Subunit structure of 27 S thyroid iodoprotein.

The dissociation of thyroid 27 S iodoprotein by sodium dodecyl sulfate (SDS) and by succinic anhydride was investigated by means of ultracentrifugation and polyacrylamide gel electrophoresis. The iodoprotein obtained from either a human or hog was dissociated into three kinds of subunits (S-19, S-17 and S-12) by SDS treatment. At increased concentrations of SDS, the S-12 subunit was predominant among the dissociation products. The succinylation of 27 S iodoprotein showed essentially the same dissociation pattern as in the case of SDS treatment. This dissociation products of the protein preparations of different animals were qualitatively the same as those of thyroglobulin of the respective animals, confirming the hypothesis that 27 S iodoprotein was composed of two molecules of thyroglobulin. However, the extent of dissociation of 27 S iodoprotein measured by S-12 formation showed higher resistancy of the protein to the dissociating agents than that of thyroglobulin. The contents of sialic acid and hexose as well as iodoamino acids of 27 S iodoprotein were found to be the same as, or not far from, those of thyroglobulin; The dissociability and chemical composition of 27 S iodoprotein was discussed with reference to the subunit structure of the protein.     

Non-enzymatic formation of sulfenyl iodide by solubilized thyroid particulate protein with peroxidase activity

Thyroidal particulate protein with peroxidase activity has been studied to determined wetherr it could be induced to form a sulfenyl iodide, postulated as a reactive intermediate in the iodination of tyrosine. The protein was solubilized with digitonin and purified by tryptic digestion and filtration through Sephadex G-200. When supplemented with H₂O₂ it catalyzed the oxidation of guaiacol iodide or thiourea at 37°C. With iodide as substrate the product was an iodoprotein. In the absence of H₂O₂ the protein did not bind ¹³¹I^? or thio[¹⁴C] urea unless it first had been dialyzed against [³⁶Cl] chlorinated buffer. During dialysis a portion of the ³⁶Cl from the dialysis medium was bound by protein. Subsequent binding of iodide or thiourea was accompanied by loss of protein-bound ³⁶Cl.Addition of iodide to dialyzed protein at 4°C resulted in formation of a yellow compound with maximum absorbance at 355 nm. It was postulated to be a sulfenyl periodide on the basis of its absorption spectrum and its behavior with thio[¹⁴C] urea and 2-mercaptoethanol. The stability of the colored species was dependent on temperature and concentration of iodide. Disappearance of color as the solution was warmed was accompanied by formation of iodo-protein. Predialysis of the protein against p-chloromercuribenzoate, but not 2-mercaptoethanol or bisulfite, prevented the formation of the yellow proteiniodide species, indicating that a reactive sulfhydryl group was involved in the reaction. It was concluded that a particulate protein closely asscociated with thyroid peroxidase could be induced by non-enzymatic means to form a species which has properties consistent with those of a sulfenyl iodide. Further investigation will be required to determine whether the same protein-iodine species can be identified during the peroxidase-catalyzed oxidation of iodide.     

Intracellular and extracellular sites of iodination in dispersed hog thyroid cells.

Iodination and hormone synthesis has been studied in isolated hog thyroid cells in suspension. We characterized three iodination processes by use of pharmacological agents. (1) Intracellular iodination dependent on active iodide transport, which was inhibited by NaClO4 or ouabain, but not by catalase. This iodination was linear for 6h with no apparent Km for iodide of 1.5 muM, was stimulated by thyrotropin or N6O2'-dibutyryladenosine 3':5'-cyclic monophosphate, yielded mostly iodinated thyroglobulin and was efficient for tetraiodothyronine synthesis. (2) Extracellular iodination, which was sensitive to catalase, but not to NaClO4 or ouabain. This iodination plateaued after 2h and the apparent Km was 16.5 muM. This process was insensitive to thyrotropin and dibutyryl cyclic AMP. The major products were iodoprotein other then thyroglobulin and iodolipid and the yield of tetraiodothyronine was low. (3) Intracellular iodination from passively diffused iodide, which was not sensitive to inhibitors. Other characteristics of passive intracellular iodination were intermediate between active intracellular iodination and extracellular iodination. The fact that the three processes are inhibited by similar concentrations of methimazole, and their apparent Km values, when corrected for the concentrating effect of iodide trapping, are all of the same order as the Km of purified thyroid peroxidases, suggest that although their locations are different, the enzymic systems involved are identical. These results show that, besides an extracellular site of iodination, dispersed thyroid cells process an intracellular site of iodination with biochemical characteristics of physiological relevance.     

Change of the protein composition of the thyroid colloid during treatment with propylthiouracil and thyroxine A microgel electrophoretic study of single rat thyroid follicles

Propylthiouracil and thyroxine were given daily to rats for 4 weeks. Samples of colloid were collected in vivo from the superficial thyroid follicles during this period and their protein composition was analysed by gel electrophoresis.It was observed that the aggregates of thyroglobulin, i.e., the 27-S thyroid iodoprotein and the heavier fractions, were reduced to 50% after 1 week and were almost absent after 2 weeks.A faster migrating thyroglobulin fraction was observed in the samples of colloid and in the homogenate of the whole gland after 48 h of treatment. During the following period of treatment there was an increase in the relative amount of the faster migrating thyroglobulin fraction compared to 19-S thyroglobulin in the colloid, the former comprising approx. 75% of the globulins after 4 weeks, It can be concluded that propylthiouracil inhibits the formation of the 27-S iodoprotein and that a structurally altered and iodine-poor thyroglobulin fraction is accumulated in the follicle lumen.     

Effects of Chronic Basic Fibroblast Growth Factor Administration to Rats with Partial Flmbrial Transections on Presynaptic Cholinergic Parameters and Muscarinic Receptors in the Hippocampus: Comparison with Nerve Growth Factor

Abstract: The present study compares the effects of chronic administration of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on various hippocampal cholinergic parameters in rats with partial unilateral fimbrial transections. Lesions resulted in marked reductions of several presynaptic cholinergic parameters: choline acetyltransferase (ChAT) activity (by 50%), [³H]-acetylcholine ([³H]ACh) synthesis (by 59%), basal and ve-ratridine (1 μM)-evoked [³H]ACh release (by 44 and 57%, respectively), and [³H]vesamicol binding site densities (by 35%). In addition, [³H]AF-DX 116/muscarinic M₂ binding site densities were also modestly decreased (by 23%). In contrast, [³H]pirenzepine/muscarinic M₁ and [³H]AF-DX 384/muscarinic M₂/M₄ binding site densities were not altered by the lesions, nor were they affected by any of the treatments. Intracerebroventricular administration of bFGF (10 ng, every other day, for 21 days) partially prevented the lesion-induced deficit in hippocampal ChAT activity, an effect that was not markedly different from that measured in the NGF-treated (1 μg intracerebroventricularly, every other day, for 21 days) rats. In rats treated with a combination of bFGF and NGF, ChAT activity was not different from that in rats treated with the individual factors alone. In contrast, the lesion-induced deficits in the other cholinergic parameters were not attenuated by bFGF treatment, although they were at least partially prevented by NGF administration. To determine whether higher concentrations of bFGF are necessary to affect cholinergic parameters other than hippocampal ChAT activity, rats were treated with 1 μg (every other day, 21 days) of the growth factor. In this group of rats, detrimental effects of bFGF, manifested by an increased death rate (46%), and marked reductions in body weight of the survivors, were observed. In addition, this concentration of bFGF appeared to exacerbate the lesion-induced reduction in [³H]ACh synthesis by hippocampal slices; [³H]ACh synthesis in lesioned hippocampi represented 36 and 52% of that in contralateral unlesioned hippocampi for the bFGF-treated and control groups, respectively. In conclusion, although bFGF administration attenuates the deficit in hippocampal ChAT activity induced by partial fimbrial transections, this does not appear to translate into enhanced functional capacity of the cholinergic terminals. This is clearly in contrast to NGF, which enhances not only hippocampal ChAT activity, but also other parameters indicative of increased function in the cholinergic terminals.     

Change of the protein composition of the thyroid colloid during treatment with propylthiouracil and thyroxine: a microgel electrophoretic study of single rat thyroid follicles

Prophylthiouracil and thyroxine were given daily to rats for 4 weeks. Samples of colloid were collected in vivo from the superficial thyroid follicles during this period and their protein composition was analysed by gel electrophoresis. It was observed that the aggregates of thyroglobulin, i.e., the 27-S thyroid iodoprotein and the heavier fractions, were reduced to 50% after 1 week and were almost absent after 2 weeks. A faster migrating thyroglobulin fraction was observed in the samples of colloid and in the homogenate of the whole gland after 48 h of treatment. During the following period of treatment there was an increase in the relative amount of the faster migrating thyroglobulin fraction compared to 19-S thyroglobulin in the colloid, the former comprising approx. 75% of the globulins after 4 weeks, It can be concluded that propylthiouracil inhibits the formation of the 27-S iodoprotein and that a structurally altered and iodine-poor thyroglobulin fraction is accumulated in the follicle lumen.     

Inhibition of nucleic acid synthesis in Ehrlich tumor cells by periodate-oxidized adenosine and adenylic acid

Periodate-oxidized adenosine and AMP were inhibitory to both RNA and DNA synthesis in Ehrlich tumor cells in culture. With periodate-oxidized adenosine, the inhibition of RNA synthesis paralleled the inhibition of DNA synthesis. Periodate-oxidized AMP, however, was more inhibitory to DNA synthesis than to RNA synthesis. With both compounds, there was a decrease in the conversion of [¹⁴C]cytidine nucleotides to [¹⁴C]deoxycytidine nucleotides in the acid-soluble pool. The borohy-dride-reduced trialcohol derivative of the periodate-oxidized adenosine compound was not inhibitory to DNA or RNA synthesis in the tumor cells. The incorporation of [³H]uridine into 28S and 18S ribosomal RNA was inhibited by both periodate-oxidized adenosine and AMP, but the incorporation of [³H]uridine in 45S, 5S, and 4S RNA was essentially unaffected by these compounds. Periodate-oxidized adenosine inhibited Ehrlich tumor cell growth in vivo.     

Hybridizable ribonucleic acid of rat brain

1. Cerebral RNA of adult and newborn rats was labelled in vivo by intracervical injection of [5-³H]uridine or [³²P]phosphate. Hepatic RNA of similar animals was labelled by intraperitoneal administration of [6-¹⁴C]orotic acid. Nuclear and cytoplasmic fractions were isolated and purified by procedures involving extraction with phenol and repeated precipitation with ethanol. 2. The fraction of pulse-labelled RNA from cerebral nuclei that hybridized to homologous DNA exhibited a wide range of turnover values and was heterogeneous in sucrose density gradients. 3. Base composition of the hybridizable RNA was similar to that of the total pulse-labelled material; both were DNA-like. 4. Pulse-labelled cerebral nuclear RNA hybridized to a greater extent than cytoplasmic RNA for at least a week after administration of labelled precursor. This finding suggested that cerebral nuclei contained a hybridizable component that was not transferred to cytoplasm. 5. The rates of decay of the hybridizable fractions of cerebral nuclei and cytoplasm were faster in the newborn animal than in the adult. Presumably a larger proportion of labile messenger RNA molecules was present in the immature brain. 6. Cerebral nuclear and cytoplasmic RNA fractions from newborn or adult rats, labelled either in vivo for periods varying from 4min. to 7 days or in vitro by exposure to [³H]-dimethyl sulphate, uniformly hybridized more effectively than the corresponding hepatic preparation. These data suggested that a larger proportion of RNA synthesis was oriented towards messenger RNA formation in brain than in liver.     

9 S thyroid particulate iodoprotein.

Particulate iodoproteins have been studied in the rat thyroid gland using the isotopic 125I equilibration method. Pulse experiments were also performed with a second isotope, 131I. Labelled iodoproteins, both soluble and solubilized by digitonin from the thyroid particulate material, were analyzed by sucrose-gradient ultracentrifugation. 1. At isotopic equilibrium and irrespective of the iodide content of the diet two particulate iodoproteins, with sedimentation coefficients of 27 and 19 S, were solubilized by digitonin. In addition a 9 S iodoprotein was also present but its proportion varied markedly with the iodine content of the diet: it accounted for 50-60% of the label found at the particulate level when the dialy diet of iodine was high (75-500 mug/day) but was almost absent when the diet was only 2 mug/day. 2. Most of this 9 S protein was found in the 600-15 000 times g particulate pellet, i.e. a fraction enriched in lysosomes and phagolysosomes. 3. The iodoamino acid composition of the 9 S fraction was very similar to that of the 19 S particulate thyroglobulin: its hormone content was 19%. 4. The double precipitation technique showed that the 9 S fraction is immunochemically related to thyroglobulin. 5. Pulse experiments showed that the 9 S particulate iodoprotein was slowly labelled by 131I. 6. The amount of 9 S iodoprotein was increased by thyrotropin (30-40% increase versus control experiments 5 min after thyrotropin injection). These properties of the 9 S particulate iodoprotein are discussed in relation to the assumption that it might be a product of partial proteolysis of thyroglobulin after endocytosis and partial digestion by the phagolysosomes.     

DNA synthesis in developing two-cell mouse embryos

Mated CF1 (Carworth) female mice were sacrificed at 2 hr intervals between 29 and 43 hr after human chorionic gonadotrophin (HCG) administration. One- and two-cell eggs were incubated in [³H]thymidine for 1 hr. Labeled two-cell embryos were first observed at 31 hr and reached a maximum number at 35 hr. The S period is approximately 6 hr in duration. Although both blastomeres were labeled in most cases, embryos with only one labeled blastomere were more numerous at later times. In vitro labeling was corroborated by injecting [³H]thymidine directly into the isthmic portion of the oviduct. Embryos usually complete the second cleavage division 18–20 hr after onset of DNA synthesis. The cell cycle at the two-cell stage is thus characterized by a G₁ of close to 1 hr, a 6 hr S, and a G₂ of about 12 hr.Embryos developing in vitro frequently fail to progress beyond the two-cell stage. The block is not due to absence of DNA synthesis since these embryos were found to incorporate [³H]thymidine.     

Binding of ricin A chain to rat liver ribosomes: Relationship to ribosome inactivation

Ricin A chain was radioactively labeled using reductive alkylation, lactoperoxidase catalyzed iodination, and reaction with iodoacetamide or N-ethylmaleimide (NEM). The inhibition of cell-free rat liver protein synthesis by the modified A chains and the ribosome binding characteristics of each of the labeled derivatives was examined. [³H] NEM was found to quantitatively react with the A chain sulfhydryl group normally involved in a disulfide bond with the B chain in intact ricin. Labeling the protein with [³H] NEM had no effect on the in vitro inhibition of protein synthesis by the A chain. [³H] NEM-labeled A chain binds to rat liver ribosomes in a manner which is dependent on the concentrations of NaCl and Mg²⁺. At optimal Mg²⁺ concentration (5.5 mM), A chain binding to ribosomes is saturable and fully reversible either by dilution of the reaction mixture or by addition of unlabeled A chain. At 5.5 mM Mg²⁺, A chain was found to bind to a single site on rat liver ribosomes with a dissociation constant of 6.2 X 10^?8 M. [³H] NEM-labeled A chain did not bind to isolated 40S ribosomal subunits and bound to 60S ribosomal subunits with a 1 : 1 molar stoichiometry and a dissociation constant of 2.2 X 10^?7 M. The relationship between ribosome binding and A chain inhibition of eucaryotic protein synthesis is discussed.     

Immunochemical and immunohistochemical studies on the 27 S iodoprotein of dog thyroid with reference to thyroglobulin-like reaction of the parafollicular cells.

Our earlier finding that the thyroglobulin-like material responsible for the immunoreaction of parafollicular cells obtained in peak I fraction of Bio-Gel A-5m was followed up in the present study by an investigation of the immunochemical and immunohistochemical reactions of 27 S iodoprotein which was the most prominent material in the peak I fraction. The antibody was raised against completely purified 27 S iodoprotein which was obtained as follows: Thyroglobulin was extracted from dog thyroids and chromatographed initially on Bio-Gel A-5m and then on Bio-Gel A-50m. The area of 27 S migrated as a single bank on polyacrylamide gel slab electrophoresis. This was cut and eluted. Anti-27 S antiserum showed the same immunochemical patterns to 27 S and 19 S as anti-19 S antiserum with three different immunochemical methods: double diffusion test, one dimensional and two dimensional immunoelectrophoresis. The immunoperoxidase reactions of the anti-27 S antiserum and anti-19 S antiserum were restricted to follicular cells and luminal colloids. No reaction of the parafollicular cells was obtained by these antisera. Thus, 27 S iodoprotein shared common immunochemical and immunohistochemical properties with 19 S thyroglobulin. It was concluded that 27 S iodoprotein was not responsible for the thyroglobulin-like reaction of the parafollicular cells.     

Corticosterone binding by rat brain cytosol

Significant quantities of corticosterone were associated with macromolecules of the brain cytosol following intrathecal administration of [³H]corticosterone to adrenalectomized rats. Fifteen times more steroid was found associated with proteins from adrenalectomized rats than from control animals or adrenalectomized animals pretreated with corticosterone. Pretreatment of adrenalectomized rats with 11-dehydrocorticosterone, Cortisol and cortisone decreased the amount of [³H]corticosterone found associated with protein, whereas progesterone, oestradiol and testosterone did not interfere with the association of [³H]corticosterone with macromolecules of the cytosol. Further evidence for protein-steroid interaction was obtained by incubating [³H]corticosterone (B), [³H]cortisol (F), or 11-[³H]deoxycortisol (S) with brain cytosols. The degree of binding was in the order B > F > S.     

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.     

Renal glomerular collagen synthesis in streptozotocin diabetes Reversal of increased basement membrane synthesis with insulin therapy

The effect of diabetes and insulin on basement membrane synthesis in vitro be renal glomeruli obtained from normal and diabetic rats was determined. Four groups of experimental animals were used: age-matched controls; streptozotocin-diabetic; and streptozotocin-diabetic treated with insulin for half or all of the duration of diabetes. Isolated glomeruli were incubated with [¹⁴C]-lysine and the radioactive lysine and hydroxylysine in glomerular proteins were measured. [¹⁴C]Lysine incorporation and hydroxy[¹⁴H]lysine synthesis were elevated in diabetic glomeruli. Progressive diminution in ¹⁴C-labelled protein and hydroxy[¹⁴C]lysine formation was observed in incubations containing glomeruli from insulin-treated diabetic rats, with greater reversal toward normal following longer periods of exogenous insulin administration. Basement membrane synthesis, determined by the appearance of labelled hydroxylysine in membranes obtained from sonicated glomeruli, was increased in diabetic preparations. Reversal of these changes toward normal values was observed in glomeruli from rats treated with insulin immediately following induction of diabetes. The results indicate that basement membrane synthesis is increased in renal glomeruli from streptozotocin-diabetic rats, and that this process is restored toward normal with continuous insulin therapy.     

Differences in iodinated peptides and thyroid hormone formation after chemical and thyroid peroxidase-catalyzed iodination of human thyroglobulin

The distribution of iodine among the polypeptides of human goiter thyroglobulin (Tg) was examined. Tg was iodinated in vitro with ¹³¹I to levels of 2 to 84 gram atoms (g.a.)/mol using thyroid peroxidase (TPO) or a chemical iodination system. The samples were reduced, alkylated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two low-molecular-weight peptides appeared preferentially in radioautograms of the sodium dodecyl sulfate (SDS) gels of TPO-iodinated samples. Iodination of these peptides increased sharply in the TPO-treated Tg as the level of total iodine/ molecule rose. Radioiodine was incorporated into these same gel regions in the chemically treated Tg, but only after much higher levels of total iodination were reached. Differences in iodoamino acid distribution were also noted between the chemically and enzymatically iodinated thyroglobulins. In the chemically iodinated samples, little thyroxine (T₄) was synthesized, even at high iodine levels. In the TPO-treated samples only small amounts of T₄ were seen below 14 g.a. total I/mol, while at or above that level of iodination T₄ formation increased sharply. To examine the coupling process, Tg was chemically iodinated, excess I^? removed, and the samples treated with TPO and a H₂O₂-generating system in the absence of iodide. Radioautograms obtained from SDS-polyacrylamide gels of reduced and alkylated protein from such coupling assays showed an increase in the level of iodine in the low-molecular-weight peptides after TPO treatment. Thyroxine production also increased with TPO treatment. The addition of free DIT (a known coupling enhancer) to the [¹³¹I]Tg/TPO incubation increased both the production of T₄ and the amount of iodine in the smaller polypeptides. Two-dimensional maps prepared from CNBr-digested TG showed differences between the coupled and uncoupled samples. Our observations confirm the importance of the lowmolecular-weight peptides derived from Tg in thyroid hormone synthesis. At total iodine levels above 14 g.a./mol Tg in enzymatically treated samples there is selective incorporation of iodine into both the low-molecular-weight polypeptides and into thyroid hormone.     

PRODUCTION OF MEMBRANE WHORLS IN RAT LIVER BY SOME INHIBITORS OF PROTEIN SYNTHESIS

Inhibitors of protein synthesis capable of differential effects on nascent peptide synthesis on membrane-bound and free polyribosomes were employed to investigate the structure and function of cellular membranes of liver. The formation of membranous whorls in the cytoplasm and distension of nuclear membranes were induced by inhibitors of protein synthesis (i.e., cycloheximide and emetine) which predominantly interfere with nascent peptide synthesis on membrane-bound polyribosomes in situ. Other inhibitors of protein synthesis such as puromycin and fusidic acid, which inhibit nascent peptide synthesis on both free and membrane-bound polyribosomes, and chloramphenicol, which inhibits mitochondrial protein synthesis, did not induce these alterations. Cycloheximide, puromycin, and chloramphenicol produce some common cellular lesions as reflected by similar alterations in morphology, such as swelling of mitochondria, degranulation of rough endoplasmic reticulum, and aggregation of free ribosomes. The process of whorl formation in the cytoplasm, the incorporation of [³H]leucine and of [³H]choline into endoplasmic reticulum and the total NADPH-cytochrome c reductase activity of the endoplasmic reticulum were determined. During maximum formation of membranous whorls, [³H]leucine incorporation into cytoplasmic membranes was inhibited, while [³H]choline incorporation into these structures was increased; maximum inhibition of protein synthesis and stimulation of choline incorporation into endoplasmic reticulum, however, preceded whorl formation. Cycloheximide decreased the activity of NADPH-cytochrome c reductase of rough endoplasmic reticulum, but increased NADPH-cytochrome c reductase activity of smooth endoplasmic reticulum. In addition, cycloheximide decreased the content of hemoprotein in both the microsomal and mitochondrial fractions of rat liver, and the activities of mixed function oxidase and of oxidative phosphorylation were impaired to different degrees. Succinate-stimulated microsomal oxidation was also inhibited. The possible mechanisms involved in the formation of membranous whorls, as well as their functions, are discussed.     

Properties of γ-aminobutyric acid synthesis by rat renal cortex

Substantial synthesis of γ-aminobutyric acid occurs in rat renal cortex. Renal glutamate decarboxylase activity (24.3±2.9 (S.E.) nmols/mg protein per h) is 15% of that in brain; renal γ-aminobutyric acid content (39.5±5.3 (S.E.) nmols/g wet wt.) is 5% of the whole brain concentration. Properties of glutamate decarboxylase were studied in homogenates of rat renal cortex and rat brain under conditions for which γ-aminobutyric acid formation from [2,3-³H]glutamate and CO₂ release from [1-¹⁴C]glutamate were equal. Several properties of renal glutamate decarboxylase distinguish it from the corresponding brain enzyme: (1) renal glutamate decarboxylase is selectively inhibited by cysteine sulfinic acid (K_i = 5·10^?5 M) ; (20 renal glutamate decarboxylase is less sensitive (K_i = 3–5·10^?5 M)_to inhibition by aminooxyacetic acid than is the brain enzyme (K_i = 1·10^?6 M); (3) brain but not renal glutamate decarboxylase activity can be substantially stimulated in vitro by the addition of exogenous pyridoxal 5′-phosphate; (4) renal glutamate decarboxylase is significantly decreased in renal cortex from rats on a low-salt diet. Proximal tubules are enriched in glutamate decarboxylase compared to the activity in whole renal cortex or glomeruli (42, 22 and 14 nmols/mg protein per h, respectively). We speculate that renal γ-aminobutyric acid synthesis does not reflect the presence of GABAergic renal nerves, but may serve a function in proximal tubular cells.     

Effects of ADP, ethanol and acetaldehyde on the relaxing complex of human muscle and its adsorption by polystyrene particles.

Protoplasts of Saccharomyces strain 1016 took up [³H]glucosamine in the presence of an energy source; mannose was chosen to minimize randomization. It accumulated in the soluble intracellular pool primarily as UDP-N-acetyl[³H]glucosamine along with a small amount of [³H]glucosamine 6-phosphate. The antibiotic tunicamycin (TM) at 10 μg/ml did not affect the levels of these metabolites or inhibit the formation of the Nacetylglucosamine polymer, chitin, but did prevent the incorporation of [³H]glucosamine into mannan peptides and the synthesis of invertase. In vitro incorporation of [¹⁴C]mannose from GDP-[¹⁴C]mannose into mannan in a membrane preparation was not sensitive to 100 μg of TM/ml. TM appears to inhibit an N-acetylglucosaminyl transferase essential for glycoprotein biosynthesis. Binding of [³H]TM reflects its association with the plasma membrane fraction. This material could be recovered in an unaltered form by extraction with chloroform/methanol. If 0.2% phosphatidyl choline or phosphatidyl serine was added simultaneously with the [³H]TM, the binding of [³H]TM was greatly reduced, and the inhibitory effects of TM on protoplasts were prevented; however, addition of phospholipid 20 min later did not eliminate the inhibition, although about 80% of the bound [³H]TM was removed. TM interacts with lipophilic membrane components as well as inhibiting glycoprotein synthesis.