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.     

Related calcium-binding proteins map to the same subregion of chromosome 1q and to an extended region of synteny on mouse chromosome 3

The serum protein cystic fibrosis-associated antigen (CFAG), present at elevated levels in CF homozygotes and heterozygotes, is now known to consist of two distinct but related subunits (calgranulins A (CAGA) and B (CAGB)). Both show similarity to the S100-related calcium-binding proteins. We have previously assigned CAGA to human chromosome 1q12-q21 and demonstrate here that the cDNA probe for CAGB cosegregates with it in our somatic cell hybrid panel. cDNA probes for the related genes calcyclin (CACY) and a mouse placental protein (18A2, suggested name Capl) enabled us to confirm and refine the in situ hybridization result assigning CACY to chromosome 1q21-25 and to demonstrate that both genes cosegregate with CAGA and CAGB. Capl was mapped to a region of chromosome 3 in the mouse using the BXD recombinant inbred strain mice where the p11 protein (calpactin light chain Cal1l), another S100 family member, has been localized. Cacy is shown to be within 8 kb of Capl in the mouse genome.     

Characterization of recombinant human renin: Kinetics,pH-stability, and peptidomimetic inhibitor binding

The kinetic behavior andpH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P₆-P₃ renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to renin. ThepH-stability profile for renin, measured with the substrate at 25°C, indicated a broad plateau of stability betweenpH 6.0 and 10.0. Analysis of thepH-activity profile of renin for the substrate indicated a minimumK _m (1.8 µM) atpH 7.4 and a maximumV _m betweenpH 7.4 and 8.0. The thermodynamics of the binding of a novel, soluble, peptidomimetic inhibitor to renin indicated it is possible to retain the tight-binding characteristics and enthalpy contributions to binding of larger peptide-derived inhibitors, while reducing inhibitor size and entropic contributions to binding. A novel derivative of the fluorogenic substrate, containing a 3-methyl histidine substitution at the P₂ site, was used to test the recent hypothesis that renin functions by virtue of substrate-directed catalysis.     

Aberrant production and regulation of proopiomelanocortin-derived peptides in ectopic Cushing's syndrome

A 64 year old woman with a pancreatic islet cell tumor developed Cushing's syndrome. Glucocorticoid secretion did not decrease after low or high dose dexamethasone administration, and the Cushing's syndrome was cured by removal of tumor tissue. Immunohistochemistry and radioimmunoassays revealed the presence of immunoreactive ACTH, beta-endorphin and alpha-MSH in the tumor cells. Gel-permeation chromatography confirmed that beta-endorphin was the predominant opioid peptide produced by the tumor. The tumor was shown to contain a single 1.2 kilobase RNA species which hybridized to a 32P human POMC-cDNA; this POMC RNA was identical in size to that isolated from a normal human pituitary. In dispersed monolayer culture, CRF failed to elicit ACTH release from the tumor cells, but dexamethasone caused a paradoxical increase in ACTH secretion in vitro. This study demonstrates that aberrant regulation of POMC synthesis and peptide processing can be seen in tumors which synthesize a POMC RNA identical in size to that made in the pituitary gland.     

A Continuous Fluorescence Assay of Renin Activity

A sensitive fluorescence assay that employs a new fluorogenic peptide substrate has been developed to continuously measure the proteolytic activity of human renin. The substrate, DABCYL-gaba-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-EDANS, has been designed to incorporate the renin cleavage site that occurs in the N-terminal peptide of human angiotensinogen. The assay relies upon resonance energy transfer-mediated, intramolecular fluorescence quenching that occurs in the intact peptide substrate. Efficient fluorescence quenching occurs as a result of favorable energetic overlap of the EDANS excited state and the DABCYL absorption, and the relatively long excited state lifetime of the EDANS fluorophore. Cleavage of the substrate by renin liberates the peptidyl-EDANS fragment from proximity with the DABCYL acceptor, restoring the higher, unattenuated fluorescence of the EDANS moiety. This leads to a time-dependent increase in fluorescence intensity, directly related to the extent of substrate consumed by renin cleavage. The kinetics of renin-catalyzed hydrolysis of this substrate have been shown to be consistent with a simple substrate inhibition model with a substrate K_m 1.5 μM at physiological pH; Cleavage of the substrate occurs specifically at the Leu-Val bond and corresponds to the renin cleavage site of angiotensinogen, as reported earlier. In this report, we describe in detail the synthesis of the fluorogenic renin substrate and its application in assays of renin activity. Assay sensitivity has been evaluated by a series of enzyme dilution experiments using the continuous assay format, showing that the assay can detect renin as low as 30 ng/ml after a incubation of only 3-5 min. It was estimated that with extended incubation time (2-3 h) the assay can detect renin at 0.5 ng/ml concentration level. An automated, high throughput fluorometric renin assay has been developed for a 96-well microtiter-plate fluorescence reader, which is useful for studies of enzyme inhibitors and enzyme stability.     

Substrate requirements for ErmC' methyltransferase activity.

ErmC' is a methyltransferase that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics by catalyzing the methylation of 23S rRNA at a specific adenine residue (A-2085 in Bacillus subtilis; A-2058 in Escherichia coli). The gene for ErmC' was cloned and expressed to a high level in E. coli, and the protein was purified to virtual homogeneity. Studies of substrate requirements of ErmC' have shown that a 262-nucleotide RNA fragment within domain V of B. subtilis 23S rRNA can be utilized efficiently as a substrate for methylation at A-2085. Kinetic studies of the monomethylation reaction showed that the apparent Km of this 262-nucleotide RNA oligonucleotide was 26-fold greater than the value determined for full-size and domain V 23S rRNA. In addition, the Vmax for this fragment also rose sevenfold. A model of RNA-ErmC' interaction involving multiple binding sites is proposed from the kinetic data presented.     

In vitro augmentation of lymphocyte sheep cell rosette formation by leukocyte dialysates.

We have studied in vitro the effects of leukocyte dialysates containing transfer factor on the formation of sheep cell rosettes by human lymphocytes. Dialysate had no effect on the toral rosettes, but increased the number of rapidly forming ("active") rosettes. This was due to an increased affinity of the lymphocytes for the sheep red cell. Trypsin-treated lymphocytes regained the ability to form rosettes more rapidly when cultured with leukocyte dialysates than with control media. These experiments suggest that leukocyte dialysate acts to increase the number or arrangement of sheep cell receptors on the lymphocyte surface.     

Crossflow microfiltration of animal cells

Laminar shear is the primary mechanism of cell damage, limiting flow rate (and hence flux) in crossflow microfiltration of animal cells. Sensitivity to hydrodynamic and interfacial stress is reduced by the addition of 0.1% Pluronic polyol. A critical average wall shear rate of 3000 s(-1) (above which damage occurs) is found for several cell types, including mammalian and insect cells. Hydrodynamic stress also limits the maximum tip speed in a rotary lobe pump to less than 350 cm/s. Turbulent flow in the recirculation loop piping at Reynolds numbers of up to 71,000 does not cause cell damage. Maximum sustainable flux decreases with cell concentration and increases with cell size (in qualitative agreement with the hydrodynamic lift model). A flux of 30 to 75 L/m(2) h (depending on cell size) can be sustained during 20-fold concentration from 2.5 x 10(6) cells/ml, while maintaining high cell viability.