ReducedS-adenosylmethionine: Protein-lysineN-methyltransferase activity (protein methylase III) in shiverer mutant mouse brain

Mice with the dysmyelinating mutation shiverer were studied by measuring the activity of two protein methylases and myelin marker enzymes in the brain. It was observed thatS-adenosylmethionine: protein-lysineN-methyltransferase (protein methylase III, EC. 2.1.1.43) activity is significantly reduced in phenotypically affected homozygous shiverer (shi/shi) mutant mouse brain compared to the unaffected heterozygous littermate brain. This reduction in enzyme activity is manifested mainly by reduced formation of trimethyllysine during the in vitro methylation of histone. In contrast, myelin marker enzymes such as 2,3-cyclic nucleotide 3-phosphohydrolase and 5-nucleotidase as well asS-adenosyl-methionine: protein-carboxylO-methyltransferase (protein methylase II, EC. 2.1.1.24) activities were not significantly affected in these strains of mice.     

In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) fromEuglena gracilis [J. Biol. Chem.,260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as -N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as thein vitro substrate. Presently, histone H1 has been purified fromEuglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. TheEuglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higherR _f value than the other histones H1 in acid/urea gel electrophoresis. When theEuglena histone H1 was [methyl-³H]-labeledin vitro by a homologous enzyme (one of the twoEuglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C₁₈ columns on HPLC, we demonstrated thatEuglena histone H1 contains approximately 9 mol% of -N-methyllysines (1.40, 1.66, and 5.62 mol% for -N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of -N-methyllysines in histone H1.     

The Lipid Composition of Urodele Myelin Which Lacks Hydroxycerebroside and Hydroxysulfatide

The concentrations of cerebrosides and sulfatides were measured in the nervous systems of urodeles and related orders with a high performance liquid chromatographic technique. The peripheral and central nervous systems of all three urodele species, Necturus maculosis (mud puppy, a salamander), Notophthalmus viridescens (eastern red spot newt), and Desmognathus ochropheus (mountain salamander), were found to be completely devoid of alpha-hydroxy fatty acid-containing cerebrosides and sulfatides. All species of reptiles and fish classes close to urodeles contain these galactolipids. The levels of nonhydroxy fatty acid-containing cerebrosides and sulfatides are essentially similar in both urodeles and reptiles. Myelin isolated from Necturus spinal cord had a specific density of 1.07, lighter than mammalian myelin. Except for the absence of hydroxycerebrosides and hydroxysulfatides, the lipid composition of Necturus spinal cord myelin is essentially similar to that of frog and rat myelin. The fatty acids of nonhydroxycerebrosides are rich in monounsaturated homologs of C22-C25, and the sphingoid base consists of both sphinganine and sphingosine. Electron microscopic examination of the sciatic nerve showed that the general structure and interlamellar distances of salamander and newt myelin are identical to those of frog, chameleon, and rat. Necturus myelin, therefore, can be used as a model for the study of the functional and structural role of hydroxygalactolipids.     

Feeding and growth of larval herring,Clupea harengus,in relation to density of copepod nauplii

Synopsis Feeding and growth rates of 1–3 wk old herring larvae from four different stocks were compared in laboratory experiments (8°C). For most of the larval groups, feeding rate was saturated at nauplii (Acartia tonsa, nauplii stages 3–5) densities over 301^–1 (5 g d.w. 1^–1). Specific growth rate increased asymptotically with nauplii density, and reached about 6% d^–1 at densities over 120 nauplii 1^–1. The growth rates attained in the laboratory were similar to field measured growth rates of similarly aged herring larvae at comparable food densities. Since food particles were homogenously distributed in the laboratory tanks, patches of dense plankton concentrations are, thus, apparently not necessary for larval growth and survival in the sea. Growth efficiency differed between larval groups, with large sized larvae being the most efficient in transforming ingested matter into growth. The difference probably relates to different sizes rather than to the different geographical origins of the larvae.     

Tubular Transport Maxima of PAH and Diodrast Measured Individually in the Aglomerular Kidney of Lophius, and Simultaneously as Competitors Under Conditions of Equimolar Loading

The maximal tubular transfer rates (Tm) of both p-aminohippurate (PAH) and diodrast (3,5-diiodo-4-pyridone-N-acetic acid or iodopyracet) were found to be fixed and reproducible when measured separately in Lophius (goosefish) under standard laboratory conditions. Expressed on a molar basis Tm_PAH was four times Tm_D. However, when these transport competitors were presented simultaneously in equimolar concentrations with the plasma levels of each sufficiently high enough to saturate the carrier system, the relative rates of excretion were reversed with the diodrast transfer rate then four times that of PAH. The combined rate of excretion was far below Tm_PAH alone, and roughly equal to Tm_D. Interaction with a common carrier was indicated by the gradations in degree of inhibition which resulted when plasma concentration ratios of diodrast to PAH were extended from 0.1 to 3.2, and PAH transfer rates expressed as percentage of Tm_PAH were correspondingly depressed from 17 to 1.0 per cent respectively. These observations again point up the inverse relationship between transfer rate and competitive effectiveness which exists for members of a series of substances actively transported by a common mechanism. It appears that carrier affinity and dissociation characteristics may be quite different for various compounds in a series, and also that these parameters may vary significantly from species to species.     

Biochemical Studies on Amphibian Metamorphosis : I. The effect of thyroxine on protein synthesis in the tadpole

Thyroxine has been shown to accelerate the synthesis of carbamyl phosphate synthetase in the liver of Rana catesbeiana. Stimulation of carbamyl phosphate synthetase synthesis by thyroxine appears to be relatively specific because of the following observations: (1) succinoxidase activity decreased during the time that carbamyl phosphate synthetase increased; (2) liver catalase responded more slowly than carbamyl phosphate synthetase to thyroxine; (3) the ratio of biochemical changes/morphological changes was greatly altered during thyroxine-induced metamorphosis. The relationships between the concentration of thyroxine and (1) temperature; (2) duration of exposure of the tadpole to thyroxine; and (3) the activity of carbamyl phosphate synthetase during the induced synthesis of carbamyl phosphate synthetase by thyroxine are discussed. Chloramphenicol and thiouracil partly counteracted the effect of thyroxine on the synthesis of carbamyl phosphate synthetase.     

Expression of prostaglandin endoperoxide synthase-1 in a baculovirus system.

A cDNA coding for ovine prostaglandin endoperoxide (PGH) synthase-1 was used to construct a recombinant baculovirus which was expressed in Spodoptera frugiperda (Sf9) insect cells. Two proteins reactive with anti-PGH synthase antibody were produced. A larger protein (Mr = 72,000) coelectrophoresed with native enzyme; a smaller, more abundant protein (Mr = 66,000) was unglycosylated enzyme. About 90% of both the immunoreactivity and the cyclooxygenase activity were present in a low speed (10(5) g x min) pellet; variable but low peroxidase activities were observed in this fraction. The specific cyclooxygenase activity of solubilized PGH synthase-1 from Sf9 cells was 56 units/mg versus 112 units/mg for the same cDNA expressed in cos-1 cells. The baculovirus-insect cell system is not ideal for generating large amounts of active PGH synthase-1 apparently because of inefficient N-glycosylation.     

Comparative studies on S-adenosyl-l -methionine binding sites of protein N-methyltransferases, using 8-azido-S-adenosyl-l -methionine as photoaffinity probe

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-l-[methyl-³H]methionine (8-N₃-Ado[methyl-³H]Met), the binding sites for S-adenosyl-l-methionine (AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC2.1.1.23); AdoMet:histone-arginin N-methyltransferase (EC2.1.1.23); and AdoMet:cytochromec-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.     

Effect of light intensity and ammonium-N on carotenogenesis ofTrentepohlia odorata andDunaliella bardawil

AxenicTrentepohlia odorata was cultured at three different NH₄Cl levels (3.5 × 10^–2, 3.5 × 10^–3, 3.5 × 10^–4 M) and three different light intensities (48, 76, 122 µmol m^–2 s^–1). Chloride had no effect on growth over this range of concentration. High light intensity and high NH₄Cl concentration enhanced the specific growth rate. The carotenoid content increased under a combination of high light intensity and low N concentration. WhenD. bardawil was exposed to the same combination of growth conditions, there was an increase in its carotenoid content. The light saturation and the light inhibition constants (K _s andK _i, respectively) for growth, and the saturation constant (K _m) for NH₄Cl were determined. TheK _s andK _i values were higher inT. odorata (66.7 and> 122 mol m^–2 s^–1, respectively) than inD. bardawil (5.1 and 14.7 µmol m^–2 s^–1, respectively). TheK _m value determined at 122 µmol m^–2 s^–1, however, was lower inT. odorata (0.048 µM) than inD. bardawil (0.062 µM).Author for correspondence