Metabolite and enzyme contents of freeze-clamped liver of the marine fish Stenotomus chrysops

Hepatic metabolites and enzymes in the marine fish, scup or porgy (Stenotomus chrysops), were determined in freeze-clamped tissue taken either within a day of removing fish from their natural habitat or after scup were held in captivity for 6-8 months. The same determinations were made for liver from fed or 48 hr-starved rats (Mus norvegicus albinus). Compared with rat liver, both groups of fish had, per gram of liver, higher contents of AMP, inorganic phosphate, glucose, glucose-6-phosphate, malate, glutamate and NH4+. ATP was lower in fish liver, and ADP, lactate and pyruvate contents were similar in rats and fish. Fish held in captivity had significantly lower pyruvate, alpha-ketoglutarate, and cytosolic free NAD+/NADH and higher cytosolic free NADPH/NADP+. These decreases were similar to those seen when starved rats were compared with fed ones. In scup liver, glucose-6-phosphate dehydrogenase was 3-8 times, malic enzyme about 2 times, and alanine aminotransferase 2-4 times higher than those activities in rat liver. Those results and a higher cytosolic free NADPH/NADP+ are consistent with the liver being the major site of lipogenesis in fish.     

Detection of albumin mRNAs in rat liver by in situ hybridization: usefulness of paraffin embedding and comparison of various fixation procedures

Our aim was to define optimal conditions for efficient and reproducible albumin mRNA detection in rat liver by in situ hybridization. We used an albumin-specific [3H]-labeled cDNA probe with a specific activity of 6-8.10(6) cpm/microgram DNA. In situ hybridization is as efficient on paraffin sections as on cryostat sections for detecting albumin mRNAs. Perfusion fixation with a 4% paraformaldehyde solution results in homogeneous RNA retention within tissue blocks, in contrast with immersion fixation, which yields heterogeneous RNA preservation. Comparison of immersion fixation with three different fixatives (paraformaldehyde, ethanol-acetic acid, and Bouin's fixative) shows that the highest level of hybridization signal is obtained with paraformaldehyde. Ethanol-acetic acid and Bouin's fixative appear less efficient for albumin mRNA detection. Loss of mRNAs within liver tissue blocks over time is largely although not completely prevented by paraffin embedding.     

Pinocytic uptake and intracellular degradation of N-(2-hydroxypropyl)methacrylamide copolymers a potential drug delivery system

Synthetic ¹²⁵I-labelled N-(2-hydroxypropyl)methacrylamide copolymers containing four different, potentially degradable peptidyl side chains were incubated with rat visceral yolk sacs cultured in vitro. All copolymers were captured by fluid-phase pinocytosis and three of the side chains were susceptible to lysosomal hydrolysis, resulting in release of [¹²⁵I]iodotyrosine back into the culture medium. Uptake and degradation was completely inhibited by 2,4-dinitrophenol. The thiol-proteinase inhibitor leupeptin did not affect the rate of pinocytosis, but caused different degrees of inhibition of hydrolysis depending on side chain composition.     

Effects of mutation on the downfield proton nuclear magnetic resonance spectrum of the 5S RNA of Escherichia coli

The imino proton spectra of several mutants of the 5S RNA of Escherichia coli are compared with that of the wild type. Three of the variants discussed are point mutations, and the fourth is a deletion mutant lacking bases 11-69 of the parent sequence, all obtained by site-directed mutagenesis techniques. The spectroscopic effects of mutation are limited in all cases, and the differences between normal and mutant spectra can be used to make or confirm the assignments of resonances. Several new assignments in the 5S spectrum are reported. Spectroscopic differences due to sequence differences permit the products of single genes within the 5S gene family to be distinguished and their fates followed by NMR.     

Isolation and characterization of an insulin-degrading enzyme from Drosophila melanogaster

An insulin-degrading enzyme (IDE) from the cytoplasm of Drosophila Kc cells has been purified and characterized. The purified enzyme is a monomer with an s value of 7.2 S, an apparent Km for porcine insulin of 3 microM, and a specific activity of 3.3 nmol of porcine insulin degraded/(min.mg). N-Terminal sequence analysis of the gel-purified enzyme gave a single, serine-rich sequence. The Drosophila IDE shares a number of properties in common with its mammalian counterpart. The enzyme could be specifically affinity-labeled with [125I]insulin, has a molecular weight of 110K, and has a pI of 5.3. Although Drosophila Kc cells grow at room temperature, the optimal enzyme activity assay conditions parallel those of the mammalian IDE: 37 degrees C and a pH range of 7-8. The Drosophila IDE activity, like the mammalian enzymes, is inhibited by bacitracin and sulfhydryl-specific reagents. Similarly, the Drosophila IDE activity is insensitive to glutathione as well as protease inhibitors such as aprotinin and leupeptin. Insulin-like growth factor II, equine insulin, and porcine insulin compete for degradation of [125I]insulin at comparable concentrations (approximately 10(-6) M), whereas insulin-like growth factor I and the individual A and B chains of insulin are less effective. The high degree of evolutionary conservation between the Drosophila and mammalian IDE suggests an important role for this enzyme in the metabolism of insulin and also provides further evidence for the existence of a complete insulin-like system in invertebrate organisms such as Drosophila.     

A comparative study of the characteristics of eIF-2 and eIF-2-ancillary factor activities from yeast Saccharomyces cerevisiae and rabbit reticulocytes

The characteristics of yeast eukaryotic initiation factor 2 (eIF-2) and Co-eIF-2A have been studied and compared with those of the corresponding factors from rabbit reticulocytes. 1) Unlike eIF-2r, purified eIF-2y did not contain bound GDP. 2) Purified eIF-2y preparation contained GTPase activity and dephosphorylated GTP to GDP. 3) An anti-eIF-2r preparation which predominantly precipitated the gamma-subunit (Mr 54,000) of eIF-2r also precipitated the larger subunit (Mr 54,000) of eIF-2y. 4) Unlike eIF-2r, ternary complex formation by eIF-2y was not inhibited by Mg2+. 5) Both Co-eIF-2A20y and Co-eIF-2r significantly enhanced Met-tRNAf binding to eIF-2y and, again, Mg2+ did not have any effect on this stimulated Met-tRNAf binding to eIF-2y. 6) Both Co-eIF-2A20y and Co-eIF-2r were similarly effective in stimulating Met-tRNAf binding to eIF-2r in the absence of Mg2+. However, in the presence of Mg2+, Co-eIF-2A20y was significantly less effective than Co-eIF-2r as Co-eIF-2A20y did not promote displacement of GDP from eIF-2r X GDP. 7) eIF-2y bound [3H]GDP and this binding was significantly enhanced in the presence of Mg2+. Also, [3H]GDP in the preformed eIF-2y X [3H]GDP complex was rapidly exchanged with exogenously added unlabeled GDP in the presence of Mg2+. Co-eIF-2A20y had no effect on GDP binding to eIF-2y nor on GDP exchange reactions. 8) Reticulocyte heme-regulated protein synthesis inhibitor, which phosphorylated almost completely (in excess of 80%) the alpha-subunit (Mr 38,000) of eIF-2r, also phosphorylated similarly the smaller subunit (Mr 36,000) of eIF-2y. However, such phosphorylation had no significant effect on ternary complex formation, GDP binding, and GDP exchange reactions.     

Chicken riboflavin-binding protein. cDNA sequence and homology with milk folate-binding protein

The Rd gene is expressed in the livers and oviducts of laying hens and codes for the riboflavin-binding protein (RfBP) of egg yolk and egg white. A lambda gt11 cDNA library derived from chicken oviduct poly(A)+ RNA was screened with polyclonal rabbit antiserum to chicken RfBP. Positive clones were isolated and rescreened with a mixed oligonucleotide probe corresponding to residues 20-25 of the mature protein. The largest cDNA clone (969 base pairs) was subcloned into plasmid pIBI21, and the nucleotide sequence was determined by the dideoxynucleotide method. This clone contained the entire coding region for RfBP. The published amino acid sequence of the mature protein was confirmed. In addition, the following 17-residue signal peptide was deduced: Met-Leu-Arg-Phe-Ala-Ile-Thr-Leu-Phe-Ala-Val-Ile-Thr-Ser-Ser-Thr-Cys. Unexpectedly, the nucleotide sequence codes for 2 adjacent arginine residues at the carboxyl terminus that are not observed in the mature protein. The amino acid sequence of RfBP is homologous with bovine milk folate-binding protein. Eight of the nine pairs of cysteines involved in disulfide bonds in RfBP are conserved in folate-binding protein, as are all of the tryptophan residues. Sequence identity between homologous regions of these two vitamin-binding proteins is more than 30%.