Structure of a porcine relaxin inactive on the mouse pubic ligament

In addition to B31 (CM-a) and B28 (CM-B) relaxins, acid-acetone extracts of ovaries of pregnant sows contain a third major relaxin species (relaxin C). The major components of relaxin C possess about half the activity of CM-a or CM-B in the guinea pig palpation assay, but are completely inactive in the mouse pubic ligament assay. Its uterotrophic and protein anabolic effects in ovariectomized, estrogen-primed mice, however, are comparable to those of CM-B. Sequence analysis indicates that the two major components of relaxin C, like the other porcine relaxins, consist of two polypeptide chains linked by disulfide bonds. The shorter (A) chains are identical to the A chains of the other porcine relaxins, except for the absence of the N-terminal arginine residue. The B chains display microheterogeneity; the B sequences of the two predominant species are the same as those of the other porcine relaxins through B25, but terminate at valine residue B25 or serine residue B26, respectively.     

Rabbit muscle phosphofructokinase. Modification of molecular and regulatory kinetic properties with the affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine.

The affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine modifies rabbit muscle phosphofructokinase to the extent of one group/subunit. Modification appears to occur at a binding site specific for AMP, cyclic AMP, and ADP, i.e. those adenine nucleotides which are activators under conditions where regulatory kinetic behavior is obtained. The consequences of the modification are consistent with the model proposed previously for correlation between the pK of specific ionizable groups, regulatory kinetic behavior, ligand binding, and the reversible cold inactivation of the enzyme (Frieden, C., Gilbert. H. R., and Bock, P. E. (1976) J. Biol. Chem. 251, 5644-5647). Thus, the modification shifts the apparent pK of the essential ionizable groups from 6.9 to 6.4 at 25 degrees C, with the result that regulatory kinetic behavior at pH 6.9 and 25 degrees C is lost. Furthermore, the apparent affinity of a site (other than the active site) for ATP, as measured by ATP-dependent quenching of intrinsic protein fluorescence at pH 6.9 and 25 degrees C, is decreased by the modification. Regulatory kinetic behavior for both substrates is obtained with the modified enzyme at a lower pH, consistent with the downward shift in the pK of the ionizable groups, but sensitivity to cAMP activation is abolished by the modification. The loss of regulatory kinetic behavior upon modification of sulfhydryl groups does not appear to be the same as that due to modification by the affinity label.     

In vitro regeneration of apomictic bluestem grasses

Explants from immature inflorescences of four genotypes of Old World bluestem grasses, (Bothriochloa spp.), produced callus tissue on Linsmaier and Skoog (RM) and 1/2 Murashige and Skoog (1/2 MS) media containing high levels of growth regulators. Callus masses were composed of two distinct tissue types, one a compact, white, embryogenic portion (E calli), the other soft, translucent, gelatinous and nonembryogenic (NE calli). When transferred to medium with a reduced level of 2,4-D, and/or supplemented with zeatin, E callus underwent further organization culminating in shoot production. Light and scanning electron microscopy confirmed the embryogenic pathway of differentiation. Genotype significantly affected callus induction frequency and the number of plants regenerated. The RM medium induced more explants to initiate callus compared to the 1/2 MS medium. Age of the inflorescence explant, as indicated by size, was critical for callus induction. Inflorescences with racemes 8 mm in length were superior to older ones. Five-hundred-twenty-two plantlets were regenerated and grown to maturity.     

Adenosine deaminase: viscosity studies and the mechanism of binding of substrate and of ground- and transition-state analogue inhibitors

We have studied the effects of viscosogenic agents, sucrose and ficoll, on (1) the hydrolysis of adenosine and of 6-methoxypurine riboside catalyzed by adenosine deaminase and (2) the rates of association and dissociation of ground-state and transition-state analogue inhibitors. For adenosine, Vmax/Km is found to be inversely proportional to the relative viscosity with sucrose, an agent affecting the microscopic viscosity, while no effect is found with ficoll, an agent affecting the macroscopic viscosity. Viscosogenic agents have no effect on the kinetic constants for 6-methoxypurine riboside. Thus, the bimolecular rate constant, Vmax/Km = 11.2 +/- 0.8 microM-1 s-1, for the reaction with adenosine is found to be at the encounter-controlled limit while that for the reaction with the poor substrate 6-methoxypurine riboside, 0.040 +/- 0.004 microM-1 s-1, is limited by some other process. Viscosity-dependent processes do not make a significant (less than 10%) contribution to Vmax. The dissociation constants for inhibitors are unaffected by viscosity. The ground-state analogue inhibitor purine riboside appears to bind at a rate comparable to that of adenosine. However, the slower rates of association (0.16-2.5 microM-1 s-1) and dissociation (5 X 10(-6) to 12 s-1) of transition-state analogue inhibitors are affected by the viscosity of the medium to approximately the same extent as the encounter-controlled rates of association and dissociation of adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant human prorenin from CHO cells: Expression and purification

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from 1–5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P₆-P₃ sequence of human angiotensinogen.