Separation of recombinant human growth hormone from Escherichia coli cell pellet extract by capillary zone electrophoresis.

Free zone capillary electrophoresis (FZCE) was used to resolve recombinant human growth hormone (rhGH) and its variants from very crude mixtures of Escherichia coli (E. coli) cell paste extract. The methodology employs a phosphate deactivated fused-silica capillary and a 250 mM phosphate (pH 6.8), 1% (v/v) propylene glycol buffer with a high field strength of 600 V/cm. Resolution of rhGH and its variants from very crude mixtures did not change after 80 injections for the PSC capillaries. Bare silica and PVA coated capillaries had a more limited lifetime when injected with the same crude mixtures (10 to 30 injections). This FZC method provides a very powerful tool for assessing rhGH modification during the fermentation and isolation of rhGH that is not possible with other current analytical techniques.

Characterisation of the processing by human neutral endopeptidase 24.11 of GLP-1(7-36) amide and comparison of the substrate specificity of the enzyme for other glucagon-like peptides.

The post-secretory processing of the potent insulinotropic peptide hormone, GLP-1(7-36)amide, probably involves one or more of a small group of membrane-bound ectopeptidases. Reported here, is the characterisation of the endoproteolysis of human GLP-1(7-36)amide by the recombinant human form of neutral endopeptidase (NEP) 24.11, which is one of the best characterised and widely-distributed of ectopeptidases and is involved in the processing of other peptide hormones. The products of the limited endoproteolysis were characterised by mass and primary structure following fractionation using high performance liquid chromatography. The rate of this endoproteolysis by NEP 24.11 was estimated and compared to that of GLP-1(7-36)amide-related peptides. GLP-1(7-36)amide appears to be good substrate for NEP 24.11 with most, but not all potential target bonds being cleaved. Also, the structurally-related peptides, secretin and glucagon appear to be good substrates whereas GIP and exendin-4 are very poor substrates. That the GLP-1(7-36)amide super-agonist, exendin-4 is a poor substrate for NEP 24.11 is significant for the possible use of this peptide as a prototype for the development of clinically-useful peptide agonists. Further studies should reveal whether NEP 24.11 is important for the metabolic clearance of GLP-1(7-36)amide and will be highly relevant for the attempts to realise the suggested therapeutic value of GLP-1(7-36)amide.

Protein sorting by high performance liquid chromatography. 2. Separation of isophosphorylates of recombinant human DNase I on a polyethylenimine column.

Anion exchange HPLC with a polyethylenimine (PEI) column separates recombinant human deoxyribonuclease I (rhDNase) glycoforms according to the extent and positions of phosphorylation of mannose residues in N-linked oligosaccharides. The separation provides a selectivity unavailable by anion exchange HPLC with other columns or by isoelectric focusing gel electrophoresis and can be used to quantify the phosphate content of preparations of rhDNase. Tryptic mapping of fractions collected from the column and treated with alkaline phosphatase was used to identify the sites of phosphorylation. Unnatural forms of rhDNase, bearing oligosaccharide structures at only one of the two sites of glycosylation, were prepared by cleaving the phosphate-containing high mannose and hybrid structures from the purified isophosphorylates fractionated on the PEI column. The separation of rhDNase isophosphorylates on the PEI column mimics the relative affinities for the mannose 6-phosphate receptor that traffics acid hydrolases to lysosomes and provides a useful example of protein sorting by biomimetic interaction chromatography.

Metabolism of endothelin-1 and big endothelin-1 by recombinant neutral endopeptidase EC.3.4.24.11.

1. Inhibitors of neutral endopeptidase EC.3.4.24.11 (NEP) have been shown to attenuate the hypertensive effect of big-endothelin-1 (BET-1) in rats. To determine whether NEP converts BET-1 to endothelin-1 (ET-1), the effect of a recombinant NEP (rNEP) on BET-1 and on ET-1 was assessed in vitro. 2. Incubation of [125I]-ET-1 with 1 microgram ml-1 of rNEP resulted in degradation of the peptide within minutes. Increase in the amount of rNEP to 10 micrograms ml-1 led to total cleavage of [125I]-ET-1 within seconds. 3. Phosphoramidon (10 microM) or SQ-28,603 (100 microM) totally suppressed the degradation of [125I]-ET-1 by rNEP. 4. The degradation of [125I]-BET-1 by either 1 or 10 micrograms ml-1 of rNEP was much slower than that of [125I]-ET-1. Again, both phosphoramidon and SQ 28,603 protected the peptide from degradation. 5. Intact [125I]-ET-1 was not observed when [125I]-BET-1 was incubated with rNEP. 6. These data show that neutral endopeptidase EC.3.4.24.11 is not an endothelin converting enzyme.

Administration of recombinant enkephalinase (neutral endopeptidase) prevents capsaicin-induced miosis in the rabbit eye in vivo.

Substance P induced a dose-dependent contraction of iris sphincter muscles when applied in the presence of atropine to the isolated rabbit iris in vitro as evidenced by a decreased pupil diameter. Pretreatment of the iris with 20 micrograms of recombinant enkephalinase (neutral endopeptidase; EC 3.4.24.11) totally abolished the contractile response to substance P. Injection of 10 micrograms of capsaicin into the anterior chamber of atropine-treated rabbit eyes in vivo induced an immediate and intense miosis. Injection of 100 micrograms of recombinant enkephalinase, 1 or 5 min before capsaicin injection, significantly inhibited this miosis. This effect of enkephalinase was totally abolished by preincubating the enzyme with thiorphan, a high-affinity enkephalinase inhibitor. These results show that enkephalinase, which is known to hydrolyze substance P in vitro with high efficiency, also hydrolyzes endogenously released substance P in vivo. Furthermore, our results suggest that enkephalinase application may represent a novel therapeutic approach to treat substance P-mediated pathologies.

Recombinant human enkephalinase (neutral endopeptidase) prevents cough induced by tachykinins in awake guinea pigs.

To determine whether recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11) prevents cough induced by exogenously applied and endogenously released neuropeptides, we measured cough responses to aerosolized solutions of substance P or of capsaicin for 2 min in random-source guinea pigs before or after exposing them to aerosolized recombinant human enkephalinase. Substance P (10(-16) M) increased coughing compared with its vehicle. Enkephalinase (120 micrograms) inhibited cough induced by subsequent exposure to substance P compared with the response to substance P alone, but after further exposure to the enkephalinase inhibitor leucine-thiorphan (10(-5) M), substance P increased cough significantly. Similar results were obtained for capsaicin-induced cough. In pathogen-free guinea pigs, after they inhaled inactive recombinant enkephalinase (33 micrograms), capsaicin (10(-13) M) increased cough significantly. In contrast, after they inhaled active recombinant enkephalinase (33 micrograms), capsaicin increased cough only slightly. These results suggest that aerosolized enkephalinase reaches the sites of release or actions of endogenous neuropeptides and, by degrading them, prevents cough induced by their release. Furthermore, these studies suggest that recombinant enkephalinase might be useful in the treatment of cough and other symptoms of diseases involving peptides cleaved by this enzyme.

Preferential cleavage of amino- and carboxyl-terminal oligopeptides from vasoactive intestinal polypeptide by human recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11).

Human recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11) cleaved synthetic vasoactive intestinal peptide (VIP1-28) with time-and peptidase concentration-dependence, which left less than 30% intact after 30 micrograms was incubated at 37 degrees C with 0.1 micrograms and 10 micrograms of peptidase for 120 min and 15 min, respectively. The rank order of relative rates of peptidolysis amino-terminal to hydrophobic amino acids was Ala4 and Val5 greater than Tyr22 and Ile26 much greater than Leu13 and Met17. The many effects of VIP1-28 on epithelial cell and leukocyte functions thus may be influenced by degradation of the mediator by enkephalinase at the surface of target cells.

Neuropsychologic symptoms in obstructive sleep apnea improve after treatment with nasal continuous positive airway pressure.

To describe the affective changes associated with sleep restoration we assessed psychologic symptoms using the Profile of Mood States questionnaire before and two months after treatment with nasal continuous positive airway pressure (NCPAP) in seven men with obstructive sleep apnea (OSA). The results were compared with those of a control group of patients with OSA who did not receive NCPAP. Two of six mood factors, depression and fatigue, improved significantly following treatment with NCPAP. Total Mood Disturbance (TMD) score was used to assess global mood differences. The mean TMD score for the patients before treatment was 1.7 and during treatment decreased to -7.6 (p less than 0.05). This mean decrease of 9.3 in the TMD score implies generalized improvement in mood. These findings support the opinion that sleep fragmentation and abnormalities of respiration during sleep are at least partially responsible for affective changes seen in sleep apnea. These psychologic disturbances improve after treatment with NCPAP.

Acute generalized myoclonus following buspirone administration.

The authors present a case of a 62-year-old woman who was hospitalized with severe medical problems that included congestive heart failure secondary to mitral stenosis and atrial fibrillation, coronary artery disease, chronic renal failure, and a recent history of a right cerebral lacunar infarction. She also had a 2-year history of anxiety and depression, manifested in the hospital by frequent crying spells, sleeplessness, and ruminating about her illnesses. The patient received buspirone 5 mg three times a day for her anxiety and depression. Approximately 12 hours after her first dose, she developed dramatic myoclonus, dystonias, and akathisia. She was given 25 mg of intramuscular diphenhydramine and 1 mg of intramuscular benztropine mesylate, which resulted in little relief; however, 1 mg clonazepam caused both the myoclonic jerks and dystonias to resolve completely.

2-[(4-Azido-2-nitrophenyl)amino]ethyl triphosphate, a novel chromophoric and photoaffinity analogue of ATP. Synthesis, characterization, and interaction with myosin subfragment 1.

A facile and high-yield synthesis of a new ATP analogue, 2-[(4-azido-2-nitrophenyl)amino]ethyl triphosphate (NANTP), is described. NANTP and ATP are hydrolyzed by skeletal myosin subfragment 1 (SF1) at comparable rates in the presence of Ca2+, Mg2+, or NH4+-EDTA. NANTP is also cleaved but less readily by mitochondrial F1-ATPase and by (Na+ + K+)-ATPase from dog brain and hog kidney. F-Actin markedly activates NANTP cleavage by SF1 in the presence of Mg2+, suggesting that the diphosphate product NANDP is slow to be released from the enzyme. [alpha-32P]NANDP binds to a single site on SF1 (KA = 1 X 10(6) M-1) with an affinity identical with that of ADP. The absorption maximum of NANDP was shifted from 474 to 467 nm upon binding to SF1, suggesting that the purine binding site has a dielectric constant of about 45. NANDP was trapped in nearly stoichiometric amounts at the active site by cross-linking SH1 and SH2 with N,N'-p-phenylenedimaleimide (pPDM) or by chelation with cobalt (III) phenanthroline [Wells, J., & Yount, R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966]. The trapped [beta-32P]NANDP X SF1 complex, like the comparable ADP X SF1 complex, was stable for days at 0 degree C and could be purified free of extraneous analogue by ammonium sulfate precipitation and gel filtration. Photolysis of the purified complex gave greater than 50% covalent incorporation of the trapped NANDP into the 95-kilodalton (kDa) heavy chain of SF1. Limited trypsinization and analysis by gel electrophoresis showed that greater than 95% of the bound label was associated with the 25-kDa NH2-terminal peptide. Without trapping, NANDP labeling of SF1 was nonspecific and was not prevented by addition of a large excess of ATP. This new approach of trapping photoaffinity analogues by cross-linking agents before photolysis may prove to be of general usefulness in increasing the specificity and extent of labeling of enzymes that undergo substrate-induced conformation changes.

Gizzard Ca2+-independent myosin light chain kinase: evidence in favor of the phosphorylation theory.

Limited digestion of calmodulin (CaM)-dependent myosin light chain kinase from turkey gizzard with alpha-chymotrypsin in the presence of bound CaM generated an 80,000-dalton kinase fragment that was fully active in the absence of Ca2+. This kinase catalyzed specific Ca2+-independent phosphorylation of the 20,000-dalton light chain of myosin using isolated light chains, intact myosin, and actomyosin. Phosphorylation of myosin in the absence of Ca2+ allowed us to dissociate myosin phosphorylation from other potential Ca2+-dependent regulatory mechanisms, thus permitting an evaluation of the postulated central role of myosin phosphorylation in the regulation of smooth muscle contraction. Ca2+-independent myosin phosphorylation was found to cause loss of Ca2+ sensitivity of 1) actin-activated myosin ATPase activity in a crude actomyosin preparation, and 2) tension development in skinned smooth muscle fibers in the absence of Ca2+. Myosin phosphorylation is, therefore, the key event in actin activation of ATPase activity and initiation of contraction in skinned chicken gizzard fibers.

Phosphorylation-dependent activated tension in skinned gizzard muscle fibers in the absence of Ca2+.

A Ca2+-insensitive myosin light chain kinase was prepared and used in experiments with skinned gizzard fibers. In the absence of Ca2+, this kinase activated isometric force development. The force development was associated with phosphorylation of the 20,000-dalton myosin light chains and addition of Ca2+ did not activate the fibers further. Pretreatment of the fibers with Ca2+-insensitive myosin light chain kinase and adenosine 5'-O-(3-thiotriphosphate) in the absence of Ca2+ caused thiophosphorylation of the light chains and, on the addition of ATP, an activation of isometric tension was observed. The subsequent addition of Ca2+ did not increase force development. A comparison of Ca2+-activated tension in the skinned gizzard muscle fibers with Ca2+-insensitive myosin light chain kinase-activated tension suggests that the phosphorylation of the myosin light chains is the dominant factor in the development of tension in smooth muscle.