Properties of detergent-solubilized and membranous (Ca2+ + Mg2+)-activated ATPase from sarcoplasmic reticulum as studied by sulfhydryl reactivity and ESR spectroscopy. Effect of protein-protein interactions

(1) Sulfhydryl reactivity and electron spin resonance spectra of nitroxide maleimide spin labels, covalently attached to sarcoplasmic reticulum ATPase, were examined on both detergent-solubilized and membranous material. Monomeric and oligomeric ATPases were prepared by the use of dodecyloctaethylene glycol monoether as a solubilizing detergent. (2) Immediately after solubilization, the reaction curve of nonomeric ATPase with 5,5'-dithiobis(2-nitrobenzoate) was characterized by positive cooperativity (S-shaped as a function of time). In contrast, the SH reactivity of both oligomeric and membranous ATPases obeyed usual first-order kinetics and could be analyzed in terms of three classes of reactive site. All enzymatically active ATPase preparations responded to addition of ADP with a decrease in SH reactivity. During enzymatic inactivation of monomeric ATPase, the SH-modification rate was dramatically enhanced with loss of cooperative features. Ca2+ removal from the high-affinity sites stimulated SH reactivity before inactivation had taken place. (3) ESR spectroscopy indicated less motional constraints on monomeric than on oligomeric and membranous ATPases. Arrhenius plots of ESR spectral parameters suggest a conformational transition in both membranous and solubilized ATPases at about 22 degrees C. The transition was also present in EGTA-, but not in heat-inactivated ATPase. Although SH reactivity of monomeric ATPase was dramatically enhanced by EGTA inactivation, the results of ESR, circular dichroism and analytical ultracentrifugation experiments indicate limited conformational changes induced by EGTA treatment. (4) The data indicate marked differences in the properties of monomeric ATPase on the one hand and oligomeric and membranous enzymes on the other hand. They are consistent with previous functional evidence for the presence of ATPase in an associated state in the membrane (M?ller, J.V., Lind, K.E. and Andersen, J.P. (1980) J. Biol. Chem. 255, 1912-1920).     

A dnaB analog ban, specified by bacteriophage P1: genetic and physiological evidence for functional analogy and interactions between the two products.

Summary Bacteriophage P1 has been shown previously to determine a product ban than can substitute in DNA replication for the protein specified by cistron dnaB of Escherichia coli. However, ban product furnished by P1 bac prophage (ban constitutive) substitutes only poorly for DNA replication in the absence of dnaB product in a strain bearing an unsuppressed amber mutation, dnaB266, as shown by the cryosensitivity of the dnaB266 (P1 bac) lysogen and its unability to support growth. An additional mutation (termed crr) in the P1 bac prophage has been obtained which confers cryoresistance to the sup ⁺ dnaB266 (P1 bac crr) lysogen and restores its ability to support growth. ban product produced in P1 bac crr lysogen fulfills all dnaB roles in vivo, especially in the various instances in which ban product expressed in P1 bac lysogens does not. The ban product is expressed constitutively in P1 crr prophage. The crr-1 mutation is tightly linked to the bac-1 and ban-1 mutations and is dominant over crr ⁺. The nature of the crr mutation is discussed: two hypotheses are considered, that of a mutation in the ban gene rendering the ban product more active or that of a site mutation in the ban operon increasing the level of ban expression. Expression of ban product (wild type or altered) leads to interactions with the variously altered dnaB product. Both positive and negative interactions are described. Genetic results presented here suggest that ban and dnaB subunits interact to form hybrid dnaB-like molecules; the average composition of which depends on the relative quantities of ban and dnaB subunits in the cell.     

Effect of aging on the morphology and function of the thyroid gland of the cream hamster

Summary The effect of aging on the morphology and function of the thyroid gland of the cream hamster was studied by light and electron microscopy coupled with autoradiography or histochemistry.Morphologically, aging induces an accumulation of lysosomal dense bodies and a loss of the phagocytosis of colloid droplets after stimulation with TSH. Iodine uptake and organification occur normally and thyroglobulin synthesis, estimated by autoradiography with ³H-leucine, is not different from that observed in young animals. The basal T₄ and T₃ plasma levels are lower in the old animals. A low iodine diet administered for several months prevents the age related accumulation of lysosomal dense bodies. Hormone secretion seems to proceed by two different mechanisms; phagocytosis of colloid droplets, the classical mechanism that decreases with age, and an additional mechanism, probably micropinocytosis, that is maintained during the whole lifespan.Presented at the First French Congress of Endocrinology, Montpellier, September 1980Work was performed under contracts n 3.4523.79 and n 3.4512.80 of the Fonds de la Recherche Scientifique Médicale, thanks to a grant of the Ministère Belge de la Politique Scientifique (Action concertée) and with the help of the Fondation Interuniversitaire pour la Recherche du Processus du Vieillissement, Belgium     

Further observations on cell wall morphogenesis and polysaccharide arrangement during plant growth

Summary The three-dimensional arrangement of the polysaccharide chains in cell walls was investigated, using ultracryotomy and cytochemistry, in order to test the validity of the previously postulated ordered fibril hypothesis and to analyze the characteristics of the primary wall morphogenesis.Both in mung bean hypocotyl (Phaseolus aureus) and pea root (Pisum sativum) cultured in defined conditions, cell to cell endogenous specificity is marked by differences in the numbers of layers, thickness, rhythm and direction of deposition. The occurrence of bow-shaped arrangements and of strata of orientation intermediate between the main crisscrossed multifibrillar layers suggests that the sequential changes of the morphogenetic activity of the cells is progressive. The twisted polysaccharide disposition evokes certain mesomorphic states; a part of the mechanism responsible for the wall arrangement may result from a self-assembly process as in the orientation of the molecules in a liquid cristal. This possibility finds experimental support in the fact that a three-dimensional association of the hemicellulose chains spontaneously appears when precipitated in acellular conditions.Polysaccharide removal associated with shadowing indicates that the ordered disposition within the wall is extensively altered by even a slight extraction. These data may invalidate diverse results which are generally brought forward to explain the wall organization during growth.     

ADP-ribosylation of brain synaptosomal proteins correlates with adenylate cyclase activation

ADP-ribosylation of the adenylate cyclase $\text{G}\text{F}$ regulatory subunit by cholera toxin is a major tool for the study of this enzyme. Investigation of the brain enzyme has been hampered up to now by the failure to demonstrate cholera toxin-dependent ADP-ribosylation of membrane-bound proteins. Synaptosomes prepared by flotation from fresh brains homogenized in the presence of protease inhibitors yielded membranes of which several proteins could be ADP-ribosylated by the toxin. The same membranes subjected to mild proteolysis could not be ADP-ribosylated. Adenylate cyclase activation and ADP-ribosylation were simultaneous processes. The major labeled species was of 47,000 M_r. It was solubilized by Lubrol-PX, together with other labeled polypeptides. As analyzed on sucrose gradients, the 47,000 M_r protein was found both in the 3S region, and in the adenylate cyclase containing fraction (9.1S).     

Steroid glucuronides: Human circulatory levels and formation by LNCaP cells

We studied the relationship between circulating androsterone glucuronide, androstane-3,17β-diol glucuronide and androstane-3β,17β-diol glucuronide concentrations and adrenal as well as testicular C-19 steroids in men. Among the three 5-reduced steroid glucuronides, androsterone glucuronide is the predominant C-19 steroid measured in plasma and its levels are markedly elevated compared to those of the non-conjugated steroid. The marked rise in testosterone during puberty was strongly correlated with the increase in both androsterone glucuronide and androstane-3,17β-diol glucuronide, thus suggesting that testicular C-19 steroids are the main precursors of the steroid glucuronides. We also found that the presence of testicular androgen in plasma contributes to approx. 70% of plasma androsterone glucuronide and androstane-3,17β-diol glucuronide. Our data suggest that the adrenal C-19 steroids remaining in circulation after castration in men are converted into potent androgen which are then glucuronidated by UDP-glucuronyltransferase. We also demonstrated that the human prostate cell line LNCaP is capable of converting to a large extent androstenedione into androsterone glucuronide. Our data further confirm that glucuronidation is a major pathway of steroid metabolism in steroid target tissues.     

Experimental silver bioaccumulation in the polychaetePomatoceros triqueter (L.)

Summary The tubicolous polychaetePomatoceros triqueter was exposed for 6–7 weeks to 200 or 400 g · l^–1 silver introduced as the nitrate into sea water. Survival conditions and mortality were evaluated and silver bioaccumulation analysed by atomic absorption spectrometry. Characteristic morphological lesions were recognized. Histopathologic examination was performed on paraffin or semi-thin sections and at the ultrastructural level. Histochemical examination mainly concerned the metals, reducing groups and sulfur-containing proteins. Microanalytical study involved the use of a wavelength-dispersive X-ray spectrometry microprobe and ion microanalyzer, and the use of an energy-dispersive X-ray spectrometry microprobe at the ultrastructural level. Our results emphasize the role of the branchial crown for metal penetration. Its cuticle accumulates silver as a metal, in particulate form. The internal accumulation of mainly extracellular deposits concerns the basement membranes and connective tissue present in the axis of the branchial crown filaments, or surrounding the nephridial pouches and the gut sinus. The carrier role of the closed vascular system is suggested by ultrastructural observations. The silver route from transepithelial uptake to nephridial excretion involves at least two intracellular transits, plus the vascular mesothelium. Nephridia play a role in silver storage (lysosomes) and elimination (concretions). In all parts internal to the crown cuticle, silver is at least partly associated with protein SH-groups (metallothionein-like); deposits can be enriched with silver sulfide and metallic silver.     

Synthesis of Tricyclic Phenylpyrrole Lactams, New Models of Antitubulin Agents

The tricyclic lactams 3 and 4 having a phenylpyrrole framework have been prepared from the arylpyrroles 5 and 16 respectively. These structural analogs of rhazinilam 1 present, like the latter, an interesting antitubulin and cytotoxic activity.     

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.