Kinetics of a model of autocatalysis, coupling of a reaction in which the enzyme acts on one of its substrates.

A global kinetic analysis is presented of a model of an enzyme autocatalytic process, to which a reaction is coupled, in which the enzyme acts upon one of its substrates. The kinetic equations of both the transient phase and the steady state are derived for this mechanism. In addition, we determine the corresponding kinetic equations for several particular cases which are characterized by certain relations between the rate constants. Finally, a kinetic data analysis is proposed for one of these particular cases. It can easily be extended to any of the other cases.     

Sequence and characterization of Bacillus subtilis CheW.

A Bacillus subtilis open reading frame (ORF) encoding a predicted polypeptide of 156 amino acids was subcloned and sequenced. The polypeptide was found to be homologous to CheW of Escherichia coli, sharing 28.6% amino acid identity. The ORF was verified by using a bacteriophage T7 expression system in E. coli. The gene was inactivated by insertion of a nonpolor chloramphenicol acetyltransferase cassette in its N-terminal region. In the absence of chemoeffectors, the mutant displayed a smooth swimming bias, with some tumbling. The CheW- mutant was defective on swarm plates but was complemented by a plasmid that expressed wild type CheW. Addition of attractant or repellent to the CheW- mutant resulted in transient smooth swimming or tumbling, respectively. However, capillary assays revealed that chemotaxis was substantially impaired in the mutant strain.     

Naturally occurring benzodiazepines in human milk

The presence of benzodiazepine-like molecules was detected radioimmunologically in the plasma and milk of 12 women and in the plasma of 9 men. All subjects were non-users of benzodiazepines. The concentration of these biological materials expressed as diazepam equivalents per mL amounted to 2.54 +/- 0.74 ng in male plasma; to 2.20 +/- 0.35 ng in female plasma and to 1.91 +/- 0.54 ng in milk. Further investigation of the active compounds in milk permitted the unequivocal identification of diazepam, both free and bound to a presumably protein carrier and, at least, three more benzodiazepine-like molecules. Their origin either from dietary sources or as a result of endogenous biosynthesis is still unclear.     

Thyroid hormones control lipid composition and membrane fluidity of skeletal muscle sarcolemma.

Sarcolemma membrane lipid phase of skeletal muscles of hyperthyroid animals was compared to that of control (euthyroid) ones. Hyperthyroidism caused 15% decrease in cholesterol and 70% increase in the phospholipid content of the membrane. This was accompanied by the alterations in proportions between individual phospholipid classes, and was followed by changes in the composition of phospholipid fatty acids. The calculated fatty acid unsaturation index was higher for membrane lipid phase of hyperthyroid animals than of euthyroid ones. Thyroxine-induced alterations in the lipid composition of sarcolemma caused changes in the membrane fluidity and the activity of calmodulin-stimulated (Ca(2+)-Mg(2+)-ATPase. Measurements of the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene indicated that the lipid phase transition of membrane vesicles occurred at 25.9 degrees C and at 28.9 degrees C for preparations isolated from hyperthyroid and euthyroid rabbits, respectively. Arrhenius plot break-point temperature for CaM-stimulated (Ca(2+)-Mg(2+)-ATPase activity was lower in membrane preparations isolated from hyperthyroid (26.9 degrees C) than from euthyroid ones (30.0 degrees C). Thus, the increase of the membrane fluidity presumably caused that the enzyme was characterized by the lower activation energy value. This phenomenon may be viewed as a supplementary mechanism for activation of the enzyme by thyroid hormones to previously reported elevation of the amount of (Ca(2+)-Mg(2+)-ATPase protein exerted by hyperthyroidism (Famulski et al. (1988) Eur. J. Biochem., 171, 363-368; Famulski and Wrzosek (1988) in The Ion Pumps-Structure, Function and Regulation (Stein, W.D., ed.), pp. 355-360, Alan R. Liss, New York).     

Taurine release associated to volume regulation in rabbit lymphocytes.

Rabbit lymphocytes exposed to hyposmotic media first swell and then recover their initial volume within 6 min. During volume recovery, free amino acids (FAA) decrease from 451.1 to 208 nmoles/mg protein. Taurine was the dominating FAA, accounting for 70% of the FAA pool. The time course of 3H-taurine release induced by hyposmolarity followed that of volume recovery. Efflux of 3H-taurine in an 8 min period was 17.8% (of total labeled taurine accumulated during loading) in an isosmotic medium. Reducing osmolarity to 0.87, 0.75, 0.62, and 0.5 increased this release to 24.8%, 38.1%, 56.4% and 70.9%, respectively. The volume-sensitive release of 3H-taurine was unaffected by omission of external Na+ or Ca++ and was reduced by 23% in the absence of Cl-. It was unaffected by agents disrupting the cytoskeleton or by tetraethylammonium, barium, quinidine, and gadolinium, but was 26% reduced by DIDS. Taurine release was inhibited at 4 degrees C, but was unchanged at 15 degrees C or 25 degrees C. An involvement of FAA, particularly taurine, in lymphocyte volume regulation is suggested.     

In vivo and in vitro effects of temperature on monoamine oxidase activity in brain and other tissues of the goldfish. Carassius auratus L

1. The maximum velocity (Vmax) and apparent Michaelis constant (Km) of brain and liver monoamine oxidase (MAO) in goldfish were different in fish acclimated to 22 degrees C and to 7 degrees C ambient temperature. 2. In brain, Vmax and Km were dependent upon incubation temperature, but both parameters were lower in 7 degrees C, adapted fish over most of the incubation temperature range. 3. The values obtained for Km showed a plateau at incubation temperatures at and below 25 degrees C for warm water fish, and at and below 20 degrees C for cold water fish. The activation energy of brain MAO was lower in fish adapted to the colder water. 4. These results show that goldfish MAO displays changes in functional activity in response to a change in environmental temperature. Apparently the purpose of this adaptation is to compensate for a reduction in enzyme concentration.     

Microfluorometric filter determination of DNA in sucrose density gradients

A simple filter method for the fluorometric estimation of DNA in alkaline and neutral sucrose gradient fractions with DABA·2HCl is discussed. Alpha-450 membrane filters of regenerated cellulose (Gelman Instrument Co., Ann Arbor, Michigan) were used. In the proposed method washing of the DNA precipitate as well as the reaction with DABA·2HCl were performed directly on the filters, thus avoiding repeated washing and centrifugations of DNA precipitates applied hitherto in analogous fluorometric techniques. A good coincidence of the results concerning localization of DNA sedimentation profiles determined by radioisotopic and fluorometric methods was obtained. The method is very convenient for DNA estimation in alkaline and neutral sucrose density gradient fractions obtained by ultracentrifugation of DNA of nonproliferating cells where DNA labeling is very difficult, and in the case of human lymphocytes even impossible without stimulation for blastic transformation. Its other advantages are a considerably simplified procedure and a higher precision with respect to other fluorometric methods for determination of DNA.     

Trypanosoma cruzi: defined medium for continuous cultivation of virulent parasites.

A liquid medium was developed for the continuous cultivation of Trypanosoma cruzi. Among the several highly purified macromolecules tested only bovine liver catalase, horseradish peroxidase, lactoperoxidase, and bovine hemoglobin supported the continuous growth, at high yield, of mice-virulent Trypanosoma cruzi; other hemoproteins were inactive. Bovine liver catalase showed optimal Trypanosoma cruzi growth-promoting activity, parasites reaching 20 × 10⁶ parasites/ml (95% epimastigotes) at about 10 days in most of the 45 subpassages to date. Furthermore, this protein in the incubation medium provided all the amino acid requirements of actively growing parasites, thus eliminating the need for exogeneous free amino acids. Additional experiments revealed that the hemoprotein's growth-promoting activity was independent of any enzymatic activity and that reconstituting the exact protein composition by means of exogeneous amino acids did not support parasite multiplication, suggesting the importance of the primary structure of the active proteins for growth-promoting activity. These active macromolecules supported the multiplication of five different strains of Trypanosoma cruzi, but did not support Leishmania brasiliensis or Leishmania mexicana proliferation, suggesting species specificity.