K-252a, a novel microbial product, inhibits smooth muscle myosin light chain kinase

Effects of K-252a, (8R*, 9S*, 11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta [cde]trinden-1-one, purified from the culture broth of Nocardiopsis sp., on the activity of myosin light chain kinase were investigated. 1) K-252a (1 x 10(-5) M) affected three characteristic properties of chicken gizzard myosin-B, natural actomyosin, to a similar degree: the Ca2+-dependent activity of ATPase, superprecipitation, and the phosphorylation of the myosin light chain. 2) K-252a inhibited the activities of the purified myosin light chain kinase and a Ca2+-independent form of the enzyme which was constructed by cross-linking of myosin light chain kinase and calmodulin using glutaraldehyde. The degrees of inhibition by 3 x 10(-6) M K-252a were 69 and 48% of the control activities with the purified enzyme and the cross-linked complex, respectively. Chlorpromazine (3 x 10(-4) M), a calmodulin antagonist, inhibited the native enzyme, but not the cross-linked one. These results suggested that K-252a inhibited myosin light chain kinase by direct interaction with the enzyme, whereas chlorpromazine suppressed the enzyme activation by interacting with calmodulin. 3) The inhibition by K-252a of the cross-linked kinase was affected by the concentration of ATP, a phosphate donor. The concentration causing 50% inhibition was two orders magnitude lower in the presence of 100 microM ATP than in the presence of 2 mM ATP. 4) Kinetic analyses using [gama-32P]ATP indicated that the inhibitory mode of K-252a was competitive with respect to ATP (Ki = 20 nM). These results suggest that K-252a interacts at the ATP-binding domain of myosin light chain kinase. The direct action of the compound on the enzyme would explain the multivarious inhibition of myosin ATPase, of superprecipitation, and of the contractile response of smooth muscle.     

Low-density lipoprotein receptor activity in the guinea pig adrenal cortex. II. Zonal response to aminoglutethimide and 17 alpha-ethinylestradiol

Low-density lipoprotein (LDL) receptor activity and the concentration of cholesterol were measured in the outer (glomerulosa/fasciculata) and inner (reticularis) zones of the adrenal cortex of the guinea pig to examine the relation between cholesterol content and LDL receptor activity. While the concentration of cholesterol was 2-3-times higher in the outer cortical zone, the maximum high-affinity binding capacity for LDL was essentially the same for the two zones, or slightly higher for the inner zone. Adrenocorticotrophic hormone (ACTH) caused a significant increase in LDL receptor activity only in the outer zone, but led to a reduction in the cholesterol content in both adrenocortical zones. The treatment of animals with 17 alpha-ethinyl-estradiol also resulted in a reduction of cholesterol in both adrenocortical zones, but an increase in LDL receptor number only in the outer zone. The latter effect was partially reversed by the administration of dexamethasone. Aminoglutethimide, which was used in a dose that did not block steroidogenesis but did block the hydrolysis of cholesteryl esters in response to ACTH, did not prevent the ACTH-induced increase in LDL receptor number in the outer zone. Thus, the number of LDL receptors was increased in the zona fasciculata by ACTH in the absence of a reduction in cellular cholesterol content, while the number of LDL receptors in the zona reticularis was not increased by ACTH even in the face of a reduction in cellular cholesterol. Exclusive of the experiments employing aminoglutethimide, when the cellular cholesterol content was plotted against LDL binding activity, an excellent inverse correlation was revealed for the zona fasciculata, but essentially no correlation was noted for the zona reticularis. It is concluded that the outer and inner cortical zones of the guinea pig adrenal are quite distinct in the nature of their LDL receptor activity and regulation: the LDL receptor of the outer zone appears to function in a way similar to what has been reported for the whole adrenal cortex of other species in that receptor number correlates with tissue cholesterol content and is primarily regulated by ACTH; the LDL receptor number of the inner zone, however, does not correlate with tissue cholesterol content and is apparently not regulated by ACTH.     

Acrosomal actin bundles of Asian and American horseshoe crab sperm differ in protein composition

Acrosomal actin bundles were isolated from the sperm of horseshoe crabs from four different sources, three from Asia and one from North America, and their protein constituents and structures were compared. The bundle from the American Limulus polyphemus sperm was composed of actin and two associated proteins of MW 95,000 and MW 52,000, as reported previously (Tilney, L. G. (1975) J. Cell Biol. 64, 289-310). However, those from the three Asian species (Tachypleus tridentatus, T. gigas, and Carcinoscorpius rotundicauda) were composed only of actin and the protein of MW 95,000. Electron microscopic and optical diffraction studies indicated that both the helical structures and the interfilament spacing of the actin filaments composing the acrosomal bundle were indistinguishable among the four species. These results suggest that the MW 95,000 protein crosslinks actin filaments in the bundle. Moreover, they support the idea that Limulus and the three Asian species have evolved independently from a common ancestor.     

Enhancement of actin-activated myosin ATPase by an 84K Mr actin-binding protein in vertebrate smooth muscle

A Ca2+-dependent actin-severing 84K Mr protein prepared from bovine aorta caused four-fold activation of smooth muscle actin-activated myosin ATPase at a 1/10(2) molar ratio to actin in the presence of tropomyosin and light chain kinase-calmodulin in a Ca2+-dependent manner, while it inhibited it markedly at a 1/5 molar ratio. Purified actin-tropomyosin filaments under the experimental ATPase conditions were distributed in a range of more than 10 micron in length and the addition of the 84K Mr protein changed the filament length to around 1 micron at a 1/10(2) molar ratio to actin or less than 50 nm at a 1/5 molar ratio in the presence of Ca2+. However, the apparent length of actin filaments alone does not appear to be responsible for the activation of ATPase activity, since in the absence of tropomyosin, the ATPase activation was much less in spite of actin filament length changes. These results indicate the possibility that the 84K Mr protein plays an important role with tropomyosin in at least in vitro smooth muscle actin-myosin interaction.     

Heptapeptide toxin production during the batch culture of two Microcystis species (Cyanobacteria)

Changes in the content of cyclic heptapeptide hepatotoxins called microcystins were investigated during batch culture of two Microcystis species using high performance liquid chromatography. After adsorption to ODS-silica gel cartridges and elution with methanol, the toxins were analyzed and quantified by HPLC. 35 μg per 100 mg dry cells of microcystin-RR, 34 μg of -YR and 43 μg of -LR were present at the beginning of the exponential growth phase of M. viridis. Microcystin-RR increased markedly towards the end of the exponential phase with the maximum content of 112 μg per 100 mg cells was measured at the late stage of the exponential phase. A remarkable increase of microcystin-YR from 130 μg per 100 mg cells to 1020 μg was observed during the exponential phase of a highly toxic strain of M. aeruginosa. However no clear differences were found in the pattern of change among the three toxins during the growth course.     

Enhancing effect of wortmannin on muscarinic stimulation of phospholipase D in rat pheochromocytoma PC12 cells.

Wortmannin, a specific inhibitor of myosin light chain kinase (MLCK), enhanced carbachol-induced formation of [3H]phosphatidylethanol ([3H]PEt), a marker of phospholipase D (PLD) activity, in [3H]palmitic acid-labeled PC12 cells. The apparent EC50 value was 1.5 microM, and the effect was maximal at 3 microM and slightly attenuated at higher concentration. Wortmannin alone had no significant effect on [3H]PEt formation. The enhancing effect of wortmannin was observed at the initial increasing phase of [3H]PEt formation but not at the subsequent plateau phase. Wortmannin enhanced also phorbol ester-induced PLD activation. Although the precise mechanism remains to be clarified, these results suggest that MLCK may be involved in PLD regulation in PC12 cells.     

Comparison of axonemal proteins from two kinds of Tetrahymena. I. Different characteristics of dyneins in heat stability

Tetrahymena thermophila could still swim after incubation of the cell body at 40°C for 30 min, whereas Tetrahymena pyriformis did not show any motility after the treatment. Turbidity measurements revealed that axonemes of T. pyriformis lost ATP-dependent sliding activity by the heat treatment, whereas those of T. thermophilia still had the activity under the same conditions. In connection with this difference in susceptibility to high temperature, the biochemical characteristics of dyneins were compared between the two species of Tetrahymena. Axonemal dyneins from the two species had significant vanadate-sensitive ATPase activity even after the heat treatment. Native gel electrophoresis and the following two-dimensional electrophoresis showed that the outer arm dynein of T. thermophilia is more stable in maintaining native configuration than that of T. pyriformis against the heat treatment, although both treated dyneins keep three (α, β and γ) subunits. Analysis by peptide mapping demonstrated that β- and γ-subunits of the outer arm dynein are considerably different in amino acid sequences between the two species. These results imply that dynein of T. thermophilia changed their amino acid sequences and biochemical characteristics to adapt to high temperature.