converts the carboxyamide group of protein-bound asparagine to the corresponding primary amine, the amount of MAPs susceptible to be acted upon by 0.05 M NH₄OH became greatly reduced. Finally, acidic slab gel electrophoresis of membrane-bound MAPs, carboxyl-[³H]-methylated by the membrane-bound PCMT, revealed the presence of about 12 radioactive protein bands, ranging in MW from under 20 KDa to about 90 KDa.     

The selective inhibitory effect of NH4Cl on estrogen-stimulated rat uterine in vitro RNA synthesis

Estrogen-stimulated in vitro RNA synthesis in rat uterine nuclear-myofibrilar fractions, isolated uterine nuclei, and nucleoli was selectively inhibited by NH₄Cl (400 μmoles) when added prior to the start of a 37 °C incubation. This inhibitory effect was not observed if the salt was added after the start (as early as 1 min) of a 37 °C incubation. The removal of NH₄Cl from reaction mixtures not yet incubated at 37 °C further reduced in vitro RNA synthesis in both control and hormone-treated nuclei. The data suggest that NH₄Cl (400 μmoles) added prior to, but not after, the start of a 37 °C incubation inhibited estrogen-stimulated rat uterine nucleolar RNA polymerase activity perhaps by removing a protein component(s) which is necessary for hormonal stimulation.     

Latent acetylcholinesterase in secretory vesicles isolated from adrenal medulla

A new procedure is described for the preparation of highly purified and stable secretory vesicles from adrenal medulla. Two forms of acetylcholinesterase, a membrane bound form as well as a soluble form, were found within these vesicles. The secretory vesicles, isolated by differential centrifugation, were further purified on a continuous isotonic Percoll? gradient. In this way, secretory vesicles were separated from mitochondrial, microsomal and cell membrane contamination. The secretory vesicles recovered from the gradient contained an average of 2.26 μmol adrenalin/mg protein. On incubation for 30 min at 37°C in media differing in ionic strength, pH, Mg²⁺ and Ca²⁺ concentration, the vesicles released less than 20% of total adrenalin. Acetylcholinesterase could hardly be detected in the secretory vesicle fraction when assayed in isotonic media. However, in hypotonic media (<400 mosmol/kg) or in Triton X-100 (0.2% final concentration) acetylcholinesterase activity was markedly higher. During hypotonic treatment or when secretory vesicles were specifically lyzed with 2 mM Mg²⁺ and 2 mM ATP, adrenalin as well as part of acetylcholinesterase was released from the vesicular content. On polyacrylamide gel electrophoresis this soluble enzyme exhibited the same electrophoretic mobility as the enzyme released into the perfusate from adrenal glands upon stimulation. In addition to the soluble enzyme a membrane bound form of acetylcholinesterase exists within secretory vesicles, which sediments with the secretory vesicle membranes and exhibits a different electrophoretic mobility compared to the soluble enzyme. It is concluded, that the soluble enzyme found within isolated secretory vesicles is secreted via exocytosis, whilst the membrane-bound form is transported to the cell membrane during this process, contributing to the biogenesis of the cell membrane.     

The gelation of deoxyhemoglobin S in erythrocytes as detected by transverse water proton relazation measurements

At 37 °C, when samples of blood, washed erythrocytes, or isolated hemoglobin from individuals with sickle cell disease are deoxygenated, the transverse water proton relaxation time is sharply decreased. In similar samples from normal adults homozygous for hemoglobin A, only a slight decrease in t₂ is observed upon deoxygenation at 37 °C. In samples containing deoxyhemoglobin S the value of t₂ increases as the temperature is decreased from 37 °C to 4 °C, in contrast to samples containing oxyhemoglobin S, oxyhemoglobin A, or deoxyhemoglobin A where t₂ decreases as the temperature decreases. It is suggested that this decrease in t₂ observed in samples of deoxyhemoglobin S at 37 °C is the result of an increase in the amount of preferentially oriented water at macromolecular interfaces which occurs under conditions known to produce deoxyhemoglobin S gelation. Conditions which reverse deoxyhemoglobin S gelation such as lowering the temperature to 4 °C decrease the amount of preferentially oriented water which results in an increase in the value of t₂. Thus, measurement of the transverse water proton relaxation time can be used to monitor the gelation of deoxyhemoglobin S inside the erythrocyte.     

Co-clustering and internalization of low-density lipoproteins and alpha 2-macroglobulin in human skin fibroblasts

The endocytosis of low density lipoprotein (LDL) and α₂-macroglobulin (α₂M) has been examined simultaneously in human skin fibroblasts. Incubation of cells at 4 °C with rhodamine-α₂M and LDL plus [(dichlorotriazinyl)amino]fluorescein-anti-LDL gave a weak fluorescence for α₂M and a brighter, clustered fluorescence for LDL. Following warming to 37 °C, LDL and α₂M were observed to be coincident within endocytotic vesicles in the cell. By electron microscopy, LDL-ferritin and α₂M-colloidal gold were present in the same coated pit at 4 °C. After 7 min at 37 °C, both ligands were observed in the same receptosome. Pretreatment of fibroblasts at 37 °C with 200–300 μM dansylcadaverine or 50 mM methylamine blocked clustering and internalization of both LDL and α₂M. Bacitracin (5 mg ml^?) blocked clustering and endocytosis of α₂M, but not of LDL. These data indicate that both LDL and α₂M are processed via the same endocytotic pathway in skin fibroblasts.     

Hydrolysis of adenylyl imidodiphosphate in the presence of Na+ + Mg+ by (Na+ + K+)-activated ATPase

(1) Contrary to what has usually been assumed, (Na⁺ + K⁺)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na⁺ + Mg²⁺ to ADP-NH₂ and P_i. The activity is ouabain-sensitive and is not detected in the absence of either Mg²⁺ or Na²⁺. The specific activity of the Na⁺ + Mg²⁺ dependent AdoPP[NH]P hydrolysis at 37°C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K⁺. The activity is not stimulated by K⁺, nor can K⁺ replace Na⁺ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na⁺ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5–9 mM. The K_m value for AdoPP[NH]P is 17 μM. At 0°C and 21°C the specific activity is 2 and 14%, respectively, of that at 37°C. AMP, ADP and AdoPP[CH₂]P are not detectably hydrolysed by (Na⁺ + K⁺)-ATPase in the presence of Na⁺ + Mg²⁺. (2) In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37°C of 0.0006, 0.006 and 0.07 h^?1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg²⁺ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h^?1 at 37°C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. (3) A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na⁺ + Mg²⁺ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0°C. On the other hand, determination of affinity in the (Na⁺ + K⁺)-ATPase reaction by competition with ATP at low protein concentrations (μg/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37°C.     

Serial passage of Heliothis zea singly embedded nuclear polyhedrosis virus in a homologous cell line

This paper describes the replication and serial passage of Heliothis zea nuclear polyhedrosis virus (NPV) in a H. zea cell line. It was demonstrated that long-term serial passages of the H. zea NPV in homologous host cell culture decreased both the total number of polyhedral inclusion bodies (PIBs) produced and the infectivity of the supernatant as measured by TCID₅₀. The growth curve indicated that infectious material was released from cells 24 hr postinfection (p.i.) and approached a maximal titer 3 days p.i. The kinetics of H. zea NPV decay at 4°, 27°, and 37°C were determined. Infectivity was not detected after 3 weeks at 37°C, but approximately 10^3.5 TCID₅₀/ml activity was still present after 3 and 8 weeks storage at 27° and 4°C, respectively. Electron microscopy confirmed the presence of single embedded virions in the inoculated cells.     

Growth at 37 degrees C of the EGs strain of the amoeboflagellate Naegleria gruberi containing viruslike particles. I. Nuclear changes

Naegleria gruberi amoebae, EG_s strain, containing viruslike particles (VLP) were grown at temperatures of 21° and 37°C. At 21°C, the amoebae displayed the morphological structures associated with development of the VLP's. At 37°C, however, gross morphological modifications and new structures appeared. When amoebae were at 37°C for less than 12 hr, nuclei were found to have a larger number of VLP's than amoebae at 21°C. Exposure of the amoebae to the higher temperature for 12–24 hr resulted in a scarcity of particles. Large bundles of microtubulelike fibrils were present in the nucleoplasm of amoebae at 37°C, and, in addition, the nuclei showed degenerative modifications. The fibrillar changes were not due to the elevated temperature alone since a substrain of EG_s (=EG_B) not infected with VLP's exhibited no nuclear modifications. It is assumed that the elevated temperature accelerated and enhanced a lytic effect of the VLP's upon the cells.     

The carbon dioxide hydration activity of brush-border carbonic anhydrase from the dog kidney

The steady-state kinetic parameters for the hydration of CO₂ catalyzed by membrane-bound carbonic anhydrase from the renal brush-border of the dog are compared with the same parameters for water-soluble bovine erythrocyte carbonic anhydrase. For the membrane-bound enzyme, the turnover number k_cat is 6.5 × 10⁵ s^?1 and the Michaelis constant is 7.5 mm for CO₂ hydration at pH 7.4 and 25 °C. The corresponding constants for bovine carbonic anhydrase under these conditions are 6.3 × 10⁵ s^?1 and 15 mm (Y. Pocker and D.W. Bjorkquist (1977)Biochemistry16, 5698–5707). The rate constant for the transfer of a proton between carbonic anhydrase and buffer was determined from the dependence of the catalytic rate on the concentration of the buffers imidazole and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes); the value of 2 × 10⁸m^?1s^?1 describes this constant for both forms of carbonic anhydrase at pH 7.4. Furthermore, the pH dependence of the initial velocity of hydration of CO₂ in the range of pH 6.5 to 8.0 is identical for the membrane-bound and soluble enzyme at low buffer concentration (1–2 mm imidazole). We conclude that the membrane plays no detectable role in affecting the CO₂ hydration activity and that the active site of the renal, membrane-bound carbonic anhydrase is exposed to the aqueous phase.     

Purification,Characterization, and Crystallization of Crocodylus siamensis Hemoglobin

Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two β chains) showed high oxygen affinity at 3.13 mmHg (P₅₀) and 1.96 (n value), and a small Bohr effect (δH⁺ = ?0.29) at a pH of 6.9–8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O₂ affinity at P₅₀ of 2.5 mmHg which may assure efficient utilization of the lung O₂ reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10–13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl₂·6H₂O] solution at a pH of 7.0–8.5.     

Differential inhibition of soluble and membrane-bound acetylcholinesterase forms from mouse brain by choline esters with an acyl moiety of an intermediate size

Differential inhibitions of soluble and membrane-bound acetylcholinesterase forms purified from mouse brain were examined by the comparison of kinetic constants such as a K _m value, a K_ss value (substrate inhibition constant), and IC₅₀ values of active site-selective ligands including choline esters. Membrane-bound acetylcholinesterase form (solubilized only in the presence of detergent) showed lower K_m and K_ss values than soluble acetylcholinesterase form (easily solubilized without detergent). Edrophonium expressed a slightly but significantly (p<0.01) higher inhibition of detergent-soluble acetylcholinesterase form than aqueous-soluble acetylcholinesterase form, while physostigmine inhibited both forms with a similar potency. A remarkable difference in inhibition was observed using choline esters; although choline esters with acyl chain of a short size (acetyl-to butyrylcholine) or a long size (heptanoyl- to decanoylcholine) showed a similar inhibitory potency for two forms of acetylcholinesterase, pentanoylcholine and hexanoylcholine inhibited more strongly aqueous-soluble acetylcholinesterase than detergent-soluble acetylcholinesterase. Thus, it is suggested that the two forms of AChE may be distinguished kinetically by pentanoyl- or hexanoylcholine.This work was supported in part by Agency for Defense Development.     

Solubilization of benzodiazepine binding site from rat cortex.

The high-affinity binding site for [³H] diazepam has been solubilized from rat brain using 0.5% Lubrol-PX. Using a polyethylene glycol (PEG)-γ-globulin assay, it has been possible to demonstrate solubilization of about 60% of the binding sites in a single step. The solubilized binding site possesses a K_D of 11 nM for [³H] diazepam compared to approximately 4 nM for the membrane-bound form, and binding is to a single class of sites. The order of potency of benzodiazepines is identical for the solubilized receptor and the membrane-bound form. Binding of [³H] diazepam is temperature dependent and higher at 4° than 37°C. Both urea and guanidine-HC1 were capable of totally inhibiting binding, and this inhibition was partly reversible; neither sulfhydryl groups nor carbohydrate moieties seem to be important for binding. γ-Aminobutyric acid which enhanced [³H] diazepam binding to membrane fractions was without effect on the solubilized binding site.     

Low and high temperature asymmetric forms of pig kidney and rabbit muscle phosphofructokinases and reversible, temperature-dependent transitions.

Previous viscometric studies from this laboratory (Johnson, C. S., Vogtmann, L., and Deal, W. C., Jr. (1976) Biochem. Biophys. Res. Commun.73, 391–395) have shown that at 3.5 ° C, pig kidney phosphofructokinase (PFK) is markedly asymmetric and rabbit muscle PFK is moderately asymmetric. The present viscometric and ultracentrifugal studies show that both enzymes are also asymmetric at near-physiological temperatures, that both exist in high-temperature and low-temperature forms, and that the high-temperature forms of both are less asymmetric and more dissociated than the low-temperature forms. The risults also show that the transitions from low- to high-temperature forms are reversible if the exposure to 35 °C is short enough that no irreversible chemical modification occurs. For pig kidney PFK, intrinsic viscosity values of 34.0, 25.6, and 13.8 ml/g were obtained at 3.5, 20 and 35 °C, respectively, whereas rabbit muscle PFK yielded values of 6.9, 6.2, and 5.2 ml/g at the corresponding temperatures. These data clearly show a decrease in asymmetry with increase in temperature. However, both enzymes are still asymmetric at the higher temperature, inasmuch as most globular macromolecules have intrinsic viscosity values in the range of 3 to 4 ml/g, regardless of molekular weight. Studies from 1 to 45 ° C at a fixed protein concentration (4.8 mg/ml) showed that pig kidney PFK has reduced viscosity values of 51.0 ml/g (low-temperature form) and 20.4 ml/g (high-temperature form) in plateau regions of the viscosity graph at the temperature extremes; the mid-point of the transition between the two forms is at about 22–24 °C. Rabbit muscle PFK at 4.2 mg/ml reproducibly gave corresponding reduced viscosity values of 6.9 and 4.8 ml/g for the low- and high-temperature forms, respectively; the transition mid-point between the two forms is at about 16 °C. The first reported sedimentation velocity studies of rabbit muscle PFK at near-physiological temperature (35 °C) show that with near-physiological protein concentration (1.25 mg/ml), the enzyme is in a much more dissociated form, s_20,w(weight average) = 14. 5 S; s_20,w(peak leading edge) = 17 S, than that previously reported at lower temperatures, s_20,w(fastest peak) = 23–30 S. Similarly, the first sedimentation studies on the pig kidney enzyme indicate a lower sedimentation coefficient at 35 ° C (s^0.39%_20,w = 48 S) than at 3.5 ° C(65 S).     

Observations on the morphology and infectivity for cattle of Babesia bovis parasites in unfed Boophilus microplus larvae after incubation at various temperatures

Dalgliesh R. J. and Stewart N. P. 1979. Observations on the morphology and infectivity for cattle of Babesia bovis parasites in unfed Boophilus microplus larvae after incubation at various temperatures. International Journal for Parasitology9: 115–120. The temperature of incubation of unfed Boophilus microplus larvae infected with Babesia bovis influenced the morphology and infectivity of the Babesia within the tick. Incubation at 37°C for 1–3 days stimulated the development of parasites morphologically similar to those usually observed in fed larvae harvested from cattle; similar forms appeared more slowly in larvae incubated at 31°C or 25°C. Extracts prepared from larvae after incubation at 37°C for 3–5 days or 30°C for 8 days were consistently infective for cattle. Prior storage of larvae at 14°C for up to 28 days enhanced the development of infectivity at 37°C; infectivity could still be produced after 65 days storage at 14°C but not after 76 days. Larvae released on a host transmitted B. bovis sooner if they had been incubated at 37°C for 4 days. It was concluded that the development of B. bovis to an infective stage in B. microplus is temperature dependent and does not require the stimulus of feeding by the host.     

Biochemical composition of temperate and Arctic populations of Saccharina latissima after exposure to increased pCO2 and temperature reveals ecotypic variation

Previous research suggested that the polar and temperate populations of the kelp Saccharina latissima represent different ecotypes. The ecotypic differentiation might also be reflected in their biochemical composition (BC) under changing temperatures and pCO₂. Accordingly, it was tested if the BC of Arctic (Spitsbergen) and temperate S. latissima (Helgoland) is different and if they are differently affected by changes in temperature and pCO₂. Thalli from Helgoland grown at 17 °C and 10 °C and from Spitsbergen at 10 °C and 4 °C were all tested at either 380, 800, or 1,500 µatm pCO₂, and total C-, total N-, protein, soluble carbohydrate, and lipid content, as well as C/N-ratio were measured. At 10 °C, the Arctic population had a higher content of total C, soluble carbohydrates, and lipids, whereas the N- and protein content was lower. At the lower tested temperature, the Arctic ecotype had particularly higher contents of lipids, while content of soluble carbohydrates increased in the Helgoland population only. In Helgoland-thalli, elevated pCO₂ caused a higher content of soluble carbohydrates at 17 °C but lowered the content of N and lipids and increased the C/N-ratio at 10 °C. Elevated pCO₂ alone did not affect the BC of the Spitsbergen population. Conclusively, the Arctic ecotype was more resilient to increased pCO₂ than the temperate one, and both ecotypes differed in their response pattern to temperature. This differential pattern is discussed in the context of the adaptation of the Arctic ecotype to low temperature and the polar night.