Enzymatic and Radioimmunoassay Procedures Combined with Electrophoresis and HPLC for the Recovery and Characterization of Substance P in Human Brain

Immunoreactive substance P was recovered from human brain (hypothalamus and substantia nigra) by acetic acid extraction, ion exchange chromatography (SP-Sephadex), molecular sieving (Sephadex C-50) and column electrophoresis in agarose suspension. The chemical nature of the active material was further studied with various biochemical techniques including agarose suspension electrophoresis, HPLC and different kinds of enzyme radioimmunoassays. By combining these techniques it was possible to confirm structure identity between the recovered active component and substance P previously isolated from bovine brain. Thus, the major activity reacting with the substance P antibodies was indistinguishable from the synthetic bovine analogue in all chromatographic systems including analytical electrophoresis at different pH:s and HPLC. Furthermore, digestion of the active material with post-proline cleaving enzyme and trypsin yielded fragments identical with those expected from the bovine peptide as confirmed by specific radioimmunoassays in conjuction with electrophoresis or HPLC. The result also indicates the usefulness of the present procedures for identifying peptides structures available only in minute amounts.     

Isoforms of a dynorphin B converting enzyme isolated by column electrophoresis in agarose suspension.

A dynorphin B converting enzyme previously purified from bovine spinal cord was subjected to column electrophoresis in agarose suspension. By this technique combined with HPLC gel filtration it was possible to resolve and recover several isoforms of the proteinase. All these isoenzymes were associated with a similar molecular size but apparently they differed with regard to their net charges. No significant difference between their inhibitory profiles or their Km values for the release of Leu-enkephalin-Arg6 from dynorphin B was observed.     

Purifcation and properties of multiple forms of brain acetylcholinesterase (EC 3.1.1.7)

—Approximately 70 per cent of the total AChE of bovine brain tissue was solubilized by repeated homogenization and centrifugation in 0.32 m sucrose containing EDTA. After ammonium sulphate fractionation, application of the enzyme preparation to an agarose affinity gel column effected a 700-fold purification. Subsequent molecular filtration separated three active forms of AChE with molecular weights of 130,000, 270,000 and 390,000 with an average specific activity of 575 mmol of acetylthiocholine hydrolysed/mg of protein/h. The complete procedure represented an approximate 23,000-fold purification of the enzyme from that in the original tissue homogenate. The three forms of AChE exhibited certain differences in properties, including apparent K_m values, pH optima and sensitivity to inhibitory agents. Ancillary studies on less purified enzyme preparations by use of polyacrylamide gel electrophoresis and isoelectric focusing techniques also suggested that brain AChE exists in multiple forms.     

Measurement of Substance P Metabolites in Rat CNS

A procedure based on ion-exchange chromatography for chemical separation and radioimmunoassays for quantitation of substance P (SP), the SP(1-7), and C-terminal fragments, respectively, has been developed. The procedure allows the determination of these fragments in the presence of large (i.e., 50- to 100-fold) excess of parent compound. The chemical identity of isolated SP and fragments was studied with preparative electrophoresis on dilute agarose gel and with HPLC. The activity identified as SP(1-7) comigrated with the authentic standard whereas practically all activity isolated as C-terminal fragments comigrated with SP(5-11). The levels of C-terminal fragments in rat brain areas rich in SP and in spinal cord were 1-2% of those of parent compound. The levels of SP(1-7) were always higher, in the spinal cord markedly higher (three to five times). Postmortem storage of samples from brain and spinal cord indicated that SP(1-7) levels fell more rapidly than those of SP or C-terminal fragments.     

Electrophoresis of opioid peptides on columns with agarose suspension

Opioid peptides have been fractionated by column electrophoresis in agarose suspension. The procedure provides a high resolution efficiency and compares favorably with high-performance liquid chromatography. Peptides were recovered from the column in 70% or higher yields when run in the femtomole to the nanomole range. By choosing different pHs it is possible to get complete resolution of a number of different endorphins. The electrophoreses were performed in volatile buffers, which are easily evaporated, and the residues are suitable for direct screening by radioreceptor- or radioimmunoassays. In combination with these assays the separation procedure makes it possible to detect and identify very low concentrations of endorphins in samples of biological origin.     

Demonstration and Distribution of Kassinin-Like Material (Substance K) in the Rat Central Nervous System

Antiserum was raised against kassinin in rabbits. Cross-reactivity with other tachykinins was determined; these included substance K (100%) and substance P (0.1%). Peptides extracted from rat brain and synthetic tachykinins were chromatographed by reverse-phase HPLC. The major peak of kassinin-like material eluted at a time different from that of synthetic kassinin, eledoisin, physalaemin, neurokinin beta, and substance P but coeluted with substance K. Measurement of kassinin-like material in macrodissected and microdissected brain regions indicated that the distribution of kassinin-like material was similar to that of substance P.     

Purification and characterization of a substance P-degrading endopeptidase from rat brain

A novel substance P-degrading endopeptidase has been solubilized with Brij 35 from a membrane fraction of rat brain and purified by a procedure involving DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G-100 gel filtration, and Mono-Q HPLC. The activity of the degrading enzyme was monitored by measuring the disappearance of substance P by means of a bioassay and HPLC. SDS-polyacrylamide gel electrophoresis under reducing conditions of the enzyme gave a single band corresponding to a molecular weight of 58,000. The molecular weight of the enzyme was estimated to be 55,000 by gel filtration and the optimum pH for its activity was 7.5.. The purified enzyme cleaved substance P at three bonds, Pro4-Gln5, Gln5-Gln6, and Gln6-Phe7, in the ratio of 2:2:3. EDTA, o-phenanthroline, and p-chloromercuribenzenesulfonic acid strongly inhibited the enzyme, while diisopropyl fluorophosphate, E-64, Z-Gly-ProCH2Cl, phosphoramidon, and captopril had little or no inhibitory effect on it. The cleavage of substance P by the rat brain synaptic membrane was also analyzed under the conditions with or without these inhibitors. The inhibitor-susceptibility of the cleavage sites suggests that the present enzyme, together with endopeptidase-24.11, is involved in the degradation of substance P in the synaptic region.     

Soluble dipeptidyl peptidase IV from terminal differentiated rat epidermal cells: purification and its activity on synthetic and natural peptides

In terminally differentiated epidermal cells dipeptidyl peptidase IV (EC 3.4.14.5) (DPP IV) is present mainly in a soluble form. We purified the enzyme from 2-day-old rat cornified cells to homogeneity by Sephadex G-200 and Mono-Q column chromatography and finally HPLC gel filtration on G3000SW. The enzyme was estimated to be Mr 190,000 by HPLC gel filtration and Mr 90,000 by sodium dodecyl sulfate-electrophoresis. The enzyme showed general properties reported for detergent-solubilized DPP IV from other tissues. It was Con A binding and almost completely inhibited by 1 mM diisopropyl fluorophosphate and Diprotin A. The pI was 5.6 and the pH optimum was 7.5. The specific activity for Gly-Pro-p-nitroanilide was 31.9 units/mg. HPLC analysis demonstrated the release of dipeptides of the N-terminal of substance P, beta-casomorphin, and their related peptides. A stoichiometric reaction of the enzyme on substance P was observed. The epidermal DPP IV had a Km of 0.3 mM and a kcat of 50.3 s-1 for substance P and the Km value decreased by shortening the peptide from the carboxyl-terminal amino acids. The enzyme hydrolyzed human and bovine beta-casomorphin with Km values of 0.025 and 0.05 mM, respectively. Shortening the bovine beta-casomorphin peptide chain did not affect enzyme affinity.     

Cation-sensitive neutral endopeptidase: isolation and specificity of the bovine pituitary enzyme

A highly purified preparation of a cation-sensitive neutral endopeptidase was obtained from bovine pituitaries. The enzyme constitutes almost 0.1% of the protein in bovine pituitary homogenates. Polyacrylamide gel electrophoresis of the enzyme showed a single protein band, and in gel filtration experiments on calibrated Sepharose 6B columns the enzyme eluted slightly ahead of thyroglobulin, suggesting an apparent molecular weight of about 700,000. Polyacrylamide gel electrophoresis in SDS-containing buffers indicated the presence of three major components with molecular weights ranging from about 24,000 to 28,000. The enzyme hydrolyzes bonds between hydrophobic and small neutral amino acids in both model synthetic substrates and biologically active peptides such as substance P, LH-RH, and bradykinin. Peptide bonds in which the carbonyl group is contributed by a glutamyl or arginyl residue are also hydrolyzed, especially if they are preceded in the sequence by hydrophobic amino acids. Leupeptin exclusively inhibited enzymatic activity toward the arginine-containing substrates. This observation, together with the high molecular weight and broad specificity of the enzyme, raised the possibility that the isolated enzyme represents a proteolytic complex composed of units with distinctly different activities. Preliminary attempts to dissociate the enzyme into catalytic units of lower molecular weight were not successful and led to loss of activity.     

Post-proline cleaving enzyme (prolyl endopeptidase) from bovine brain

A post-proline cleaving enzyme [prolyl endopeptidase, EC 3.4.21.26] was purified about 3,700-fold from an extract of bovine brain by a series of column chromatographies on DEAE-Sephadex, hydroxyapatite and PCMB-T-Sepharose, and gel filtration on Sephadex G-200 using N-carbobenzoxy-Gly-Pro-beta-naphthylamide (Z-Gly-Pro-2-NNap), thyrotropin releasing hormone (TRH) and oxytocin as substrates. The purified enzyme appeared homogeneous as judged by disc gel and SDS gel electrophoreses. The enzyme was most active at pH 7.5 and 7.2 with Z-Gly-Pro-2-NNap and TRH, respectively, and hydrolyzed peptide bonds involving Pro-X (X=amino acid, peptide, ester and amide) bonds of synthetic substrates, oxytocin, vasopressin, neurotensin, substance P, tuftsin, bradykinin, and insulin B chain. However, the enzyme was inert toward collagen, gelatin, and casein. The enzyme was completely inactivated by diisopropylphosphorofluoridate (DFP), Z-Gly-Pro-chloromethyl ketone and p-chloromercuribenzoate (PCMB), while it was not inhibited by phenylmethane sulfonylfluoride (PMSF) or metal chelators. Determination of the amino acid composition revealed that the enzyme contained 25 half-cystines. Modification of three cysteine residues of the enzyme by PCMB led to complete inactivation. The isoelectric point of the enzyme was 4.8, and the molecular weight was estimated to be 76,000 by ultracentrifugal analysis and 75,000-74,000 by both gel filtration and sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme is present as a monomer. These results indicate that the post-proline cleaving enzyme from bovine brain is very similar to those previously purified from lamb brain and kidney in its enzymatic properties, substrate specificity and physicochemical properties, in sharp contrast with the results obtained by Tate, who reported that the bovine brain prolyl endopeptidase was inert toward oxytocin, vasopressin and bradykinin.     

Purification of substance P endopeptidase (SPE) activity in human spinal cord and subsequent comparative studies with SPE in cerebrospinal fluid and with chymotrypsin

An enzyme activity capable of hydrolysing the neuroactive undecapeptide substance P (SP) between its Phe7-Phe8 residues was purified from the membrane-bound fraction of human spinal cords. The enzyme preparation yielded was compared with a previously described SP-hydrolysing enzyme from human cerebrospinal fluid (CSF) with regard to inhibition profile, protein chemical properties and kinetics. In addition, the results were compared with those of bovine pancreatic chymotrypsin (a serine protease that cleaves the carboxy-terminal side preferentially at hydrophobic amino acids). The SP peptidase activity was extracted from human spinal cords with 1% Triton X-100 in 20 mM Tris-HCI pH 7.8. After ion exchange chromatography (DEAE-Sepharose) where the enzyme activity was separated from other proteins by gradient elution, the pooled enzyme fraction was further purified by molecular sieving (Sephadex G-50). The enzyme activity was finally recovered by HPLC molecular sieving (Superdex 75 HR 10/30) using a new preparative system, AKTA-purifier, controlled by UNICORN software version 2.20.     

Solubilization of tryptophan-5-monooxygenase from the pineal glands and existance of an activating substance in the tissue extract

A particle bound tryptophan-5-monooxygenase in bovine pineal glands was solubilized by a combination of high pH (PH 8.5), high salt concentration (0.5 M-KCl or NaCl), and sonication (20 kHz, 200 W, 30min), We recovered 75% or more of the monooxygenase activity of the tissue homogenate in the 105,000 g supernatant thus confirming our previous study (Hori et al., 1976). The solubilized enzyme preparation contained an activating substance which was also particle bound and which activated the monooxygenase when preincubated together with the enzyme and dithiothreitol. The solubilized tryptophan-5-monooxygenase was purified about 6-fold over the tissue homogenate using ammonium sulphate fractionation and column chromatography on hydroxylapatite and Sephacryl S-200. The activating substance was separated from the monooxygenase during hydroxylapatite column chromatography. The activating substance was shown to be different from phospholipids, such as L-α-lysophosphatidyl choline and L-α-phosphatidyl-L-serine, and from heparin or bovine serum albumin, although bovine serum albumin significantly activated the monooxygenase. Further experiments have suggested that both the monooxygenase and the activating substance are modified by dithiothreitol and that these modified materials interact to give the active form of the monooxygenase.     

Complete purification and immunochemical analysis of S-adenosylmethionine synthetase from bovine brain

We have purified S-adenosylmethionine (AdoMet) synthetase about 3000-fold from bovine brain extract. The Km values of the enzyme for L-methionine and ATP were 10 and 50 microM, respectively. An apparent molecular mass of the enzyme was estimated to be 160 kDa by gel filtration on a Sephacryl S-200 column. Sucrose density gradient centrifugation gave a sedimentation coefficient of 8 S. Polyacrylamide gel electrophoresis of the purified enzyme in native system revealed a single protein band, whereas two polypeptide bands with molecular masses of 48 kDa (p48) and 38 kDa (p38) were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme. Antibody against bovine brain AdoMet synthetase was prepared by injecting the purified enzyme into a rabbit. Immunoblot analysis revealed that the antibody recognized both p48 and p38 in the impure enzyme preparations from bovine brain as well as in the purified enzyme. Specific antibodies against p48 and p38 were separated from the immunoglobulin fraction by an affinity purification, both of which inhibited the enzyme activity. These results indicate that AdoMet synthetase from bovine brain consists of two different polypeptides, p48 and p38.     

Immunoreactive Met-enkephalin Arg6 in rat brain, and bovine brain, gut, and adrenal

Antibodies directed against the Met-enkephalin-related hexapeptide, Met-enk Arg6, have been used in radioimmunoassays in the characterization of material in rat brain, and bovine striatum, colon, and adrenal medulla. Met-enk Lys6 reacted 0.27 relative to Met-enk Arg6, but Leu-enk Arg6 and C-terminal extensions or deletions of Met-enk Arg6 showed less than 0.02 immunoreactivity. In rat brain, the concentration of Met-enk Arg6-like immunoreactivity was less than 20 pmol X g-1 in all regions, but after trypsinization of tissue extracts there were up to 80-fold increases in immunoreactivity as a result of cleavage of C-terminally extended forms. The tryptic product eluted as Met-enk Arg6 on gel filtration. In control extracts of rat brain there were at least three immunoreactive forms of Met-enk Arg6; one eluted in the position of the hexapeptide standard on gel filtration and HPLC while the others had properties of N-terminally extended forms. In bovine striatum and colon the hexapeptide-like material predominated; but in bovine adrenal extracts, there were relatively low concentrations of the hexapeptide and, instead, the dominant immunoreactive forms corresponded to two components that were probably N-terminally extended variants. Trypsin again produced marked increases in immunoreactivity. HPLC studies indicated that Met-enk Arg6Phe7- and Met-enk Arg6Gly7Leu8-like immunoreactive peptides were important substrates in bovine brain for the production of hexapeptide immunoreactivity after trypsin. The differences in the patterns of immunoreactive forms in bovine adrenal, colon, and brain are consistent with tissue variations in the pathways of posttranslational processing of the precursor molecules.     

Purification of an endopeptidase from bovine adrenal medulla granules which cleaves in vitro at paired but not at single basic residues

An endopeptidase was isolated from bovine adrenomedullary granules by fast protein liquid chromatography, including two ion exchange, one hydrophobic interaction, and one gel filtration step. The enzyme assay was based on the HPLC detection of the degradation of the dodecapeptide BAM 12P from the sequence of proenkephalin. After a 1200-fold purification, the enzyme fraction was homogeneous on polyacrylamide gel electrophoresis. The apparent molecular weight of the monomeric protein was 68,000 and its pH optimum was 5.6, in agreement with the internal pH of the granules. The specificity of the protease was determined initially by analysis of the degradation fragments of BAM 12P which showed that cleavage occurred at the double but not at the single Arg site of BAM 12P. The cleavage pattern of other substrates showed that the enzyme also recognized other pairs of basic amino acids. The behavior of the enzyme toward specific inhibitors showed that it was an acid thiol protease different from serine proteases and from lysosomal cathepsin B. The endopeptidase may act as a maturation enzyme in vivo.     

Partial reconstitution of human RNase P in HeLa cells between its RNA subunit with an affinity tag and the intact protein components

An RNA affinity tag was incorporated into the RNA subunit of human nuclear RNase P. The tagged RNA assembled with the protein components of RNase P inside HeLa cells to generate an active enzyme. Because of the specificity of the RNA tag to streptavidin, the reconstituted complex could be separated from the native enzyme and other ribonucleoproteins (particularly RNase MRP) by streptavidin agarose chromatography and could be recovered by the eluting agent, biotin. A mutant, tagged RNase P RNA, whose P3 domain was partially replaced, could not reconstitute with the proteins to yield an active enzyme. The P3 domain, therefore, is critical for the structure and function of RNase P.     

An approach for the stable immobilization of proteins

An approach is presented for the stable covalent immobilization of proteins with a high retention of biological activity. First, chemical modification studies were used to establish enzyme structural and functional properties relevant to the covalent immobilization of an enzyme to agarose based supports. Heparinase was used as a model enzyme in this set of studies. Amine modifications result in 75-100% activity loss, but the effect is moderated by a reduction in the degree of derivatization. N-hydroxysuccinimide, 1,1,1-trifluoroethanesulfonic acid, and epoxide activated agarose were utilized to determine the effect of amine reactive supports on immobilized enzyme activity retention. Cysteine modifications resulted in 25-50% loss in activity, but free cysteines were inaccessible to either immobilized bromoacetyl or p-chloromercuribenzoyl groups. Amine reactive coupling chemistries were therefore utilized for the covalent immobilization of heparinase. Second, to ensure maximal stability of the immobile protein-support linkage, the identification and subsequent elimination of the principal sources of protein detachment were systematically investigated. By using high-performance liquid chromatography (HPLC), electrophoresis, and radiolabeling techniques, the relative contributions of four potential detachment mechanisms-support degradation, proteolytic degradation, desorption of noncovalently bound protein, and bond solvolysis-were quantified. The mechanisms of lysozyme, bovine serum albumin, and heparinase leakage from N-hydroxysuccinimide or 1,1,1-trifluoroethanesulfonic acid activated agarose were elucidated. By use of stringent postimmobilization support wash procedures, noncovalently bound protein loss. An effective postimmobilization washing procedure is presented for the removal of adsorbed protein and the complete elimination of immobilized protein loss.     

Presence and Ontogeny of Enkephalin and Substance P in the Chick Ciliary Ganglion

The avian ciliary ganglion has been reported to contain both enkephalin and substance P in preganglionic terminals. However, extensive biochemical characterization of these antigens has not been completed. Using radioimmunoassays specific for Met5- and for Leu5-enkephalin and for substance P we identified immunoreactive substances in ganglionic extracts that comigrate on HPLC columns with standard Met5- and Leu5-enkephalin and with substance P. The ontogeny of Met5-enkephalin and substance P during embryogenesis was determined in ganglionic extracts and we found that the content of Met5-enkephalin in the ganglion reached a peak at embryonic stage 37 whereas the content of substance P in the ganglion reached its maximum in the adult.     

Endorphins in human cerebrospinal fluid

Opiate activity in CSF samples drawn from patients with suspected intracranial hydrodynamic dysfunction has been fractionated on Sephadex G-10 and separated by column electrophoresis in agarose suspension. From the Sephadex G-10 chromatography two receptor active fractions (FI and FII) were recovered. Both FI and FII were further resolved by the electrophoresis. FI separated into at least four components and FII into two components. The study also includes a comparison of the endorphin concentrations in CSF (samples drawn from healthy volunteers) measured by receptorassay with those detected by radioimmunoassay of beta-endorphin, [Met]enkephalin and dynorphin, respectively. The data obtained indicated negligible quantities of the radioimmunoassayable endorphins in the total CSF opiate activity.     

Identification and Characterization of Multiple Tachykinin Immunoreactivities in Bovine Retina: Evidence for the Presence of a Putative Oxidative Inactivation System for Substance P

Tachykinin immunoreactivity has been quantified and characterized in extracts of bovine retinae by combining radioimmunoassay, gel permeation chromatography, and reverse-phase HPLC. Using an antiserum specific for the C-terminal hexapeptide amide of substance P, levels of 3.43 +/- 0.33 ng g-1 and 12.45 +/- 0.76 ng g-1 (mean +/- SD, n = 5) were measured in extracts prepared by acidified ethanol and boiling 0.5 M acetic acid, respectively. Levels of neurokinin A immunoreactivity, assayed using an antiserum cross-reacting with neurokinin A (100%), neurokinin B (50%), neuropeptide K (85%), and substance P (less than 0.1%) were 12.46 +/- 0.47 ng g-1 and 7.20 +/- 0.37 ng g-1 in the same extracts. Gel permeation chromatography identified a single substance P immunoreactant eluting with substance P standard, whereas two neurokinin A immunoreactants were resolved eluting with neuropeptide K and neurokinin A standards. Reverse-phase HPLC analysis resolved immunoreactivity eluting with substance P, neurokinin A, neuropeptide K, and neurokinin B and their respective methionine sulphoxides. The amount of immunoreactive material co-eluting with the respective sulphoxides was higher in acidified ethanol extracts, and substance P was most susceptible to oxidative modification. Subsequent incubation of synthetic substance P with dispersed bovine retinal cells resulted in rapid conversion to three metabolites identified and isolated by reverse-phase HPLC. Each had an amino acid composition identical to that of substance P, and the major product had the same retention time as substance P sulphoxide.(ABSTRACT TRUNCATED AT 250 WORDS)