Thyroglobulin interactions with thyroid plasma membranes. The existence of specific receptors and their potential role.

Thyroglobulin binds to isolated thyroid plasma membrane preparations. Binding is pH- and temperature-dependent with 10-fold better binding at pH 5.0 and 37 degrees C than at 0 degrees C and pH 6.0 through pH 7.5. Binding is, however, maximal in 90 min at all pH values and temperatures examined. Although salts can inhibit or enhance thyroglobulin binding depending on the temperature or pH, conditions approaching those of the physiological state are not inhibitory; physiological conditions do inhibit thyrotropin binding to the same membrane preparations. 125I-Labeled thyroglobulin binding is poorly reversed by unlabeled thyroglobulin at all pH values and temperatures studied; excess unlabeled thyroglobulin can, however, readily prevent binding. At pH values greater than 6.0 and at 0 degrees C, the iodine content of thyroglobulin can affect binding, and the 27 S thyroid iodoprotein is relatively ineffective in preventing the binding of the 19 S species. At pH 5.0 and 37 degrees C, there is no difference in binding of highly and less iodinated thyroglobulin, and the 27 S thyroglobulin iodoprotein is effective in preventing 19 S thyroglobulin binding. The complex nature of these results is interpreted in the light of additional data which show (i) that the thyroid membrane recognizes asialothyroglobulin and (ii) that at pH 5.0 and 37 degrees C a membrane-associated neuraminidase is activated which removes sialic acid from thyroglobulin. Vibrio cholerae neuraminidase can substitute for the endogenous neuraminidase. The receptor on thyroid membranes for asialothyroglobulin is similar to the asialoglycoprotein receptor on liver membranes (Morell, A.G., Gregoriadis, G., Scheinberg, I.H., Hickman, J., and Ashwell, G. (1971) J. Biol. Chem. 246, 1461-1467) in that sialic acid on the receptor is critical for receptor expression. It is distinct from the liver asialoglycoprotein receptor in its binding specificity and in its sensitivity to different bacterial and mammalian neuraminidase preparations. Relationships between thyroglobulin and thyrotropin receptors on thyroid membranes are explored, and the functional role of the thyroglobulin receptor is discussed.     

The effect of S-adenosyl-L-methionine (SAM) on membrane permeability in the perfused rat liver

S-adenosilmethionine is present in most human tissues and is an important factor for transmethylation, transulphuration and aminopropylation reactions. The compound improves the biological, morphological and histochemical aspects of rat liver following CCl4 intossication. At the same time has been successfully used during chronic liver disease in man. With the aim to better clarify the action mechanism of SAMe some aspects concerning its effects on cell permeability in rat liver, by using the perfusion technique, have been investigated. In particular the capacity of this compound to prevent the enzymatic loss (GPT and GOT) during liver perfusion has been studied. 30 perfusions without SAMe, as control, and 6 by infusing 2 mg of compound during the perfusion time have been accomplished. Varing the perfusion time from 0 to 120 min it has been observed that at any time the presence of the SAMe reduced by about 50% the loss of GOT. Similarly the activity of GPT ranging from 2 to 6 mU/ml indicate that no appreciable enzyme output occurs in presence of SAMe.     

Resolution and characterization of two forms of phosphoinositide-specific phospholipase C from bovine rod outer segments.

Two forms (I and II) of phospholipase C, specific for phosphatidyl inositol 4,5-bisphosphate, were resolved from bovine retinal rod outer segment (ROS) cytosol by DEAE-Sepharose column chromatography. The two isozymes showed reproducible differences in their catalytic properties in spite of similar substrate specificity and hydrolyzed specifically inositol 4,5-bisphosphate in a Ca(2+)-dependent fashion. In the presence of deoxycholate (DOC), pH optima were at 6.5 and 7.0 for phospholipase C I and II, respectively. Maximal phosphatidylinositol 4,5-bisphosphate hydrolysis rates were obtained at 10(-4) and 10(-5)M Ca2+ for phospholipase C I and II, respectively. Treatment with cAMP-dependent protein kinase did not alter either isozyme activity. Further purification steps were prevented by the extreme lability of the isozymes.     

Detection apparatus for multiple heterogeneous chemiluminescence immunoassay configurations

We describe an apparatus for measuring signals emanated from two heterogeneous chemiluminescence immunoassay (CLIA) configurations: antibody-coated polystyrene beads, in reaction tray wells, and microparticles captured by a porous matrix. An optics and fluidics design which allows the use of a common detection head for these two different assay configurations is described. The detection head moves along three Cartesian coordinates to create a localized light-tight compartment around each individual disposable reaction vessel. Reproducibility of the light seal, trigger solution delivery, and mixing is achieved for acridinium-labeled CLIA. The coated polystyrene beads configuration is tested using beta HCG, CEA, and TSH assays. The microparticle-capture configuration is tested using beta HCG and HBsAg assays. The microparticle capture CLIA has shorter incubation times and the potential for ease of automation.     

Studies on four restriction fragment length polymorphisms of the type I collagen genes in two Italian populations

Type I collagen, the most abundant of the collagen protein family, is encoded by two genes, COL1A1 and COL1A2. Two random population samples, one from central Italy and one from southern Italy, were studied for 1 restriction fragment length polymorphism (RFLP) of COL1A1 (RsaI) and 3 RFLPs of COL1A2 (EcoRI, RsaI and MspI). A considerable heterogeneity for COL1A1/RsaI was found not only between Italians and English but even among Italians. The potential usefulness of these RFLPs and haplotypes as anthropogenetic markers, particularly in distinguishing Caucasoids from Negroids, has been discussed.     

Thyroglobulin interactions with thyroid membranes. Relationship between receptor recognition of N-acetylglucosamine residues and the iodine content of thyroglobulin preparations

Bovine thyroglobulin has been subjected to sequential glycohydrolase treatment in order to define further the components of the carbohydrate chain which are important in binding of the glycoprotein to bovine thyroid membranes. Preparations of asialoagalactothyroglobulin exhibit the best binding, suggesting that exposed N-acetylglucosamine residues on the B carbohydrate chain of thyroglobulin play an important role in the interaction of thyroglobulin with the thyroid membranes. Enhanced binding of asialoagalactothyroglobulin to microsomal, lysosomal, and Golgi membranes, as well as to thyroid cells in culture, was also observed. Isopycnic rubidium chloride gradient centrifugation, a procedure used in the isolation of thyroglobulin molecules with a low iodine content, also isolates thyroglobulin molecules with a low sialic acid content and with an increased ability to interact with wheat germ agglutinin, a lectin which recognizes exposed N-acetylglucosamine residues. The studies further indicate that there is a correlation between iodine content, exposed N-acetylglucosamine residues, and the binding of thyroglobulin to thyroid membranes.     

Structural regularities in tetraether lipids of Caldariella and their biosynthetic and phyletic implications

Individual di(biphytanyl) diglycerol tetraether lipids from thermoacidophile archaebacteria of the Caldariella series, with differently cyclized biphytanyl components, are separated and shown to have structures 8–12, with the glycerol and biphytanyl components demonstrably both antiparallel and with partial assignments of stereochemistry. Tetraethers with alternative arrangements of the components are absent. The structures allow previous observations on these and related lipids to be rationalized both biosynthetically and phyletically.     

Selective 32P-labelling of individual species in a total tRNA population.

A simple procedure to label individual tRNA species in a total tRNA preparation has been developed. The principle of the method is as follows: total crude tRNA (from E. coli) is incubated in the presence of a crude aminoacyl-tRNA synthetase preparation, containing most aminoacyl-tRNA synthetases and only one specific amino acid corresponding to the tRNA species which is intended to be labelled. This achieves the purpose of charging the desired tRNA species thereby protecting its 3'OH-terminus; obviously all the other tRNA species will have a free 3'OH group. Periodate oxidation, followed by beta-elimination, destroys any free 3'OH. After deacylation of the specific aminoacylated tRNA at pH 8.8 the only free 3'OH group will be the one of the desired tRNA species. High specific activity (32P)-pCp is ligated to this 3'OH by means of T4-RNA ligase. Two-dimensional polyacrylamide gel electrophoresis (2D-PGE) and sequence analysis of the isolated tRNA show that the method is very specific. Individually labelled tRNA species can be used as probes for cloning tRNA genes.     

New, rapid methods for purifying alpha-actinin from chicken gizzard and chicken pectoral muscle

We introduce two new, rapid procedures. One is specifically designed for isolating alpha-actinin from skeletal and the other for isolating alpha-actinin from smooth muscle. Approximately 20 mg of greater than 95% pure alpha-actinin can be obtained/100 g of ground chicken pectoral muscle in just 4 days. The smooth muscle protocol yields 2.7 mg of greater than 99% pure alpha-actinin/100 g of ground gizzard after just 5 days. Differences in protein contaminants and in the extractability of alpha-actinin necessitated the development of separate isolation procedures for the two muscle types. Antibody prepared against the purified gizzard alpha-actinin reacted with alpha-actinin from skeletal, cardiac, and smooth muscle in immunodiffusion. Anti-alpha-actinin reacted only with alpha-actinin from crude extracts of skeletal and smooth muscle on Staph A gels. Anti-alpha-actinin stained Z-bands from skeletal muscle in indirect immunofluorescence microscopy and stress fibers from baby hamster kidney fibroblasts and mouse mammary epithelial cells in the characteristic punctate pattern observed by other workers (Lazarides, E., and Burridge, K. (1975) Cell 6, 289-298). These two methods for purifying alpha-actinin from skeletal and smooth muscle represent a significant improvement over that published previously.     

Intracellular disposition of [3H]-cocaine, [3H]-norcocaine, [3H]-benzoylecgonine and [3H]-benzoylnorecgonine in the brain of rats

[³H]-cocaine, [³H]-norcocaine, [³H]-benzoylecgonine and [³H]-benzoylnorecgonine were administered i.c. in equi-potent pharmacologic doses and the intracellular disposition and metabolism of each drug determined. Norcocaine and cocaine rapidly entered and egressed from the brain so that 4.8–6.1% of the radioactivity present in brain at one minute was observed at 30 minutes. The highest levels of subcellular radioactivity were generally found in the microsomal plus supernatant, followed by the nuclear and shocked mitochondrial fractions. No apparent localization of the radioactivity occured in synaptic membranes. The brain/plasma (B/P) ratio curves for cocaine and norcocaine were similar; however, the norcocaine values were considerably higher at each time interval. Benzoylecgonine and benzoylnorecgonine had higher comparative B/P ratios than cocaine or norcocaine and persisted in brain for a longer period of time so that 0.6–2.1% of the radioactivity present in brain at 1 hour was detected at 24 hours. Cocaine and norcocaine were extensively metabolized to the benzoylmetabolites. Benzoylecgonine was metabolized to benzoylnorecgonine and benzoylnorecgonine was unmetabolized. The brain disposition data and B/P ratios agreed quite well with the overall pharmacologic action of cocaine and its metabolites.