Human renal renin. Complete purification and characterization

Complete purification of human renin from noncancerous, autopsied kidneys is reported. A 480,000-fold purification was achieved to yield renin with a specific activity of 950 Goldblatt units/mg. This preparation satisfied multiple criteria of purity as tested by polyacrylamide gel electrophoresis, isoelectric focusing, specific activity, analytical ultracentrifugation, and immunodouble diffusion. The molecular weight of the pure enzyme determined by sedimentation equilibrium is 40,000. The apparent molecular weight estimated by gel filtration is 41,000. The enzyme has an isoelectric point of pH 5.7. Human renin shows an affinity for concanavalin A, suggesting the presence of carbohydrates. These properties and the amino acid composition of human renin are different from those of renin obtained from other mammalian species. Human renin antibodies prepared with the pure enzyme preparation showed negligible cross-reactivity with renin from other mammalian species. The activity with homologous human renin substrate has a pH optimum of 6, whereas with substrates from other mammalian species the optima were in higher or lower pH ranges.     

Characterization of cloned murine cytolytic T cell lines

Murine cytolytic T lymphocytes can be kept in continuous culture apparently indefinitely by repeated passage in a concanavalin A-induced growth promoting medium. Some of these long-term cell lines maintain their cytolytic activity. Starting from three such populations, several cloned cytolytic T cell lines were derived and subsequently subcloned one or more times. Considerable variation in the levels of cytolytic activity was observed between different subclones; some initially active subclones lost activity with prolonged culture. In addition, one of the clones appeared to progressively lose the relative specificity demonstrated during the earlier passages of the parent cell line.     

Flavin-dependent substrate photo-oxidation as a chemical model of dehydrogenase action.

As a model of flavin-dependent biological dehydrogenation, flavin-sensitized photodehydrogenation and photodecarboxylation were studied by variation of substrate, flavin, pH and solvent. Evidence for the following rules is given. (1) When the reactive site of a photosubstrate is an alpha-carbon atom of the type CH-CO2-, decarboxylation is preferred over dehydrogenation, whereas the reverse is true for the neutral CH-CO2H. (2) Consequently these reactions do not exhibit a measurable isotope effect with C2H-CO2-, in contrast with the findings by Penzer, Radda, Taylor & Taylor [(1970) Vitam. Horm. (N.Y.) 28, 441--466], which could not be reproduced. When the substate does not contain a carboxylate group, isotope effects occur, in verification of previous reports, e.g. for benzyl alcohol C6H5-C2H20H. (3) The mechanism of flavin-sensitized substrate photodecarboxylation is assumed to consist in a primary carbanion fixation at the flavin nucleus (position 4a, 5 or 8) with concomitant liberation of CO2. This step is followed by rapid fragmentation of the adduct CH-Fl-red., provided that the substrate contains a functional and electron-donating group X, e.g. X = OH, OCH3 or NH2 (but not NH3+ !) in X CH-CO2-. (4) The minimal requirement for flavin-sensitized C-H dehydrogenation is the presence of a hydroxyl group. For example, methanol as substrate and solvent is dehydrogenated at pH sufficiently alkaline for detection of the presence of the active species CH3O-, whereas at more acidic pH substrate dehydrogenation is competing with flavin autophotolysis, which depends on the substituents in the flavin nucleus.     

Electrophoretic mobilities and diffusion coefficients of hemoglobin at high pH.

Diffusion studies by photon correlation of scattered laser light confirm the dissociation of the tetrameric form of human carboxyhemoglobin to dimers above pH 10 and provide new estimates of the subunit dissociation equilibrium constants in this pH range. Electrophoretic light-scattering experiments under the same conditions reveal that the electrophoretic mobilities of tetramers and dimers are indistinguishable to within instrumental resolution (ca. 7% in these experiments). The data imply an increase of the electrical charge on the dimer of at least 2.8 to 4.4 net negative charges upon dissociation. Mechanisms for the accumulation of negative charge by the dimer upon dissociation of the tetramer are proposed.     

N-acetylglutamate 5-phosphotransferase of Pseudomonas aeruginosa. Catalytic and regulatory properties.

Some kinetic properties of N-acetylglutamate 5-phosphotransferase (ATP: N-acetyl-L-glutamate 5-phosphotransferase EC 2.7.2.8) purified approx. 2000-fold from Pseudomonas aeruginosa have been studied. The enzyme required Mg2+ for activity. Mn2+, Zn2+, Co2+, and Ca2+, in this order, could replace Mg2+ partially. The substrate specificity was narrow: N-carbamoyl-L-glutamate and N-formyl-L-glutamate were phosphorylated, but at a lower rate than N-acetyl-L-glutamate; N-propionyl-L-glutamate was almost inactive as a substrate. dATP, but neither GTP nor ITP, could be used instead of ATP. The enzyme had a broad pH optimum from pH 6.5 to 9. Feedback inhibition by L-arginine was markedly dependent on pH. Above pH 9 no inhibition was observed. L-Citrulline was three times less potent an inhibitor than L-arginine. The enzyme showed Michaelis-Menten kinetics, even at low concentration of the second substrate. The apparent Km was 2 mM for N-acetyl-L-glutamate (at 10 mM ATP) and approx. 3 mM for ATP (at 40 mM N-acetyl-L-glutamate). In the presence of L-arginine the rate-concentration curves for N-acetyl-L-glutamate became signoidal, while no cooperativity was detected for ATP. A method was developed allowing the determination of N-acetyl-L-glutamate in the nanomolar range by means of purified enzyme.     

On the evocability of a positive oestrogen feedback action on LH secretion in transsexual men and women.

In transsexual men with homosexual behaviour and intact testicular function, as well as in homosexual men with normal gender identity, following a negative oestrogen feedback effect a delayed positive oestrogen feedback action on LH secretion was evoked. By contrast, in transsexual men with hypo- or asexuality and intact testes or hypergonadotrophic hypo- or agonadism, as well as in heterosexual men with normal gender identity, a negative oestrogen feedback effect was not followed by a positive feedback action on LH release. In transsexual women with homosexual behaviour and oligo- and/or hypomenorrhoea, only a weak or at best moderate positive oestrogen feedback action on LH release was evocable, similarly as in castrated and oestrogen-primed heterosexual men. By contrast, in a transsexual woman with bisexual behaviour and eumenorrhoea, a strong positive oestrogen feedback action on LH secretion was evocable, as well as in heterosexual women with normal gender identity.     

Expression of the argA Gene Carried by a Defective Lambda Bacteriophage of Escherichia coli

Evidence is presented that the increase in specific activity of N-acetylglutamate synthase observed upon heat induction of Escherichia coli (lambdadargA) is primarily due to a gene dosage effect.     

SOME ULTRASTRUCTURAL OBSERVATIONS ON ENDODERMAL CELL DEVELOPMENT IN ZEA MAYS ROOTS

The fine structure of primary, secondary, and tertiary stages of Zea endodermal cell development was investigated. The casparian strip formed in situ in the anticlinal walls and remained at a fixed point relative to the endodermis-pericycle boundary. The only protoplasmic structure that had a constant spatial association with the developing strip was the plasmalemma. Plasmodesmata appeared to be more numerous on the tangential walls than on radial walls; only rarely were they located in the casparian strip. The suberized lamella developed on inner and outer tangential walls before it appeared on the radial walls. No cytoplasmic organelles were found to have any particular spatial association with this layer. The suberized lamella was about 0.04 μm thick except near plasmodesmata and along the adaxial margin of the casparian strip, where it was thicker. Occasionally it failed to form along the abaxial margin of the strip. The adherent affinity between plasmalemma and casparian strip was lost after the strip was covered by suberized lamella. The secondary wall became asymmetrically thickened by differential deposition of successive lamellae. A thin layer of secondary wall material extended across the floor of each pit. Pit cavities often contained mitochondria, and plasmodesmata were restricted to the pits. The plasmodesmata were constricted where they entered the thin layer of secondary wall material and where they penetrated the suberized lamella. The various stages of cell development tended to be asynchronous. No passage cells were observed. Endodermal cell development in Zea closely resembles that described for barley.