Differences between smooth and skeletal muscle myosins in their interactions with F-actin

Myosin and F-actin were prepared from bovine carotid arterial smooth muscle and the properties of the binding of myosin to F-actin were compared with those of the binding of skeletal muscle myosin to F-actin. The following differences were observed between skeletal and smooth muscle myosins. 1. The rate of ATP-induced dissociation of arterial actomyosin was equal to that of hybrid actomyosin reconstituted from arterial myosin and skeletal muscle F-actin, but was much lower than those of skeletal muscle actomyosin and of hybrid actomyosin reconstituted from skeletal muscle myosin and arterial F-actin. 2. The amount of ATP necessary for complete dissociation of arterial actomyosin was 2 mol/mol of myosin, although it is well known that skeletal muscle actomyosin is dissociated completely by the addition of 1 mol ATP per mol of myosin. 3. Arterial actomyosin and hybrid actomyosin reconstituted from arterial myosin and skeletal muscle F-actin did not dissociate upon addition of 0.1 mM PPi, while skeletal muscle actomyosin dissociated completely. 4. In the absence of Mg2+, neither dissociation by ATP nor ATPase [EC 3.6.1.3] activity was observed with arterial actomyosin and hybrid actomyosin reconstituted from arterial myosin and skeletal muscle F-actin. On the other hand, skeletal muscle actomyosin dissociated almost completely upon addition of ATP and showed a considerably high ATPase activity. These observations reveal marked differences between myosins from skeletal and smooth muscles in their binding properties to F-actin.     

Glucans in the Cell Walls Regenerated from Vinca rosea Protoplasts

Protoplasts prepared from suspension-cultured Vinca rosea cellswere cultured for 5 days. The cell walls regenerated from theprotoplasts were mainly composed of glucans having 1,3- and1,4-linkages. To investigate the molecular species, these glucanswere separated into four fractions: EDTA (50 mM, pH 4.5)-soluble(fraction E), KOH (24%)- soluble but not precipitatable by neutralizationwith acetic acid (fraction K-S), KOH (24%)-soluble and precipitatableby neutralization with acetic acid (fraction K-P), and KOH (24%)-insoluble(fraction C). By means of sugar composition analysis, methylationanalysis, periodate oxidation and enzymatic digestion, the molecularspecies of the glucans contained in the regenerated cell wallswere deduced to be ß-1,4-glucan (cellulose) and ß-1,3-glucan.Fraction C was mainly composed of ß-1,4-glucan; ß-1,3-glucanwas mainly recovered in fraction K-P. The ß-l,3-glucanwas soluble in dilute alkali solution, but was only slightlysoluble in water. The ß-1,3-glucan had an essentiallyunbranched structure, and its weight average molecular weightestimated by gel permeation chromatography was 4.5–5.0x 10⁴. ¹ Present address: Division of Environmental Biology, NationalInstitute for Environmental Studies, Yatabe, Tsukuba, Ibaraki305, Japan (Received May 21, 1981; Accepted October 13, 1981)     

Soluble and bound forms of intracellular acid carboxypeptidase inAspergillus saitoi

The enzymological, physical, and immunological properties of soluble and bound forms of intracellular acid carboxypeptidase isolated from fresh mycelia ofAspergillus saitoi are reported. In the broken mycelia, about 60% of the total activity was found in the 2,000×g precipitate, with most of the remainder in the 100,000×g supernantant. The highly purified enzymes, I_a and I_b, from the 100,000×g supernatant were found to be homogeneous by such criteria as disc gel electrophoresis at pH 9.4 The bound enzyme, II, was solubilized from the 2,000×g precipitate by self-digestion at pH 6.4 and was highly purified by chromotography. The two forms of intracellular enzymes, the soluble enzymes (I_a and I_b) from the 100,00×g supernatant and the solubilized enzyme (II) from the 2,000×g precipitate, were closely related to, but not completely identical with, the extracellular acid carboxypeptidase.     

Dynamic states of phospholipids in Escherichia coli B membrane. Electron spin resonance studies with biosynthetically generated phospholipid spin labels

Mobility of phospholipid hydrocarbons in the Escherichia coli B membrane fractions was studied by labeling phosphatidylethanolamine or phosphatidylglycerol in situ by biosynthetic incorporation of the spin label. For this purpose, CDP-diacylglycerol spin label was synthesized from phosphatidic acid spin label and cytidine 5'-phosphoromorpholidate and purified by thin-layer chromatography. DCP-diacylglycerol spin label was then incorporated into phospholipids biosynthetically. ESR spectra of these E. coli B membrane fractions showed that phosphatidylglycerol tended to interact with membrane proteins through the mediation of Mg2+, whereas phosphatidylethanolamine had less of this tendency and was more involved in the formation of the bulk of the bilayer continuum of the membrane. These conclusions were also supported by labeling membranes with exogenous spin-labeled phospholipids, although there was some indication that exogenous phospholipids were incorporated into sites different from the sites of incorporation of phospholipids newly synthesized in situ.     

Entamoeba histolytica: localization and characterization of ca2+-dependent nucleotidases

An activity of Ca²⁺-dependent nucleotidase was detected in axenically-cultivated trophozoites of Entamoeba histolytica. The enzyme was concentrated by differential and sucrose density gradient centrifugation and catalyzed hydrolysis of nucleoside tri- and diphosphates and also thiamine pyrophosphate. Hydrolysis of nucleoside mono-phosphates was not affected by Ca²⁺. Among substrates tested, ATP was most active. Addition of Zn²⁺ or heat treatment almost abolished the enzyme activity. The enzyme exhibited almost the identical activity at acid and neutral pH. Among 6 bands isolated by polyacrylamide gel electrophoresis, 4 were stained with ATP, UTP, CTP and ADP, whereas the other 2 were stained only with ATP, UTP and CTP. The concentrated enzyme preparation, primarily composed of membrane fragments, also had activities of acid phosphatase, acid inorganic pyrophosphatase, 5'-nucleotidase and Mg²⁺-dependent ATPase. These observations suggest that E. histolytica has 2 Ca²⁺-dependent nucleotidases, i.e. one Ca²⁺-dependent ATPase and the other Ca²⁺-dependent nucleoside diphosphatase or an apyrase-like enzyme, and that these nucleotidases are at least partially associated with the plasma membrane or an organelle of lysosomal nature in this parasite.     

Structural study of the sugar chains of alpha-amylases produced ectopically in tumors

About thirty percent of two alpha-amylases produced from a serous papillary cystadenocarcinoma of the ovarium (case 1) and a bronchioloalveolar adenocarcinoma of the lung (case 2) was glycoproteins containing 1 mol of asparagine-linked sugar chain, respectively. The structures of the sugar moieties were found by sequential enzymatic degradation and methylation analysis to be as follows: [(Gal beta 1 leads to 4)0 or 1GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(3)][GlcNAc beta 1 leads to 2Man alpha 1 leads to 3(6)]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc and [(Gal beta 1 leads to 4)0 or 1GlcNAc beta 1 leads to 2Man alpha 1 leads to 6][NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc. Structures of asparagine-linked sugar chains were the same in the tumors of cases 1 and 2 and were incomplete in comparison with those of the parotid amylase.     

One step purification procedure of elastase from pancreatic powder by affinity chromatography

Pancreatic elastase was isolated from acetone powder of porcine pancreas by a one step purification procedure on a trialanyl CH-Sepharose 4B affinity column. This column exhibited affinity not only for active elastase but also for trypsin and chymotrypsin which were present in the same pancreatic powder. However, as the extent of affinity toward elastase is considerably higher, the proper conditions were determined with which the adsorbed elastase was isolated in a highly purified form. The yield of elastolytic activity ranged from 60–85% and the purified elastase was shown to be one component by polyacrylamide disc electrophoresis.     

Control of concanavalin A receptor mobility by cytoplasmic actin in human tumour cells.

Tissue culture monolayers of seven human intracranial tumours comprising 2 astrocytomas, 3 meningiomas, 1 secondary squamous cell carcinoma and 1 secondary adenocarcinoma were examined by a double immunofluorescent staining technique to demonstrate Concanavalin A (Con A) surface receptors and cytoplasmic actin in the same cell. Tumour cells, treated with fluoresceinisothiocyanate-labelled Con A (FITC-Con A) showed staining in cell margins or in a random distribution over the cell surface. Incubating the cells with FITC-Con A at 37 degrees for increasing periods of time resulted first in staining of clusters and later of perinuclear globules. Cells, pretreated with 4% paraformaldehyde at 4 degrees for 10 min or with cytochalasin B at 37 degrees for 30 min showed staining restricted to cell margins. In the cytochalasin B-treated cells, the peripheral staining was in the form of coarse clusters. Double fluorochrome studies showed that the anti-actin antibody (AAA) staining occurred in sites closely related to those stained by FITC-Con A both in untreated as well as in cytochalasin B-treated cells. The findings suggest that Con A receptors, as an example of a stable cell membrane determinant in human tumour cells, are associated with actin and that their mobility on the cell surface is dependent on an intact cytoplasmic actin system.