The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum.

1. Liver and bone alkaline phosphatase isoenzymes were solubilized with the zwitterionic detergent sulphobetaine 14, and purified to homogeneity by using a monoclonal antibody previously raised against a partially-purified preparation of the liver isoenzyme. Both purified isoenzymes had a specific activity in the range 1100-1400 mumol/min per mg of protein with a subunit Mr of 80,000 determined by SDS/polyacrylamide gel electrophoresis. Butanol extraction instead of detergent solubilization, before immunoaffinity purification of the liver enzyme, resulted in the same specific activity and subunit Mr. The native Mr of the sulphobetaine 14-solubilized enzyme was consistent with the enzyme being a dimer of two identical subunits and was higher than that of the butanol-extracted enzyme, presumably due to the binding of the detergent micelle. 2. Pure bone and liver alkaline phosphatase were used to raise further antibodies to the two isoenzymes. Altogether, 27 antibody-producing cell lines were cloned from 12 mice. Several of these antibodies showed a greater than 2-fold preference for bone alkaline phosphatase in the binding assay used for screening. No antibodies showing a preference for liver alkaline phosphatase were successfully cloned. None of the antibodies showed significant cross-reaction with placental or intestinal alkaline phosphatase. Epitope analysis of the 27 antibodies using liver alkaline phosphatase as antigen gave rise to six groupings, with four antibodies unclassified. The six major epitope groups were also observed using bone alkaline phosphatase as antigen. 3. Serum from patients with cholestasis contains soluble and particulate forms of alkaline phosphatase. The soluble serum enzyme had the same size and charge as butanol-extracted liver enzyme on native polyacrylamide-gel electrophoresis. Cellulose acetate electrophoresis separated the soluble and particulate serum alkaline phosphatases as slow- and fast-moving forms respectively. In the presence of sulphobetaine 14 all the serum enzyme migrated as the slow-moving form on cellulose acetate electrophoresis. Monoclonal anti-(alkaline phosphatase) immunoadsorbents did not bind the particulate form of alkaline phosphatase in cholestatic serum but bound the soluble form. In the presence of sulphobetaine 14 all the cholestatic serum alkaline phosphatase bound to the immunoadsorbents. 4. The electrophoretic and immunological data are consistent with both particulate and soluble forms of alkaline phosphatase in cholestatic serum being derived from the hepatocyte membrane.     

Interaction of glucocorticoid receptor from rat liver with protamine and arginine

The nontransformed glucocorticoid receptor (GR) from rat liver was found to bind to protamine-Sepharose and could be recovered by a salt gradient without a change in molecular configuration. The nontransformed GR also bound to arginine-Sepharose, but the transformed GR did not bind to either resin. Ligand-free GR interacted with both resins and was eluted without loss of its steroid binding ability. The bindings of GR to protamine- and arginine-Sepharose were saturable. The apparent dissociation constants of GR on protamine-Sepharose varied from 0.34 nM (−molybdate) to 0.68 nM (+10 mM molybdate) and those on arginine-Sepharose were 1.99 nM (− molybdate) and 0.65 nM (+ 10 mM molybdate), respectively. The maximum binding capacity was achieved by arginine-Sepharose in the absence of molybdate. Higher salt concentrations (0.5 M NaCl) were required to elute GR from protamine-Sepharose than from arginine-Sepharose (approx 0.03 M NaCl). However, the effectiveness of several salts for the elution of GR was consistent in both resins as follows; MgCl₂ = CaCl₂ = Na₂WO₄ > (NH₄)₂SO₄ = Na₂MoO₄ > arginine-HCl > lysine-HCl > KCl = NaCl. These results suggest that GR interacts with arginine residues in protamine. Chromatography using these resins resulted in 7–10-fold purification of occupied and unoccupied nontransformed GRs.     

The Effects of Detergents on the Composition of Postsynaptic Densities

A method of purifying postsynaptic densities (PSD) of Cohen et al. (1977) has been modified, primarily by the substitution of octyl glucoside as the detergent used to solubilize synaptosomal fractions. Subsequent extraction with other detergents resulted in the selective removal of specific polypeptides. In particular sulphobetaine 3-14 removed most of the beta-tubulin but not alpha-tubulin. Sodium N-lauroyl sarcosinate completely destroyed the structural integrity of the PSD when the in vitro formation of intermolecular disulphide bonds was minimized. These results suggest that the structure of PSDs is more labile than previously thought and demonstrate a technique for further examining their composition.     

Purification and characterization of a multicatalytic proteinase from crustacean muscle: comparison of latent and heat-activated forms

A high-molecular-weight (Mr 740,000) multicatalytic proteinase (MCP) was purified over 3100-fold from soluble extracts of lobster claw and abdominal muscles. The enzyme was extracted from muscle in a latent state; brief (3 min) heating of an ammonium sulfate fraction (45-65% saturation) at 60 degrees C irreversibly activated the proteinase while denaturing about 55% of the protein. MCP was further purified by chromatography on two sequential arginine-Sepharose columns and a Mono Q column with a yield of 60%. About 1.12 mg MCP was obtained for every 100 g tissue. In addition to [14C]methylcasein, the MCP hydrolyzed synthetic peptide substrates of trypsin and chymotrypsin at pH 7.75. Serine protease inhibitors (diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, aprotinin, benzamidine, soybean trypsin inhibitor, chloromethyl ketones), leupeptin, antipain, hemin, sulfhydryl-blocking reagents (N-ethylmaleimide, mersalyl acid, p-chloromercurisulfonic acid, iodoacetamide) suppressed activity while Ep-475, a specific inhibitor of cysteine proteinases, had no effect, suggesting the MCP is a serine proteinase with one or more cysteine residues indirectly involved in catalysis. The latent MCP was purified using the same procedure as that for the active form, except that thermal activation was omitted. The elution characteristics of latent MCP from the arginine-Sepharose and Mono Q columns were identical to those of active MCP. Since the purified latent form could still be activated by heating, activation did not involve denaturation of an endogenous inhibitor or substrate. Subunit compositions of both forms were identical in two-dimensional polyacrylamide gels; each was composed of eight polypeptides with molecular weights between 25,000 and 32,500 and a ninth polypeptide with a molecular weight of 41,000. Electron microscopy of negatively stained material showed that each form was a cylinder-shaped particle (approximately 10 x 15 nm) consisting of a stack of four rings with a hollow center; no differences in shape, dimensions, or submolecular structure were observed. These results suggest that activation probably involved small conformational changes rather than covalent modifications or rearrangement of subunits within the complex.     

A carnitine/acylcarnitine translocase assay applicable to biopsied muscle specimens without requiring mitochondrial isolation.

A simple method for assaying the mitochondrial carnitine/acylcarnitine translocase of muscles that needs only few milligrams of fresh tissue is described. The procedure involves monitoring of the sulphobetaine (an inhibitor of the translocase)-sensitive acetylation of sub-saturating concentrations of carnitine in the medium, linked to the oxidation of [2-14C]pyruvate in the presence of malonate. Conditions affecting the reliability of the outlined procedure and the ancillary information to be collected, namely the activities of pyruvate oxidase system and carnitine acetyltransferase, for detecting possible deficiency of the translocase are described, together with data on the translocase activity in human skeletal muscle, in rat red and white skeletal muscles and in rat heart. The concepts outlined should allow development of assays of other mitochondrial transporters that also would require neither isolation of mitochondria nor availability of a large quantity of tissue, both of which are otherwise needed at present.     

Purification of glandular kallikrein in maxillary mucosa from humans suffering from chronic inflammation

Glandular kallikrein was purified from human maxillary mucosa with chronic inflammation and its biochemical properties were characterized. The purification procedure consisted of extraction with 3 mol/l KCl, saturation of ammonium sulfate (66%), DEAE-Sepharose, arginine-Sepharose and Sephadex G-150 chromatographies. Maxillary mucosa with chronic inflammation contains considerable activity of glandular kallikrein, which is a serine protease with limited proteolytic activity and its biochemical properties resemble those of pancreatic kallikrein.     

Role of polycationic C-terminal portion in the structure and activity of recombinant human interferon-gamma

Purified recombinant human interferon-gamma, produced in Escherichia coli, was digested with trypsin under mild conditions, resulting in a preparation containing approximately 90% of a Mr = 15,800 protein and 10% of a 14,400 protein. The Mr = 15,800 protein has an intact N terminus and the Mr = 14,400 protein lacks 14 N-terminal residues. Both proteins lack C terminus of approximately 13 residues. This preparation containing the Mr = 15,800 and 14,400 proteins was identical with the intact protein with respect to conformation and dimerization, as analyzed by circular dichroism and gel filtration. However, the antiviral activity of this preparation was 1000-fold lower than that of the intact molecule. Since the majority of this preparation is the Mr = 15,800 protein, these results suggest that the C terminus does not affect the protein conformation and self-association, but greatly alters antiviral activity.     

Purification and characterization of a new arginine carboxypeptidase in human serum

A carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates is present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its unstability at room temperature and at 37 degrees C, we named it unstable carboxypeptidase (carboxypeptidase U). Carboxypeptidase U was partially purified from fresh human serum by chromatography on DEAE-cellulose and Mono-Q sepharose and was found to be a 435 kDa protein. We compared this enzyme with carboxypeptidase N, purified from human serum by a two-step affinity chromatography on arginine-Sepharose 4B, followed by ion-exchange chromatography on Mono-Q sepharose. Carboxypeptidase U cleaves hippuryl-L-arginine and hippuryl-L-lysine, but at a different relative rate than carboxypeptidase N, and has no esterase activity on hippuryl-L-argininic acid. Its activity was inhibited by o-phenanthroline, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, CoCl2, 2-mercaptoethanol, dithiothreitol and 4-chloromercuribenzoic acid. These characteristics differentiate carboxypeptidase U from carboxypeptidase N and other known carboxypeptidases.     

Proteins in Intercellular Washing Fluids from Leaves of Barley (Hordeum vulgare L.)

Primary leaves of barley were detached, infiltrated with variousbuffers, and centrifuged to yield ‘intercellular washingfluid’ (IWF). Effective pH control of the IWF was obtainedonly with Tris, among all buffers tried. In these liquids, upto 30 proteins were detected by gradient gel electrophoresis.Intracellular protein from injured cells at the cut ends ofleaves was present in IWF but did not contribute significantlyto the total protein recovered in this liquid. The yield ofprotein in the IWF depended on the buffer used for infiltrationand on the concentration of the buffer. Higher concentrationsof buffer yielded more protein. In other experiments leaves were infiltrated with Tris, centrifuged,and then infiltrated a second time with this buffer containingvarious concentrations of the zwitterionic detergent CHAPS,a sulphobetaine derivative of cholate. Gel electrophoresis ofthe IWF obtained after the second centrifugation revealed protein‘bands’ not detected when the detergent had beenomitted from the infiltration buffer. The electrophoretic patternsof protein ‘bands’ in the gels differed dependingon the CHAPS concentration used for infiltration. The effect of CHAPS on plasmalemma integrity was studied byobserving infiltrated tissue with the electron microscope andby treating isolated protoplasts with the detergent. After infiltrationwith CHAPS at 0.6 mM or 2.0 mM no plasmalemma breaks were detectedin leaves, and isolated protoplasts survived exposure to CHAPSat these concentrations for 2 h without bursting. Evidently,CHAPS at these low concentrations did not destroy the integrityof the plasmalemma; the additional protein recovered in theIWF under these conditions probably originated in the cell wall.Infiltration of leaves with 6.0 mM CHAPS resulted in breaksof the plasmalemma, in tissue collapse and leaf tip necrosis.Isolated protoplasts burst within minutes after being exposedto CHAPS at this concentration. Key words: Cell wall permeability, Intercellular space, Detergent, CHAPS, Protoplasts     

Biosynthesis of glycogen in Neurospora crassa. Existence of a glucoproteic intermediate in the initiation process.

A soluble enzyme preparation (20,000 X g supernatant fraction), prepared from the mycelia of wild-type Neurospora crassa, was capable of transferring [14C]glucose from UDP-[14C]glucose into both trichloroacetic acid (TCA)-soluble and TCA-insoluble macromolecule products in the absence of added primer. These reactions did not require either high concentrations of salts or any other chemical reagents. Two labeled products were formed; one was a glycogen-like polysaccharide and the other was a glycoprotein with glucosyl chains bound to protein through an acid-labile bond. After mild treatment of the glucoprotein with acid, the product liberated from the protein behaved as a mixture of malto-oligosaccharides and alpha-1,4-glucan with branches. The carbohydrate moiety of the glucoprotein seemed to be released upon prolonged incubation with the enzyme preparation. The glucan thus liberated from the glucoprotein may serve as a primer for the glycogen synthase. The results obtained are therefore suggestive of the existence of a glucoproteic intermediate in the initiation of glycogen biosynthesis.     

Polyamine metabolism in McCoy cells: II. Cellular origin of excreted polyamine conjugated proteins

Incubation of McCoy cultures with medium containing 14C-putrescine resulted in the incorporation of 14C-polyamine into intracellular proteins. A greater than 100,000 dalton 14C-polyamine conjugated protein was present in the McCoy cell lysate supernatant (CLSP). CLSP was heterogeneous containing proteins with pIs ranging from 4.55 to 5.50. The major proteins had pIs of 4.55 and 5.20. Electrophoresis of solubilized McCoy cell lysate pellet revealed a 14C-polyamine conjugated protein peak with Mr approximately or equal to 70,000 (CLPP). Both CLSP and CLPP contained bound polyamine. The major CLSP polyamine was spermidine while spermine exceeded the other two polyamines (putrescine and spermidine) in CLPP. About 25% of the polyamines associated with CLSP and CLPP were covalently bound with the exception of CLSP putrescine where 62.1% was covalently bound. Results suggested the presence of a polyamine protein isopeptide bond in CLSP. Sephadex gel filtration of cultured medium resulted in the identification of two macromolecular polyamine-containing fractions (MP1, Mr greater than 100,000 and MP2, M = 60,000-70,000). Antibody raised in rabbits against a membrane-organelle preparation cross-reacted with Sephadex gel filtration derived MP1 but not with peak MP2 suggesting that MP1 but not MP2 might be a membrane constituent. Antibody raised against medium polyamine conjugated protein peak 2 (MP2) cross-reacted with the cell lysate supernatant indicating that MP2 was present in the cytosol. It did not cross-react with the cell lysate pellet preparation. Antibody against MP2 also formed a precipitation band with MP1 indicating that there might be a common antigenic site on MP1 and MP2.     

Axoplasmic transport of a brain-specific soluble protein

The rate and extent of axoplasmic transport of the brain-specific soluble protein (14-3-2 protein) has been investigated in the avian visual system. 1-day-old chicks were injected monocularly with tritiated proline. Incorporation of the isotope into the 14-3-2 protein synthetized within the retina of the injected eye, as well as the appearance of the labeled protein in the optic lobes was determined at 6 h and 6 days. These time periods were chosen to distinguish between the rapid and slow phases of axoplasmic flow. Following preparation of high-speed supernatant fractions, dialysis, chromatography on Sephadex G-150 and immunoprecipitation with specific antiserum, identification of the labeled 14-3-2 protein was carried out by sodium dodecylsulfate-polyacrylamide gel analysis of the radioactive immunoprecipitates. 6 days after isotope administration, approx. 8% of the 14-3-2 protein synthesized in the chick retina had been transported to the contralateral optic lobe. By contrast, at 6 h no labeled 14-3-2 protein was detectable. Thus, transport of this neuronal protein appears to be a relatively slow process with little or no rapid component.     

Production and preliminary characterization of monoclonal antibodies to rat plasma fibronectin

We have produced several monoclonal antibodies which appear to be directed against different antigenic determinants of rat plasma fibronectin. Fibronectin was purified from rat plasma by affinity chromatography on gelatin-Sepharose and arginine-Sepharose columns. Mice were immunized and hybridomas were prepared by fusing spleen cells with Sp2/0-Ag14 myeloma cells using poly(ethylene glycol). Three hybridomas (RFN1, RFN2 and RFN3) were selected for characterization. All are IgG molecules, one is IgG2a, one IgG2b and one IgG1. Titers of ascites fluids produced using these hybridomas range from 102 400 to greater than 409 600. The antibodies cross-reacted to different degrees with human fibronectin. Rat fibronectin was radioactively labeled and cleaved using human polymorphonuclear leukocyte elastase. Four major peptides, Mr approx. 160 000, 140 000, 60 000 and 30 000 were produced. Each of the hybridoma antibodies immunoprecipitated different elastase peptides. RFN1 precipitated the Mr 160 000 peptide, RFN2 precipitated the Mr 160 000 and the Mr 140 000 peptide and RFN3 precipitated the Mr 60 000 peptide as well as low molecular weight material migrating at the buffer front. These antibodies will be useful in studies of structure/function relationships of rat fibronectin.     

Tubulin and tubulin-colchicine complex bind to brain microsomal membrane in vitro

Brain tubulin was labeled in vitro by post-translational incorporation of [14C]-tyrosine or in vivo by intra-cranial injection of [3H]-leucine. The labeled protein was purified by ion-exchange chromatography. After incubating at 37 degrees C with a microsomal membrane preparation from rat brain, part of the labeled soluble tubulin became sedimentable at high-speed centrifugation. This was independent of the native configuration of tubulin, the state of tyrosination of the COOH-terminus, or the presence of 100 microM colchicine in the mixture. In addition, the double-labeled tubulin-colchicine complex obtained from the binding of [3H]-colchicine to [14C]-tyrosinated tubulin, bound to the membrane preparation to the same extent as [14C]-tyrosinated tubulin. The data show that either tubulin or the complex resulting from its binding to colchicine distributed between the soluble and the membrane fractions when mixed at 37 degrees C with a microsome preparation. Seemingly, the site for colchicine binding to tubulin needs not to be free for the protein-membrane association.     

CHOLINE ACETYLTRANSFERASE–EVIDENCE FOR ACETYL TRANSFER BY A HISTIDINE RESIDUE

Abstract— Choline acetyltransferase from bovine brain has been extensively purified to a specific activity of 2.5 μmol ACh/min mg protein. Attempts to isolate an acetyl enzyme intermediate after incubation of the enzyme with [1-¹⁴C]acetyl-CoA were unsuccessful. Such an intermediate could only be isolated using a 30-fold less purified enzyme preparation. The protein, binding ¹⁴C in this preparation, did not correspond to choline acetyltransferase as shown by disc-electrophoresis. The highly purified enzyme could, however, be labelled when choline acetyltransferase was immobilized on a mercuribenzoate sepharose gel and incubated with [1-¹⁴C]acetyl-CoA. Subsequently, the immobilized labelled enzyme or the labelled enzyme which had been released by cysteine from the gel. formed ACh after incubation with choline. The labelling and the following formation of [¹⁴C]ACh was pH dependent.
Masking htstidine residues of the enzyme with diethylpyrocarbonate almost abolished the labelling of the immobilized enzyme and completely abolished the formation of [¹⁴C]ACh. Enzyme inhibited with 5.5'-dithiobis(2-nitrobenzoate) was partially reactivated when the thionitrobenzoatederivative was cleaved by KCN treatment to a thiocyanatederivalive. A reaction mechanism for ChAT is proposed based on the present data.     

Axoplasmic transport of a brain-specific soluble protein.

The rate and extent of axoplasmic transport of the brain-specific soluble protein (14-3-2 protein) has been investigated in the avian visual system. 1-day-old chicks were injected monocularly with tritiated proline, Incorporation of the isotope into the 14-3-2 protein synthesized within the retina of the injected eye, as well as the appearance of the labeled protein in the optic lobes was determined at 6 h and 6 days. These time periods were chosen to distinguish between the rapid and slow phases of axophlasmic flowmfollowing preparation of high-speed supernatant fractions, dialysis, chromatography on Sephadex G-150 and immunoprecipitation with specific antiserum, identification of the labeled 14-3-2 protein was carried out by sodium dodecylsulfate-polyacrylamide gel analysis of the radioactive immunoprecipitates; 6 days after isotope administration, approxo% of the 14-3-2 protein synthesized in the chick retina had been transported to the contralateral optic lobe. By contrast, at 6 h no labeled 14-3-2 protein was detectablemthus, transport of this neuronal protein appears to be relatively slow process with little or no rapid component.     

Some properties of phosphatidylcholine exchange protein purified from beef liver

A phospholipid exchange protein has been purified 2680-fold from beef liver. The assay of the exchange activity of the protein was based on the transfer of [¹⁴C]phosphatidylcholine from microsomes labeled with [¹⁴C]phosphatidylcholine to liposomes. The homogeneity of the protein has been established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoelectrophoresis and isoelectric focusing. The protein has a molecular weight of approximately 22000 and an isoelectric point of 5.8. The amino acid composition has been determined. The protein contains one disulfide bridge and has glutamic acid as the N-terminal amino acid. Phospholipid, tentatively identified as phosphatidylcholine, was found to be present in the protein preparation. The protein stimulated specifically the exchange of phosphatidylcholine between mitochondria and microsomes from rat liver.     

A method for the fractionation of the high-mobility-group non-histone chromosomal proteins

A method is given for the preparation of four non-histone chromosomal proteins, one of which, protein 14, hitherto has not been isolated. The method also enables the preparation of histone H1 in gram quantities. The four non-histone chromosomal proteins so prepared are all polar molecules over 50% of each being composed of acidic and basic amino acids. It is also shown that protein 14 can be prepared from calf thymus without prior isolation of chromatin.     

Reconstitution studies of the human erythrocyte nucleoside transporter

The human erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide (Mr 45,000-66,000) on the basis of reversible binding and photoaffinity labeling experiments with the nucleoside transport inhibitor, nitrobenzylthioinosine (NBMPR). In the present study, the NBMPR-binding protein was extracted from protein-depleted human erythrocyte "ghosts" with Triton X-100 and reconstituted into soybean phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes exhibited nitrobenzylthioguanosine (NBTGR)-sensitive [14C]uridine transport. A partially purified preparation of the NBMPR-binding protein, consisting largely of band 4.5 polypeptides, was also shown to have nucleoside transport activity. This band 4.5 preparation exhibited a 10-fold increase in uridine transport activity and a 7-fold increase in NBMPR-binding activity relative to the crude membrane extract. Uridine transport by the reconstituted band 4.5 preparation was saturable (apparent Km = 0.21 mM; Vmax = 9 nmol/mg of protein/5 s) and was inhibited by dipyridamole, dilazep, adenosine, and inosine. The vesicles reconstituted with the band 4.5 preparation also exhibited stereospecific glucose transport which was inhibited by cytochalasin B, but unaffected by NBTGR. In contrast, cytochalasin B was a poor inhibitor of NBTGR-sensitive uridine transport. These experiments implicate band 4.5 polypeptides in both nucleoside and sugar permeation.     

Adenosine 3'':5''-cyclic monophosphate-dependent protein kinase in brown fat from newborn rabbits. Changes in the binding of adenosine 3'':5''-cyclic monophosphate after preincubation of the tissue with noradrenaline or incubation of the enzyme with adenosine triphosphate.

The equilibrium binding of cyclic AMP to a 150-fold purified preparation of protein kinase, when expressed as the reciprocal of bound against the reciprocal of free cyclic AMP, gave a plot consisting of two straight lines. The values of apparent Kb given by these lines were lowered by preincubating the intact tissue with noradrenaline or incubating the enzyme preparation with Mg2+ plus ATP. This effect was reversed by incubating the preparation (which contained some phosphatase impurities) with Mg2+ alone. None of these procedures affected the maximal binding of cyclic AMP. During incubation of the enzyme with Mg2+ plus ATP, the terminal phosphoryl group was incorporated into protein, over 40% being present in the kinase itself. This phosphate was removed during incubation of the preparation with Mg2+ alone. The validity of expressing cyclic AMP binding as a double-reciprocal plot is discussed, and the experimental plots are compared with those derived theoretically. The results suggest that protein kinase in brown fat is present in two forms, one with an apparent Kb for cyclic AMP or approx. 250 nM (dephosphorylation) and one with an apparent Kb of approx. 14 nM (phosphorylated). Preincubation of the tissue with noradrenaline results in phosphorylation of the kinase and an increase from 15 to 45% in the proportion of the higher-affinity form.