Variations in the high concentrations of intracellular amino acids in the tissues of osmoconforming, euryhaline animals such as the ribbed mussel (Modiolus demissus) appear to have a major role in adjusting intracellular osmotic pressure in response to extracellular osmotic change. The experiments discussed here on the metabolic origins and fates of these amino acids are part of a continuing study on the physiological mechanisms regulating the concentrations of these amino acids. Tissues isolated from ribbed mussels adapted at 12 0100 were incubated for 8 hrs. in artificial seawater media at 12 0100 or 32 0100.
Preliminary experiments with 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), and sodium azide (NaN3) indicated that most of the oxygen consumption by ribbed mussel gill tissue is the result of mitochondrial respiration. A procedure utilizing isoosmotic sucrose, EGTA, defatted serum albumin, and HEPES as the isolation medium was devised for the preparation of fully coupled ribbed mussel gill mitochondria. Optimal rates of respiration and respiratory coupling required substrate, ADP, inorganic phosphate, and a fairly high KC1 concentration (90 mM) in the assay medium. Glutamate, proline, malate, and succinate stimulated oxygen consumption with high respiratory control indices and Pi0 of 3, 3, 3 and 2, respectively. Pyruvate was a weak stimulator of mitochondrial respiration and showed a low respiratory control index with a low P/O. Preparation of gill mitochondria in isoosmotic solutions containing high KC1 concentrations (150 mM) yielded mitochondria showing state 2 respiration, slow partially uncoupled ATP synthesis during state 3 respiration and no state 4 respiration. D-mannitol was not used in the mitochondrial isolation or assay medium because of the probable presence of a D-mannitol oxidase in these gill mitochondria.
Glycine levels in isolated ribbed mussel (Modiolus demissus) gill tissue increased slightly and decreased markedly when incubated at high and low salinities, respectively. Low levels of the enzymes involved in the biosynthesis of serine from triose phosphate intermediates, the serine hydroxymethyltransferase, and serine dehydrase were detected in gill tissue homogenates. Experiments using gill tissue incubated with (U-14C)-glycine and (U-14C)-serine indicated interconversion between serine and glycine and transfer of label to alanine, asparate, glutamate, CO2, organic acids, and protein. Glyoxylate was metabolized more slowly than glycine and was probably converted to glycine for catabolism. Studies using (1-14C)-glycine and (2-14C)-glycine with isolated gill tissue and mitochondria indicated that the mitochondrial glycine cleavage enzyme was the major route of glycine catabolism. Metabolic controls activating or inhibiting the glycine cleavage enzyme regulate tissue glycine accumulation and catabolism during hypersalinity or hyposalinity stress.
Rat serine dehydratase cDNA clones were isolated from a Agtll cDNA library on the basis of their reactivity with monospecific immunoglobulin to the purified enzyme. Using the cDNA insert from a clone that encoded the serine dehydratase subunit as a probe, additional clones were isolated from the same library by plaque hybridization. Nucleotide sequence analysis of the largest clone obtained showed that it has 1444 base pairs with an open reading frame consisting of 1089 base pairs. The deduced amino acid sequence contained sequences of several portions of the serine dehydratase protein, as determined by Edman degradation. Rat liver serine dehydratase mRNA virtually disappeared from livers of rats fed a protein-free diet for 5 days. Several genomic clones were isolated from two libraries. Determinations of the transcription start site and the structure of the 3' flanking region of the gene indicated that the coded mRNA is 1504 nucleotides long. The 5' promoter region contained a variety of sequences similar to several consensus sequences believed to be important for the regulation of specific gene expression.L-Serine dehydratase [L-serine hydro-lyase (deaminating), EC 4.2.1.13] catalyzes the a,,-elimination of L-serine to produce pyruvate and ammonia. Studies in several laboratories have shown that the rat liver enzyme also catalyzes the conversion of L-threonine to a-ketobutyrate by a mechanism identical to that converting L-serine to pyruvate (1, 2). The synthesis of the enzyme in vivo is enhanced in starvation and diabetes mellitus (3). Serine dehydratase is readily induced by the administration of various hormones that increase gluconeogenesis (4-7), and its synthesis is repressed acutely by the administration of D-glucose (5) and other sugars (8), or chronically by the feeding of diets high in carbohydrates (9). Since our studies were directed to the molecular mechanism of the regulation of the expression of the rat liver serine dehydratase gene, the determination of the structure of the protein, its mRNA, and its gene, especially of regulatory elements of the latter, became essential. Although Noda et al. (10) have reported the isolation of a cDNA clone for rat liver serine dehydratase, their clone [1000 base pairs (bp) long] does not represent the entire mRNA, since it was of insufficient size to code for the serine dehydratase subunit that has a molecular weight of 35,000 (10-12).In this communication, we report the isolation and sequence analysis ¶ of a cDNA encoding the entire amino acid sequence of the serine dehydratase subunit. RNA gel blot analysis indicated that the size of serine dehydratase mRNA is close to that of the largest cDNA isolated in this study. The determination of the exact size of DNA complementary to serine dehydratase mRNA was made by S1 nuclease and sequencing of genomic clones of the regions flanking the gene. MATERIALS AND METHODScDNA Cloning. A rat liver cDNA library constructed in Agtll phage (13) was screened for antibody-reactive plaques as described by Young and D...
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