Crystallization of mouse submaxillary gland renin

Renin isolated from the mouse submaxillary gland has been crystallized and is being subjected to X-ray diffraction analysis.The observed crystalline form has the following unit cell dimensions: $\text{a = 96.4 (1)}\text{A}\text{?}\text{, b = 104.4 (1)}\text{A}\text{?}\text{, c = 77.4 (1)}\text{A}\text{?}\text{, β = 101.2 (1) °}$, space group P2₁, Z = 8 (4 protein molecules in the asymmetric unit).Native data have been recorded at 5 Å resolution with an automatic diffractometer.     

Rapid and large-scale purification and characterization of renin from mouse submaxillary gland

Little is known about the structural basis for the highly restricted substrate specificity of renin, whose only function known to date is to hydrolyze the unique leucyl peptide bond in the prohormone angiotensinogen to form angiotensin I. Our lack of knowledge is due to our inability to purify this enzyme in a large quantity sufficient for structural studies. A two-step column chromatographic method for rapid and large-scale purification of renin from mouse submaxillary gland has been developed. It allows isolation of the enzyme in a hundreds-of-milligrams quantity at an overall yield of 60%. Single bands obtained by polyacrylamide gel electrophoresis, isoelectric focusing, and the result of ultracentrifugal studies indicated homogeneity of the product. The moelcular weight of renin was estimated to be 36,000 by sedimentation equilibrium studies in 6 m guanidine · HCl. Sedimentation velocity study gave a single sedimenting boundary with an s_20,w of 2.58 × 10^?13 s. A Stokes radius of 27 Å was obtained by gel filtration. The far ultraviolet-circular dichroism spectrum indicated a high content of β-structure (46%). In contrast to renal renin, submaxillary gland renin does not contain amino sugars or neutral sugars. No renin activity was retained by concanavalin Aagarose gels. Results of amino acid analysis, isoelectric focusing, and determination of amino-terminal residues by the dansylchloride reaction, together indicated that this protein is identical with renin A isolated previously in a much smaller quantity.     

The purification and characterization of multiple forms of mouse submaxillary gland renin

Five forms of renin, A₀, A, C, D and E, from mouse submaxillary gland were purified by a two-step procedure including chromatography on the immunoaffinity column and CM-cellulose column. Four renin fractions, A₀, A, C and E were purified to homogeneity by the criteria of polyacrylamide gel electrophoresis, analytical isoelectric focusing and Ouchterlony double immunodiffusion. All these forms of renin have molecular weights of 40 000 as determined by gel filtration on Sephadex G-100 column. No high molecular weight renin could be demonstrated. Individual renin fractions showed similar angiotensin I formation activity, 52–158 ng angiotensin I/ng protein per h. No other protease activity could be detected with hemoglobin or casein as substrate. These purified proteins showed a discrete pattern of migration under polyacrylamide gel electrophoresis. Under denaturing condition in SDS-gel electrophoresis, all but fraction D showed a protein band with a molecular weight of 30 000. Fraction D showed a major component with molecular weight of 33 000. The isoelectric points of these renin forms varied from 5.46 to 5.76. They all reacted with antibody raised against renin A and showed similar pressor response activity with 20 ng quantities of the purified proteins. The closely related characteristics of these five forms of renin were further demonstrated by their similarity in peptide mapping patterns after limited digestion with Staphylococcus aureus V8 protease. The data suggest that these proteins are homologous proteins.     

Preliminary X-ray crystallographic data on mouse submaxillary gland renin and renin-inhibitor complexes

We have crystallized renin from the submaxillary gland of male mice, both in its native state, and in binary complex with transition-state analog inhibitors. The best of the many crystal forms examined consisted of tetragonal bipyramids with space group symmetry P41 21 2 (or its enantiomorph) and unit cell dimensions a = b = 91.4 A, c = 211.6 A; alpha = beta = gamma = 90 degrees. This tetragonal form was compatible with both inhibited and uninhibited renin. Two of the inhibitors used were synthesized as iodinated analogs; their binary complexes with renin may serve as single-site rational heavy atom derivatives. X-ray data beyond 2.8-A resolution have been collected by oscillation photography using the Cornell High Energy Synchrotron Source in Ithaca, NY.     

A DNA polymorphism,consistent with gene duplication,correlates with high renin levels in the mouse submaxillary gland

The renin regulatory locus (Rnr) is a genetic element governing mouse submaxillary gland (SMG) renin levels. A 45,000 dalton polypeptide detectable after in vitro translation of mouse SMG mRNA has been identified by genetic and physical criteria as SMG renin. A cDNA recombinant clone specific for SMG renin has been isolated and used to demonstrate that the previously described genetic regulation of SMG renin levels is manifest at the level of renin mRNA concentration. The renin cDNA clone has also been used in Southern blot analyses to study the organization of homologous DNA sequences in strains carrying different alleles at the Rnr locus. Restriction digest patterns of high renin strains (Rnr^s) are characteristically distinct from patterns observed for low renin strains (Rnr^b) and are suggestive of a structural gene duplication at the chromosome 1 locus in high renin strains. However, gene dosage cannot account for the increased levels in high renin strains, since SMG renin levels in Rnr^s and those in Rnr^b may differ up to 100-fold.     

Genetic and endocrine control of renin activity in the submaxillary gland of the mouse

Basal activity of submaxillary gland (SMG) renin is high in female mice that carry the Rnr ^s allele and is induced to higher levels by treatment with dihydrotestosterone (DHT). To determine whether the difference in basal activity between high (Rnr ^s/Rnr^s) and low (Rnr ^b/Rnr^b) strains is due to enhanced sensitivity of Rnr ^s/Rnr^s strains to endogenous androgen, we first studied the effect of several types of endocrine ablation on SMG renin in young female mice, and second, we removed normal androgen receptor protein by introducing the X-linked Tfm gene. Adrenalectomy with or without castration had no effect on basal SMG renin; hypophysectomy decreased basal renin activity 400-fold but did not abolish responsiveness to DHT. Loss of androgen receptor did not affect basal renin activity but did prevent enhancement by DHT. Basal and induced renin activities in L.AKR(Alll)/Cy, a congenic strain homozygous for Rnr ^s introduced from AKR/J into the background of C57L/J, an Rnr ^b/Rnr^b type strain, are intermediate between levels observed in the original strains. We conclude that (1) the basal level of SMG renin is regulated directly or indirectly by some pituitary hormone(s) but not by androgen, (2) androgen induction of renin activity requires a normal androgen receptor, and (3) major gene(s) that regulate basal as well as induced SMG renin are in a circumscribed region of chromosome 1.This work has been aided by Grants GM26414 and AM03892 from the National Institutes of Health, a grant from the Texas affiliate of the American Heart Association, and by research contract NO1-CP33255 from the Division of Cancer Cause and Prevention, the National Cancer Institute. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Interaction of monosulfonate tetraphenyl porphyrin, a competitive inhibitor, with acetylcholinesterase

Monosulfonate tetraphenyl porphyrin (TPPS(1)) forms a 1:1 complex with electric eel acetylcholinesterase (AChE) inducing a loss in TPPS(1) absorbance at 402 nm and the appearance of a new absorbance centered at 442 nm. In the presence of AChE, the fluorescence of TPPS(1) at 652 nm is slightly narrowed, with the maximal 652 nm fluorescence shifted from 407 to 412 nm excitation wavelength. The fluorescence peak of TPPS(1) at 712 nm shifts to 716 nm in the presence of AChE. TPPS(1) is a competitive inhibitor of AChE. The addition of acetylcholine iodide (AChI) or the competitive inhibitor tetracaine to the preformed AChE-TPPS(1) complex results in a loss of the 442 nm absorbance band as the porphyrin is displaced from AChE. The absorbance peak does not decrease in the presence of procaine, a non-competitive inhibitor.     

Active site directed inactivators of mouse submaxillary renin.

The following active site directed inactivators for the pressor enzyme renin were synthesized: L-alpha-bromo-isocaproyl(BIC)-Leu-Val-Tyr-Ser-OH, L-BIC-Val-Tyr-Ser-OH, L-BIC-Leu-Val-OCH3, L-BIC-Leu-Val-OH, L-BIC-Val-Tyr-NH2, L-BIC-Val-Tyr-OCH3, L-BIC-Val-Tyr-OH, L-BIC-Leu-OCH3, L-BIC-Val-OCH3, and L-BIC-OCH3. The rate of inactivation of mouse submaxillary gland renin by these reagents was studied under a variety of conditions. L-alpha-Bromoisocaproyl-Val-Tyr-Ser-OH and L-alpha-bromoisocaproyl-Leu-Val-Tyr-Ser-OH and L-alpha-bromoisocaproyl-Leu-Val-Tyr-Ser-OH were the most efficient inactivators followed by L-alpha-bromoisocaproyl-Val-Tyr-NH2. The rates of inactivation by the first two peptides were strongly dependent on pH, being most efficient at low pH, least efficient at pH near 5.6, and becoming efficient again at neutral pH. The rate of the inactivation by L-alpha-bromoisocaproyl-Val-Tyr-NH2, in which the C-terminal carboxyl group is blocked, was only slightly dependent on pH. Complete inactivation was achieved by these three reagents. The inactivation was accompanied by incorporation of a stoichiometric quantity of the radiolabeled reagents. Based on these findings it was concluded that the inactivators reacted with a carboxyl group(s) in the active site of the renin molecule to form an esteric linkage. These data also suggest that a carcoxyl group(s) may constitute part of the catalytically essential functional groups in renin action. D-alpha-Bromoisocaproyl derivatives of the various peptides mentioned above were also prepared. These compounds were much less active than the L isomers indicating that the inactivation by the L-alpha-bromoisocaproyl peptides was a specific reaction.     

Iodide transport in isolated cells of mouse submaxillary gland

A method has been developed to isolate cells from the submaxillary gland of mouse by treatment with pronase. Three fractions of cells have been isolated having almost equal iodide concentrating activity. The isolated cells show time dependent uphill transport of iodide. The transport is substrate-saturable, having aK _m value of 0.3 μM for iodide. The transport is sensitive to antithyroid drugs, metabolic inhibitors and to some extent to ouabain. Pseudohalide such as thiocyanate competes with the transport of iodide. Thyroid hormones or thyroid stimulating hormone have no significant effect on the iodide transport in these cells.