The mechanism of Ca2+ regulation of vascular smooth muscle thin filaments by caldesmon and calmodulin

The interactions of vascular smooth muscle caldesmon with actin, tropomyosin, and calmodulin were determined under conditions in which the four proteins can form reconstituted Ca2+-sensitive smooth muscle thin filaments. Caldesmon bound to actin in a complex fashion with high affinity sites (K = 10(7) M-1) saturating at a stoichiometry of 1 per 28 actins, and lower affinity sites at 1 per 7 actins. The affinity of binding was increased in the presence of tropomyosin, and this could be attributed to a direct interaction between caldesmon and tropomyosin which was demonstrated using caldesmon cross-linked to Sepharose. In the presence of tropomyosin, occupancy of the high affinity sites was associated with inhibition of actin-activated myosin MgATPase activity. Caldesmon was found to bind to calmodulin in the presence of Ca2+, with an affinity of 10(6) M-1. The binding of Ca2+ X calmodulin to caldesmon was associated with the neutralization of inhibition of actin-tropomyosin. Ca2+ X calmodulin binding reduced but did not abolish the binding of caldesmon to actin-tropomyosin. From this data we have proposed a model for smooth muscle thin filaments in which Ca2+ regulates activity by converting the inhibited actin-tropomyosin-caldesmon complex to the active complexes, actin-tropomyosin-caldesmon-calmodulin X Ca2+ and actin-tropomyosin.     

Basic Protein Dissociating from Myelin Membranes at Physiological Ionic Strength and pH Is Cleaved into Three Major Fragments

Experiments were performed with isolated human myelin membrane preparations to analyse factors that may modulate association of myelin basic protein (MBP) with the membranes and could contribute to demyelinating processes. Transfer of membranes (5 mg protein ml-1) at 37 degrees C and pH 7.4 from a hypotonic medium, in which they were relatively stable, to one of physiological ionic strength produced three major effects: (1) initial dissociation of MBP from the membranes by a nonenzymatic process that was doubled in the presence of millimolar Ca2+/Mg2+; (2) within 10 min, the appearance in the medium of three major MBP fragments (14.4, 10.3, and 8.4 kilodaltons); and (3) progressive acidification of dissociated MBP and its fragments, probably due to deamidation. Between 1 and 6 h a steady state was apparent in which protein equivalent to 15% of the MBP originally bound to the membranes was found in the medium. The three major MBP fragments formed two-thirds of this solubilised material and appeared metabolically stable for 24 h. The kinetics of peptide formation suggested that dissociated, rather than membrane-bound, MBP was cleaved by myelin-associated neutral proteases. Two-dimensional electrophoresis and immunoblotting using two monoclonal antibodies indicated that proteolysis occurred in the vicinity of residues 35 and 75. Evidence was also obtained for removal of C-terminal arginines and relatively rapid deamidation in the C-terminal half of MBP. These modifications of MBP might also occur if extracellular fluid gained access to the compacted cytoplasmic space of the myelin sheath.     

Alpha-1 and alpha-2 adrenoceptor binding in cerebral cortex: Role of disulfide and sulfhydryl groups

The triated adrenergic antagonists Prazosin ([³H]PRZ) and Idazoxan ([³H]IDA, or RX-781094) bind specifically and with high affinity to ₁ and ₂-adrenoceptors respectively, in membrane preparations from cerebral cortex. Saturation experiments performed to determine the density of receptors and the dissociation constant (K _d) were analyzed by the methods of Eadie Hofstee, iterative modelling, and the procedure of Hill, while the specificity of the labelling was verified by displacement experiments. Since receptors are proteins, we examined the role of disulfide (–SS–) bridges and sulfhydryl (–SH) groups in the specific combination of [³H]PRZ and [³H]IDA to the ₁ and ₂-adrenoceptors. Pretreatment of the membranes with the –SS– reactive DL-dithiothreitol (DTT) or the alkylating agent N-ethylmaleimide (NEM), alone or in combination, decreased specific binding of both ligands, with only minor changes in the non-specific counts. The [³H]IDA binding (₂-sites) was more sensitive to both DTT and NEM than the [³H]PRZ sites (₂-adrenoceptors), and the initial changes induced by alkylation of the ₂-site were due to an important decrease in the affinity for [³H]IDA, as judged by the increase in theK _d. This modulation in the affinity caused by alkylation of a thiol group could explain the higher potency of the blocking agent tetramine disulfide benextramine at the ₂-site. The results provide evidence for the participation of –SS– and –SH groups in the binding site of ₁ and ₂-adrenoceptors in the cerebral cortex.     

Cholinergic neuronotrophic factors: VI. Age-dependent requirements by chick embryo ciliary ganglionic neurons

During development, ciliary ganglionic neurons become postmitotic and extend neurites in apparent independence of the presence of their future intraocular innervation targets. After reaching their peripheral innervation territory, however, these neurons become target dependent and about half of them die. We have previously reported that chick embryo intraocular target tissues contain a ciliary neuronotrophic factor (CNTF), which can be extracted and partially purified in a soluble form and which ensures near-total survival of 8-day chick embryo ciliary ganglionic neurons in monolayer cultures. In this study we have dissociated and cultured ciliary ganglia from embryonic Day (ED) 5 through 14, and examined dependence and responsiveness of their neurons to exogenously added CNTF. Two cell classes (dark and bright) could be distinguished by phase microscopy and differentially counted in cell dissociates from ED7–14, but not in ED5–6 ones. Dark cell number per ganglion increased from 6000 to 78,000 over this developmental time period. In contrast, bright cells (putative neurons) declined from a maximum of about 10,000 to 6000, suggesting a correlation with the expected neuronal cell death in vivo. Dissociated cells from ED5–14 ganglia were seeded on a polyornithine substratum coated with neurite promoting factor, cultured for 24 hr with or without added CNTF, and numerically examined for survival and neuritic development. Cultures from ED7–14 ganglia showed two cell categories: (i) flat nonneuronal elements dramatically increased in number with ganglionic age (thereby correlating with the increasing number of dark cells in the dissociates) and (ii) large, bright cells (often displaying neurite outgrowth) decreased in number in parallel with bright cell number in the dissociate. The survival of these neuronal elements was strictly dependent on exogenously added CNTF between ED7 and 10, but became progressively independent with older ages. ED14 neurons (fully capable of surviving for 24 hr without added CNTF) continued to require CNTF for neurite extension, thus displaying retained sensitivity to this factor. Although the ED5–6 cultures contained well-recognizable flat cells, the dominant category comprised cells with variable morphology, practically all of which exhibited neurite-like processes. Both the survival and neurite extension of these cells, which we tentatively interpret as immature neurons were independent of the presence of added CNTF.     

Cholinergic Neuronotrophic Factors: Fractionation Properties of an Extract from Selected Chick Embryonic Eye Tissues

Abstract: An aqueous extract derived from selected intraocular tissues of 15-day chick embryos contains a soluble macromolecular agent which is capable of ensuring the survival of 8-day chick embryonic ciliary ganglionic neurons in monolayer culture. When this ciliary neuronotrophic factor (CNTF) was concentrated using ultrafiltration and subjected to Sephadex G100 and G200 chromatography, activity was detected in most of the eluted fractions. A peak of the most active fractions was eluted in a region corresponding to a molecular weight of 35–40 ± 10³ and contained about 20-30% of the applied protein. CNTF activity bound readily to DE-52 cellulose resin at neutral pH and was eluted with NaCl in a narrow region containing about 20-40% of the applied protein. Gel electrophoretic staining profiles of the active DE52 fraction indicated considerable (but still only partial) simplification in protein composition. While significant CNTF activity losses were incurred in response to each of the above treatments, an active material could be conveniently generated in one working day in milligram amounts having a specific activity of 60,000 trophic units/mg protein. This trophic activity is in the same range as that of the only other known neuronotrophic factor, Nerve Growth Factor.     

Sequence variation in dispersed repetitive sequences in Saccharomyces cerevisiae

Two new dispersed repetitive DNA sequences related to the transposable element Tyl have been isolated from the genome of Saccharomyces cerevisiae. One sequence, designated Tyl-17, is present at about six copies per haploid genome, and one copy is located approximately 1000 base-pairs from the LEU2 locus on chromosome III. Tyl-17 is about the same size as Tyl (Cameron et al., 1979) and is flanked by δ sequences, but differs from Tyl by the presence of two large substitutions representing about 50% of the sequence. Tyl and Tyl-17 are found in a ‘head-to-head’ array in at least one cloned region of the yeast genome. Another sequence, designated Tyl-161, is situated about 9000 base-pairs from the PGK locus of chromosome III, and is structurally identical to Tyl except for the presence of a 1200 base-pair insertion near one end of the sequence element.     

Extraction of chloroplast light effect mediator(s) and reconstitution of light activation of NADP-linked malate dehydrogenase

Washed thylakoid membranes of pea (Pisum sativum var. Little Marvel), on brief exposure to zwittergent, an amphoteric detergent, lost the property of supporting the light activation of stromal NADP-linked malate dehydrogenase. But, these depleted membranes, on reconstitution with dialyzed, high-speed supernatant of the detergent extract, showed marked light activation of the enzyme when assayed in the presence of 2,6-dichlorophenolindophenol-ascorbate. The component of the high-speed supernatant which is required for light activation is sensitive to sulfite and is heat labile. The analysis of the high-speed supernatant on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two prominent polypeptides at approximately 18,000 and 36,000 daltons. The surface-specific, chloroglycoluril-mediated iodination of the washed thylakoid membranes revealed that zwittergent had extracted these two polypeptides. The results reveal that the light effect mediator (LEM) is a surface-exposed, tightly bound protein existing in the thylakoid membranes, and that it can be removed by zwitterionic detergent and used in reconstitution studies.     

Resolution of the light-dependent modulation system of pea chloroplasts

The mechanism of the light-dependent inactivation of glucose-6-phosphate dehydrogenase and the light-dependent activation of NADP⁺-malate dehydrogenase has been studied in partially purified extracts of pea (Pisum sativum) chloroplasts. Neither partially purified enzyme could be light modulated by washed thylakoids alone. However, a factor (mol. wt. 50 000) was present in the stroma which could, when added to purified enzyme and thylakoid membranes, reconstitute a light-dependent modulation of either glucose-6-phosphate dehydrogenase or NADP⁺-malate dehydrogenase. This factor, which we term protein-modulating factor, is distinct from ferredoxin-thioredoxin reductase and from thioredoxin, the factors involved in another scheme for light modulation. The scheme proposed here for light modulation involves electron transfer from Photosystem I to a membrane-bound light-effect mediator and then to the soluble protein modulating factor which modulates chloroplast enzyme activity, probably by reduction of a regulatory disulfide bond.     

Recombination and mutagenesis in rad6 mutants of Saccharomyces cerevisiae: Evidence for multiple functions of the RAD6 gene

Summary The rad6-1 and rad6-3 mutants are highly UV sensitive and show an increase in spontaneous and UV induced mitotic heteroallelic recombination in diploids. Both rad6 mutants are proficient in spontaneous and UV induced unequal sister chromatid recombination in the reiterated ribosomal DNA sequence and are deficient in UV induced mutagenesis. In contrast to the above effects where both mutants appear similar, rad6-1 mutants are deficient in sporulation and meiotic recombination whereas rad6-3 mutants are proficient. The differential effects of these mutations indicate that the RAD6 gene is multifunctional. The possible role of the RAD6 gene in error prone excision repair of UV damage during the G1 phase of the cell cycle in addition to its role in postreplication repair is discussed.