Effects of papain on isolation of single smooth muscle cells from the guinea pig longitudinal ileum.

The methods for isolation of single cells from the guinea pig longitudinal ileum were investigated with focussing on the papain concentration for the digestion of the ileum. The ileal muscle was minced. The minced muscle was loaded with fluorescent probes of calcein or fura 2, and treated with papain for 30 min at various concentrations. Papain at concentrations more than 1 U/ml reduced both calcein fluorescence and fura-2-signal evoked by carbachol. Carbachol-induced fura-2-signal was more sensitive to papain than calcein fluorescence, suggesting that proteins related to the formation of receptors are more vulnerable than membrane lipids or proteins limiting the membrane permeability upon the exposure to papain. The resultant yield of single cells was highest at 0.56 U/ml of papain without affecting calcein and fura-2 fluorescence responses, thus this concentration appeared to be appropriate for the isolation of single cells from the ileum. Single cells alive contracted dose dependently by the exposure to carbachol (0.1-10 microM) under the microscopic measurement, and were appeared to grow confluent in culture for approximately 15 days. These results suggest that the low concentration, 0.56 U/ml, of papain in the isolation medium is better to obtain functional cells from the guinea pig ileum.

9-Methyl-7-bromoeudistomin D induces Ca2+ release from cardiac sarcoplasmic reticulum.

9-Methyl-7-bromoeudistomin D (MBED), the most powerful caffeine-like releaser of Ca2+ from skeletal muscle sarcoplasmic reticulum, induced Ca2+ release from the cardiac sarcoplasmic reticulum. MBED (5 microM) and caffeine (1 mM) caused rapid Ca2+ release from the fragmented cardiac sarcoplasmic reticulum in a Ca2+ electrode experiment. [3H]MBED bound to a single class of high-affinity binding sites in cardiac sarcoplasmic reticulum membranes (Kd = 150 nM). These results suggest that MBED binds to a specific binding site on cardiac sarcoplasmic reticulum membranes to induce Ca2+ release from the cardiac sarcoplasmic reticulum. Thus, MBED is a useful probe for characterizing Ca2+ release the channels not only in skeletal sarcoplasmic reticulum but also in cardiac sarcoplasmic reticulum.

Purealin blocks the sliding movement of sea urchin flagellar axonemes by selective inhibition of half the ATPase activity of axonemal dyneins.

Ciliary and flagellar movements are explained by active sliding between the outer doublet microtubules of an axoneme via their inner and outer dynein arms. Purealin, a novel bioactive principle of a sea sponge Psammaplysilla purea, blocked the motility of Triton-demembranated sea urchin sperm flagella within 5 min at concentrations above 20 microM. In a similar concentration range, purealin blocked the sliding movement of the flagellar axonemes in vitro within a few minutes judging from the turbidity measurements. The ATPase activity of axonemes was partially inhibited by purealin in a concentration-dependent manner. The maximum inhibition reached approximately 50% at concentrations above 20 microM, indicating that half the axonemal ATPase activity is sensitive to purealin. Similar results were observed on the ATPase activity of outer-arm-depleted axonemes and that of a mixture of 21S dynein and salt-extracted axonemes. On the other hand, ATPase activity of isolated 21S dynein was not inhibited by purealin. The inhibitory action of purealin on the axonemal ATPases was reversed by dilution of purealin. The effect of purealin on the double-reciprocal plot of the ATPase activity as a function of ATP concentrations showed that the inhibition was not a competitive type. In accord with this finding, purealin did not affect the vanadate-mediated UV photocleavage of axonemal dyneins. These results suggest that purealin binds reversibly to a site other than the catalytic ATP-binding site and inhibits half the ATPase activity of axonemes. Taken together, our results suggest that purealin-sensitive ATPase activity of the dynein arms plays an essential role in generating the sliding movement of flagellar axonemes.

Partial purification of an endogenous inhibitor of muscarinic ligand binding.

An endogenous inhibitory factor (EIF alpha) to the binding of a muscarinic antagonist, [3H]N-methyl scopolamine ([3H]NMS), has been partially (12-fold) purified from the soluble fraction of the ileal longitudinal muscle of guinea-pigs using a heat-treatment, isoelectric fractionation, and DEAE-column chromatography. The EIF alpha inhibited the [3H]NMS binding to the longitudinal muscle membrane with an IC50 of 53.6 micrograms/ml. This was about 230-fold potent than the non-specific inhibition of [3H]NMS binding by bovine serum albumin (BSA). Zn2+ (0.1 mM) almost completely blocked the inhibitory activity of EIF alpha, whereas such the effect of Zn2+ was not observed in the inhibition by BSA. These results suggest that EIF alpha inhibits the [3H]NMS binding to the muscarinic acetylcholine receptor in a different manner from non-specific interaction with the receptor.

High affinity binding of 9-[3H]methyl-7-bromoeudistomin D to the caffeine-binding site of skeletal muscle sarcoplasmic reticulum.

3H-Labeled 9-methyl-7-bromoeudistomin D ([3H] MBED), the most powerful inducer of Ca2+ release from sarcoplasmic reticulum (SR), was successfully prepared with a high specific activity of 10.2 Ci/mmol. [3H]MBED bound to terminal cisternae (TC) of skeletal muscle SR in a replacable and saturable manner, indicating the existence of its specific binding site. Caffeine inhibited the [3H]MBED binding to the TC-SR membranes from skeletal muscle with an IC50 value of 0.8 mM, in close agreement with a concentration that causes Ca2+ release from SR. Scatchard analysis gave values of KD = 40 nM and Bmax = 10 pmol/mg protein. The KD value was increased by caffeine, while that of Bmax was not changed, indicating a competitive mode of inhibition. Adenosine 5'-(beta, gamma-methylene)triphosphate enhanced [3H]MBED binding, but ryanodine and Ca2+ did not affect it. [3H]MBED binding to TC-SR membranes was inhibited by procaine, a representative blocker of Ca(2+)-induced Ca2+ release channels, whereas that was not changed by Mg2+, suggesting that procaine but not Mg2+ may exert its inhibitory effect on Ca(2+)-induced Ca2+ release by affecting the caffeine-binding sites. These results suggest that MBED shares the same binding site as that of caffeine in TC-SR. The [3H]MBED is the first radiolabeled ligand for caffeine-binding sites in Ca2+ release channels and thus may provide an essential biochemical tool for elucidating this site.

Reduction in the numbers of muscarinic receptors by an endogenous protein.

The soluble fraction from the ileal longitudinal muscle of guinea pigs was examined for the presence of an endogenous modulator of muscarinic receptors. In the presence of the soluble fraction, the binding of [3H]quinuclidinyl benzilate to the membranes from the tissue was inhibited in a concentration-dependent manner. The inhibitory activity in the soluble fraction was heat stable, but was inactivated by trypsin treatment. Several protease inhibitors had no effect on the inhibitory activity. These results suggest the existence of an endogenous protein that inhibits the binding of the muscarinic ligand to the receptor. Ultrafiltration demonstrated that the protein factor had a molecular mass of more than 10,000 Da. Saturation binding and dissociation kinetic experiments indicate neither a competitive nor allosteric mode of inhibitory action and suggest that an irreversible block or internalization of muscarinic receptors is induced by the endogenous protein.

Heterogeneous contractile responses of single smooth muscle cells from guinea pig ileum.

Cells of guinea pig ileal longitudinal muscle layer were dispersed by treating the minced tissue with highly purified collagenase and papain. The cells were spindle shaped, 100 to 150 microns long and 5 to 10 microns wide. Contractility of the dispersed cells was determined by "cell adhesion method" resulting that the cells were contracted by carbachol in a dose-dependent manner, but sensitivity and degree of the contraction were different in each cell. Binding assay method resulted that Kd values did not change by treatment with collagenase and papain. These results indicate that contractility of each cell in the tissue is originally different and dispersion of the cells does not affect on the cell contractility.

9-methyl-7-bromoeudistomin D, a powerful radio-labelable Ca++ releaser having caffeine-like properties, acts on Ca(++)-induced Ca++ release channels of sarcoplasmic reticulum.

9-Methyl-7-bromoeudistomin D (MBED), a derivative of eudistomin D isolated from a marine tunicate, induced Ca++ release from the heavy fraction of fragmented sarcoplasmic reticulum (HSR) in the same way as that of caffeine, followed by spontaneous Ca++ reuptake in the Ca++ electrode experiment. The rate of 45Ca++ efflux from HSR vesicles was accelerated markedly by MBED or caffeine in a concentration-dependent manner. The 50% effective concentrations of MBED and caffeine were approximately 1 microM and 1 mM, respectively, indicating that MBED is 1000 times more potent than caffeine in HSR. Procaine, ruthenium red or Mg++ caused concentration-dependent inhibition of MBED-triggered Ca++ release from HSR. The bell-shaped profile of Ca++ dependence for MBED is very similar to that of caffeine. The caffeine-produced maximum response of 45Ca++ efflux was increased further by adenosine-5'-(beta, gamma-methyl-ene)triphosphate, whereas that was not changed by MBED. MBED also caused Ca++ release from sarcoplasmic reticulum (SR) of chemically skinned fibers. These stimulatory effects of MBED on the Ca++ release from skeletal muscle SR were almost indistinguishable from those of caffeine except the difference in potencies. The [3H]ryanodine binding to junctional terminal cisternae membranes was not inhibited by MBED or caffeine. MBED did not cause Ca++ release from the light fraction of fragmented SR and turbidity change of mitochondrial suspension. These observations suggest a most likely idea that MBED binds to the caffeine-binding site in the Ca channel protein and thus produces the potentiation of Ca(++)-induced Ca++ release from SR.(ABSTRACT TRUNCATED AT 250 WORDS)

Difference between amino acid residues in the metal-ligand environments of iron- and manganese-superoxide dismutases.

Alignment of the amino acid sequences of the Pseudomonas ovalis and Photobacterium leiognathi iron-superoxide dismutases (Fe-SODs) with the known sequences of the manganese-superoxide dismutases (Mn-SODs) shows that both types of SOD are highly homologous (33-53% identity) and share residues for the metal coordination. The amino acid residues that form the environment of the metal ions appear to be also conserved between the Fe- and Mn-SODs, except that the Phe-84 and Gln-154 in the Mn-SODs are replaced by Tyr and Ala, respectively, in the Fe-enzymes. Since this latter residue contributes to formation of the hydrophobic metal-ligand environment through hydrogen bonding with Trp-133 and Tyr-34 in the Mn-SODs, its substitution by Ala should cause different micro environments between the metal centers of the Fe- and Mn-SODs. This difference may account for the metal specificity of both types of SODs demonstrated by previous reconstitution experiments.

Amino acid sequence of iron-superoxide dismutase from Pseudomonas ovalis.

The amino acid sequence of iron-superoxide dismutase from Pseudomonas ovalis was deduced by the analyses of peptides derived from limited hydrolysis of the aminoethylated or pyridylethylated apoprotein with trypsin, Staphylococcus aureus V8 protease, and dilute acid hydrolysis. The polypeptide chain contains 195 amino acid residues and has a calculated Mr of 21,421. The sequence is highly homologous (65% identity) to the recently published sequence of the iron-superoxide dismutase from Photobacterium leiognathi. It is also homologous to the known sequences of the manganese-superoxide dismutase by sharing 33-53% identical residues. Alignment of the superoxide dismutase sequences and the available structural information from X-ray crystallography suggest that the ligands to the iron in the P. ovalis superoxide dismutase are His-26, His-74, Asp-156 and His-160, which align with the ligands to the manganese in the Thermus thermophilus manganese-superoxide dismutase. The sequence information of the P. ovalis dismutase will facilitate refinement of the X-ray crystallographic data that are now available at 2.9 A resolution.