A simple assay for methionine adenosyltransferase using cation exchange paper and liquid scintillation spectrometry

A rapid, sensitive and simple procedure for the assay of l-methionine-S-adenosyltransferase based on the use of phosphocellulose ion-exchange paper is presented. The analytical procedure may be generally useful for all enzymes where there is a large cationic charge difference between the substrates and products. An application of a simpllified counting procedure for radioisotopes on solid supports is presented.     

The mechanism of regulation of fibroblastic cell replication. III. A central role for nutrient limitation: a conditioning effect due to serum imprinting of the cell response

Haploid Saccharomyces cerevisiae cells of mating type a, but not α, produce and secrete a diffusible substance, designated a factor. The a factor transiently arrests cells of mating type α, but not a, at a very early stage of the cell cycle, prior to budding and to the initiation of DNA synthesis. While the cells are arrested at this stage, few, if any, of the functions required for the ensuing cell cycle are carried out. This stage of the cell cycle coincides with the stage at which α factor, produced by cells of mating type a, specifically arrests cells of mating type a [2]. It seems probable that the reciprocally acting a and α factors together provide the mechanism by which haploid cells are synchronized to the appropriate stage of the cell cycle as a prelude to conjugation.     

Isolation and sequence determination of a peptide located in or near the active site of bovine muscle pyruvate kinase

Bovine muscle pyruvate kinase was inactivated by treatment with trinitrobenzenesulfonic acid; approximately one trinitrophenyl group was incorporated per subunit. ADP or Mg-ADP decreased the rate of inactivation but Mg⁺⁺ alone or phosphoenolpyruvate had no effect. The inactivated protein was treated with trypsin and the trinitrophenylated peptide isolated by gel filtration. Homogeneity of the isolated peptide was shown by high voltage electrophoresis and high pressure liquid chromatography. Amino acid analysis and sequence determination revealed the presence of an acidic peptide 34 amino acids long and containing ?-trinitrophenylated lysine.     

The synthesis of 3-nonaprenyl-4-hydroxybenzoate in rat liver mitochondria: the effect of Ca2 , Mg2+, chelators, and bacitracin on the activity of p-hydroxybenzoate-polyprenyl transferase

The formation of the first intermediate in ubiquinone-9 biosynthesis, 3-nonaprenyl-4-hydroxybenzoate (NPHB), by the enzyme p-hydroxybenzoate:polyprenyl transferase, has been studied in isolated rat liver mitochondria using solanesol pyrophosphate and p-hydroxybenzoate as the substrates. Phosphate buffer (100 mm) is inhibitory but at 20 mm inhibition is not apparent compared to other buffers at the same concentration. With various buffers at low concentration (20 mm) both EDTA and Mg²⁺ stimulate formation of NPHB while Ca²⁺ inhibits. Release of Ca²⁺ inhibition can be achieved by the addition of Mg²⁺, or EDTA, or EGTA, with EGTA being less effective than EDTA. When Mg²⁺, Ca²⁺, and EDTA are present together, a two- to threefold increase in activity of the enzyme is observed. The antibiotic bacitracin inhibits the synthesis of NPHB and the inhibition is increased when divalent cations are present. EGTA is more effective than EDTA in overcoming inhibition due to bacitracin. The possibility that these effects are partially due to alteration of mitochondrial membrane conformation as well as a direct effect on the enzyme is evaluated. The possible role of polyprenylphosphates in mitochondrial membrane function is discussed.     

The surfactant Bio-Solv-BBS-3 as a scintillator in liquid scintillation counting

When the surfactant mixture Bio-Solv-BBS-3 is added to a scintillation solvent it acts as a primary scintillator in response to β emissions (and Compton electrons from γs). The fluorescence excitation threshold is higher and fluorescence yield is lower than those of the primary scintillators usually employed in scintillation counting. Presence of a surfactant in a sample containing ¹⁴C or more energetic βs will be counted at higher efficiency than would be indicated by a quench correction curve (efficiency vs sample channels ratios or external standard channels ratios) derived from standards not containing surfactant.     

Immunological studies on glucose 6-phosphate dehydrogenase of rat liver

Glucose 6-phosphate dehydrogenase (G6PD) was purified from the supernatant fraction of rat liver to a homogeneous preparation by a specific elution with substrate. A specific antibody against the purified enzyme was prepared in rabbits and was shown to completely inhibit the enzyme activity and precipitate the enzyme protein of liver supernatant. With this antiserum, liver supernatants with varying specific G6PD activities obtained under several experimental conditions and supernatants from other tissues examined all formed single precipitin lines, which fused with each other in the Ouchterlony double-diffusion system. Three interconvertible microheterogeneous forms of G6PD in liver, supernatant were immunologically indistinguishable from each other. The G6PDs in participate fractions of liver were, however, distinct from the supernatant enzyme both in inhibition of the enzyme activity and in formation of precipitation by the specific antiserum. Liver supernatant G6PD, which was inactivated with various reagents or by heating, showed a simultaneous loss of ability to form precipitin line. Aggregation and disaggregation of the dehydrogenase to the tetramer and monomer, respectively, also resulted in loss of immunological reactivity. The increase in G6PD activity in the cytoplasm of carbon tetrachloride-treated or glucose casein-refed rat liver was accompanied by a proportional increase in the quantity of immunologically reactive G6PD protein.     

Biosynthesis of ergosta-4,6,8(14),22-tetraen-3-one. A novel oxygenative pathway

Specifically (tritium) labeled precursors (VIII, X, XIV, XV, and XVI), upon feeding to Penicillium rubrum, are incorporated into ergosta-4,6,8(14),22-tetraen-3-one (IV) to the extent of 14.2, 4.5, 11.4, 16.3, and 5.5% respectively. Proof that the ergostane skeleton was incorporated intact was afforded by a chemical-biosynthetic cycle, the latter stages of which entailed reduction of isolated (IV) to ergosterone (VIII), followed by removal of the label through base-catalyzed exchange. A search of the growth medium of P. rubrum revealed the presence of nonartefactual ergosterol epidioxide (XIII) and ergosta-6,22-dien-3β,5α,8α-triol (XVIII). The incorporation data are consistent with a set of multiple pathways with no unique biosynthetic sequence apparent.     

Purification and properties of glycogen phosphorylase from Streptococcus salivarius

Glucose-grown cells of Streptococcus salivarius have been shown to contain a polyglucose phosphorylase which had maximum activity in the stationary phase of growth. Despite the fact that activity in crude cell-free extracts was two- to threefold greater in the presence of corn dextrin than with oyster glycogen, subsequent purification (200-fold) of the enzyme from the soluble fraction of the organism by protamine sulfate treatment, ammonium sulfate fractionation (30–50%), ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200 demonstrated that this dextrin/glycogen activity was associated with a single enzyme. Since glucose-grown cells of S. salivarius are known to synthesize a typical glycogen polymer, the enzyme was named: glycogen phosphorylase. The purified enzyme preparation was devoid of phosphoglucomutase and ADP-glucose pyrophosphorylase, but contained a small amount of ADP-glucose: α-1,4 glucan transferase activity. The enzyme was stable at ?10 °C in the presence of 0.2 m NaF, while the pH optimum for the enzyme was 6.0 both with glycogen and with dextrin. With the purified enzyme, corn dextrin was the best primer, both in the direction of synthesis and in the direction of phosphorolysis, being 1.8–1.9 times more effective than purified S. salivarius glycogen. When the enzyme was assayed in the direction of glycogen synthesis, a K_m value of 3.4 mm was obtained for glucose-1-P, while the values for S. salivarius glycogen, oyster glycogen and corn dextrin were 25, 42, and 40 mg/ml, respectively. In the direction of phosphorolysis, K_m values were 20 mm for P_i obtained with oyster glycogen, 25 mm for P_i with corn dextrin, and 20 mg/ml and 26 mg/ml for oyster glycogen and corn dextrin, respectively. Present data suggests no involvement of -SH groups in enzyme catalysis, while the enzyme was inhibited by divalent ions with the severest inhibition being observed with Ca²⁺, Zn²⁺ and Fe²⁺. The two ion chelators, EDTA and EGTA, had no effect on enzyme activity.     

Detection of estrus and quality of semen produced by rams with deviated penises

Nine rams had their penises deviated and three others were vasectomized. Six additional intact rams served as controls. Three of the penis-deviated rams were raddled and placed in small flocks of ewes where they proved to be satisfactory detectors of estrus.Vasectomized and penis-deviated rams each were exposed to an estrous ewe for 5 minutes. The number of mounts (mean ± SE) for vasectomized rams was 4.8 ± .5 and for penis-deviated rams was 4.3 ± .5 (P>.05). When the rams had become experienced there were very few failures to mount at least once during the 5-minute period in any of the groups.Semen collection was facilitated in penis-deviated rams. There were no significant differences (P>.05) between penis-deviated and control rams with respect to volume of the ejaculate, or motility, concentration or presence of abnormal spermatozoa. Rams with deviated penises could be useful for detecting estrus and supplying spermatozoa for experimental study or use in artificial insemination.     

Comparison of the human,canine and swine E apoproteins

A comparative study of the E apoprotein isolated from the d<1.02 lipoproteins of human, canine and swine plasma revealed that the various apo-E preparations had similar molecular weights (37,000–39,000) and had similar amino acid compositions in that glutamic acid, alanine, leucine and arginine were present in high concentrations. The various preparations showed partial immunochemical cross-reactivity, demonstrating significant sequence homology between the species. However, determination of the amino-terminal amino acid sequence by automated Edman degradation showed each apo-E was different, demonstrating that the amino-terminal portion of the E apoprotein was a variable region of this protein.     

Fatty acids diffusion in lecithin multilayers: hydration and PH effects.

The diffusion of the sodium salt of monocarboxylic fatty acids, from formate to stearate, has been studied as a function of water content and pH in lecithin--water lamellar phases. Evolution of the diffusion coefficients with increasing chain length reflects the different localizations of fatty acids in the system. From formate to butyrate, which are mainly restricted to the hydrophilic layer of the phase, diffusion rates decrease rapidly. From butyrate to stearate, fatty acids (anchored at the hydrophilic--lipophilic interface) undergo lateral diffusion and then the decrease of D with increasing chain length is much slower. The diffusion of stereate is already comparable to the diffusion of the lecithin molecule itself. The diffusion rates strongly depend upon phase hydration and pH: it is shown that both parameters control the fatty acid ionization. The variations in diffusion rates observed may be ascribed to the fact that, depending upon their state of ionization, fatty acids assume a different localization and therefore experience different interactions in the lamellar system.     

Some regulatory properties of glycogen phosphorylase from Streptococcus salivarius

Purified (200-fold) glycogen phosphorylase (EC 2.4.1.1) of Streptococcus salivarius was activated by AMP and NaF when assayed both in the direction of synthesis and in the direction of phosphorolysis. Activation by NaF + AMP was greater than the sum of their individual effects. In the direction of synthesis, the K_m for AMP was 0.25 mm and was decreased to 0.125 mm in the presence of NaF. The K_m for NaF was 0.49 m and was decreased to 0.40 m in the presence of AMP. Glycogen phosphorolysis was similarly affected by AMP and NaF, except that above a concentration of 2 mm AMP was inhibitory. The effects of AMP and NaF were reversible since preincubation with these compounds, followed by dialysis, restored activity almost to the control values although some inhibition of enzyme activity was noted with the samples preincubated with NaF. The presence of both NaF and AMP had no effect on the K_m values for glucose-1-P and glycogen in the direction of synthesis, but increased the V of the enzyme.When assayed in the absence of AMP and NaF in the direction of synthesis, the enzyme was slightly inhibited by glucose and glucose-6-P, and activated by P-enolpyruvate and ADP-glucose. In the presence of AMP and NaF, the enzyme was inhibited by glucose, glucose-6-P and ADP-glucose, but was activated by P-enolpyruvate. Fructose-1,6-P₂ had no effect on the enzyme. The enzyme was further activated in the absence of AMP and NaF by adenosine, ATP, GMP, cyclic AMP and ADP, and was slightly inhibited by GTP and GDP. In the presence of AMP and NaF, however, these compounds, with the exception of adenosine, either did not show any effect or were slightly inhibitory. Adenosine was slightly stimulatory with NaF + AMP, but not with AMP alone. In the direction of phosphorolysis, the enzyme was inhibited by glucose and ADP-glucose, and activated by P-enolpyruvate, fructose-1,6-P₂ and ATP, both in the presence and absence of AMP + NaF.     

Hormonal regulation of trehalose metabolism in the blowfly, Phormia regina: interaction between hypertrehalosemic and hypotrehalosemic hormones

Hypertrehalosemia occurs two days after cardiacectomy of adult male Phormia regina with no attendant change in fat body glycogen. In spite of this, cardiacectomized flies caused to fly for 10 min show a lower rate of haemolymph trehalose turnover, and seem to have a decreased capability for synthesizing trehalose from haemolymph glucose. Phormia brain is shown to contain a hypotrehalosemic hormone whose release depends on the integrity of the stomatogastric nervous system. It is possible that the hypertrehalosemic condition in cardiacectomized flies is a result of the absence of this hormone from the blood.     

A subnanosecond-pulse fluorometric study of the CA2+ and MG2+ induced conformational changes on S-100a protein

The fluorescence parameters of the single tryptophan residue (Trp 90 alpha) in S-100a (alpha beta) protein have been studied by steady state fluorimetry and by subnanosecond fluorescence decays excited by pulsed-picosecond laser system. At pHs 7.1 and 8.5, double exponential decays were consistently observed. At both pHs, Ca2+ and to a less extent Mg2+ ions proved to modify the percentage contribution of the two decaying species. The interest of the finding is discussed.     

Conformation in solution of coenzyme A thioesters: II. Butyryl-CoA and arylacetyl-CoA compounds

The ¹H nuclear magnetic resonance (¹H-nmr) spectra of aqueous solutions of butyryl-CoA (Bu-CoA), indoleacetyl-CoA (IA-CoA), and phenylacetyl-CoA (PA-CoA) were examined at various concentrations and temperatures and compared to spectra of acetyl-CoA (Ac-CoA) and benzoyl-CoA (Bz-CoA) in order to determine to what extent, if any, each acyl-CoA compound exists in an intramolecular folded conformation. It was found previously that Ac-CoA exists predominantly in an extended conformation, whereas Bz-CoA is folded (Mieyal et al., J. Biol. Chem.249, 2633 (1974)). The present study showed: (a_ the solution behavior of Bu-CoA was essentially indistinguishable from that of Ac-CoA; thus both of these aliphatic CoA esters probably exist as extended molecules; (b) IA-CoA does form an intramolecular adenyl-indolyl complex, but the folded/unfolded ratio is only about one-half of that for Bz-CoA at physiological temperature; (c) PA-CoA apparently exists predominantly as an unfolded molecule. The diminished tendency of PA-CoA and IA-CoA to fold is probably related to the rotational mobility about the bond adjoining the respective arylmethylene group to the carbonyl moiety of the thioester link. Concentration-independent effects of the phenyl and indolyl ring currents on the ¹H-nmr signals of particular pantotheinyl methylene groups in PA-CoA and IA-CoA, respectively, are consistent with this conclusion. The outstanding conformational difference between the closely related Bz-CoA (folded) and PA-CoA (unfolded) molecules may provide insight regarding the nature of the binding sites for these molecules on the specific N-acyltransferase enzymes for which they are the best substrates (Webster et al., J. Biol. Chem.251, 3352 (1976)).