The effect of calcium load on the calcium permeability of sarcoplasmic reticulum

The effect of intravesicular and extravesicular calcium concentration on the passive efflux from sarcoplasmic reticulum (SR) vesicles isolated from cardiac and skeletal muscle was determined by measuring net efflux of calcium after stopping pump-mediated fluxes. The apparent permeability, calculated as the passive efflux divided by the total intravesicular calcium, depended on calcium load. This dependence of the apparent permeability on calcium load could be explained by the presence of intravesicular calcium-binding sites with a dissociation constant less than 10(-3) M. When the intravesicular bound calcium was taken into account, passive calcium efflux was found to be linearly related to the difference in calcium concentration across the SR membrane. Thus the permeability of the SR membrane is independent of intravesicular and extravesicular calcium concentration in the ranges investigated. The average first order rate constant for passive calcium efflux for six preparations was 0.8 +/- 0.2 min-1 for skeletal and 0.7 +/- 0.1 min-1 for cardiac SR. The amount of intravesicular bound calcium for the same preparations was 33 +/- 6 nmol mg-1 for skeletal and 13 +/- 2 nmol mg-1 for cardiac SR. The first order rate constants were unaffected by Mg concentration between 0.1 +/- 15.1 mM and by the presence of an ATP-regenerating system. The results suggest that some minimal calcium load may be required in order to observe a substantial passive calcium efflux, the passive calcium efflux is not carrier mediated, and passive calcium efflux is not a likely route of calcium release during excitation-contraction coupling.     

In vitro stimulation of alkaline phosphatase activity in immature embryonic chick pelvic cartilage by adenosine

Cyclic AMP content in embryonic chick pelvic cartilage increases significantly as the embryo ages from 8 to 10 d. This in ovo elevation in cyclic AMP content precedes maximal cartilage alkaline phosphatase activity by some 24 h. We studied whether this temporal relationship may be causally related, using an in vitro organ culture. Incubation of pelvic cartilage from 9- and 10-d embryos in medium containing monobutyryl cyclic AMP (BtcAMP) resulted in significant increases in alkaline phosphatase activity (220 and 66 percent, respectively) as compared to that of cartilages incubated in medium alone. This stimulation was both concentration- and time-dependent with maximal response at 0.5 mM BtcAMP and 4-h incubation, respectively. Similar incubations of cartilage in medium containing 1-methyl-3-isobutyl xanthine (MIX), 0.25 mM, also resulted in increased alkaline phosphatase activity (114 percent). However, pelvic cartilage from 11-d embryos incubated in medium containing BtcAMP or MIX showed no increase in alkaline phosphatase activity. We postulated that developmental age was the factor responsible for this difference in response and that immature cartilage (that with little or no alkaline phosphatase activity) would respond to BtcAMP whereas mature cartilage (that with significant alkaline phosphatase activity) would not. This was tested by incubating end sections of 11-d cartilage, which have little alkaline phosphatase activity, and center sections, which have significantly alkaline phosphatase activity, with both BtcAMP and MIX. Alkaline phosphatase activity in end sections (immature cartilage) was stimulated by BtcAMP and MIX, whereas it was not stimulated in the center sections. Actinomycin D and cycloheximide inhibited BtcAMP and MIX stimulation of alkaline phosphatase activity. Thus, the in vitro data suggest that cyclic AMP is a mediator for the stimulation of alkaline phosphatase activity in embryonic cartilage.     

An evaluation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the identification of microsporidia

Microsporidian spore polypeptides separated with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) can be used to identify isolates of microsporidia. The spore polypeptides separated with SDS/PAGE provided unique, reproducible electrophoretic profiles which were not influenced by host species or the temperature at which the host larvae were maintained for development. Furthermore, host proteins were not detected in electrophoretic profiles of the spore polypeptides. Spore mixtures of two microsporidian species can be detected when the spore polypeptides of either or both species have been previously separated with SDS/PAGE.     

Evidence from studies with acifluorfen for participation of a flavin-cytochrome complex in blue light photoreception for phototropism of oat coleoptiles

The diphenyl ether acifluorfen enhances the blue light-induced absorbance change in Triton X100-solubilized crude membrane preparations from etiolated oat (Avena sativa L. cv. Lodi) coleoptiles. Enhancement of the spectral change is correlated with a change in rate of dark reoxidation of a b-type cytochrome. Similar, although smaller, enhancement was obtained with oxyfluorfen, nitrofen, and bifenox. Light-minus-dark difference spectra in the presence and absence of acifluorfen, and the dithionite-reduced-minus oxidized difference spectrum indicate that acifluorfen is acting specifically at a blue light-sensitive cytochrome-flavin complex. Sodium azide, a flavin inhibitor, decreases the light-induced absorbance change significantly, but does not affect the dark reoxidation of the cytochrome. Hence, it is acting on the light reaction, suggesting that the photoreceptor itself is a flavin. Acifluorfen sensitizes phototropism in dark-grown oat seedlings such that the first positive response occurs with blue light fluences as little as one-third of those required to elicit the same response in seedlings grown in the absence of the herbicide. Both this increase in sensitivity to light and the enhancement of the light-induced cytochrome reduction vary with the applied acifluorfen concentration in a similar manner. The herbicide is without effect either on elongation or on the geotropic response of dark-grown oat seedlings, indicating that acifluorfen is acting specifically close to, or at the photoreceptor end of, the stimulus-response chain. It seems likely that the flavin-cytochrome complex serves to transduce the light signal into curvature in phototropism in oats, with the flavin moiety itself serving as the photoreceptor.     

The peracid cleavage of 5-methyltetrahydrofolic acid at the C9-N10 bridge.

5-Methyltetrahydrofolate cannot be cleaved at the C⁹N¹⁰ bond by the zinc/HCl reductive or the permanganate oxidative cleavage methods. A new method has been developed to perform this cleavage, using peracetic acid in 50% trifluoroacetic acid; the cleavage is quantitative and nondestructive of γ-glutamyl peptide bonds.     

Studies on the transport mechanism of 5-methyltetrahydrofolic acid in freshly isolated hepatocytes: effect of ethanol.

The effect of ethanol on the transport of 5-methyltetrahydrofolate in freshly isolated hepatocytes in vitro resulted in about a 30% increase in accumulation of substrate. It was shown that this was not due to differences in metabolism, nor to an inhibition of efflux. Preincubation with 40 mm ethanol for 45 min resulted in a significantly increased rate of entry of 5-methyltetrahydrofolate into the cells. The stimulatory effect was specific to 5-methyltetrahydrofolate since ethanol inhibited uptake of folate and methotrexate. The increased uptake was due to metabolism of ethanol as shown by studies with pyrazole. Also, the n-alkanols, propanol through pentanol, and sorbitol but not methanol were stimulatory. Anaerobiosis and sodium azide stimulated uptake of 5-methyl-tetrahydrofolate but were inhibitory to methotrexate uptake. These data, taken together, suggest that the ethanol effect is due to increased entry of 5-CH₃-H₄PteGlu into the cells possibly as the result of an increased cellular $\text{NADH}\text{NAD}$ ratio.     

The effect of essential-fatty-acid deficiency on the activity of liver phosphatidate phosphatase in rats

The activities of the soluble and microsomal phosphatidate phosphatases (EC 3.1.3.4) are greater in the livers of essential-fatty-acid-deficient rats than in rats fed diets containing linoleic acid.     

Properties of a Solubilized Microsomal Auxin-binding Protein from Coleoptiles and Primary Leaves of Zea mays

An auxin-binding protein can be solubilized from microsomal membranes of Zea mays using either Triton X-100 extraction of the membranes or buffer extraction of the acetone-precipitated membranes. This paper describes the properties of the binding protein solubilized by these two methods. The binding is assayed by gel filtration chromatography in the presence of naphthalene [2-¹⁴C]acetic acid. Binding is rapid and reversible with an optimum at pH 5. Both preparations show similar molecular weights by gel filtration (80,000 daltons) at pH 7.6 and 0.1 molar NaCl, and both aggregate at low ionic strength. They appear to be the same active molecular species. The binding activity is destroyed by trypsin, pronase or para-chloromercuribenzoic acid, but not significantly reduced by phospholipase C, DNase, RNase, or dithioerythritol. Since saturating amounts of naphthalene acetic acid protect the molecule from inhibition by para-chloromercuribenzoic acid, it is concluded that the binding protein has a sulfhydryl group at the binding site, or protects such a group in its binding conformation. The dissociation constant of the protein for naphthalene acetic acid is 4.6 × 10⁻⁸ molar with 30 picomoles of sites per gram of tissue fresh weight. Binding constants were estimated for 13 other natural and synthetic auxins by competition with naphthalene[2-¹⁴C]acetic acid. Their dissociation constants are in general agreement with published values for their binding to intact membranes and their biological activity, although several exceptions were noted. A supernatant factor from the same tissue changes the apparent affinity of the protein for naphthalene acetic acid. This factor may be the same one as has been previously reported to alter the affinity of intact microsomes for auxin.     

Auxin receptors of maize coleoptile membranes do not have ATPase activity

Membrane-localized auxin-binding sites from coleoptiles and primary leaves of Zea mays L. which may be auxin receptors can be fully solubilized by 1 to 1.5 mg of Triton X-100 per mg of membrane protein (about 1 mg per gram of original tissue fresh weight), while 70% of the basal (Mg²⁺)-ATPase and 85% of the K⁺-stimulated (Mg²⁺)-ATPase (pH 6) remain pelletable. Gel exclusion chromatography on Bio-Gel A-1.5m indicates that the solubilized receptors occur as detergent-protein micelles of about 90,000 daltons equivalent molecular weight. Solubilized ATPase activities occur (a) as very large particles excluded from the gel, and (b) as particles of a size substantially smaller than the particles that exhibit auxin binding. The auxin-binding receptor therefore appears not to be an ATPase.