Purification of proline-rich proteins from parotid glands of isoproterenol-treated rats.

Prolonged isoproterenol treatment of rats is known to cause hypertrophy and hyperplasia of the parotid glands. Our results show that a dramatic increase in the synthesis or accumulation in the parotid glands of a series of proteins rich in proline also occurs with isoproterenol treatment. After 10 days of treatment (5 mg of isoproterenol/day) these proline-rich proteins (PRPs) comprise more than 50% of the total soluble proteins in parotid gland homogenates. The PRPs are rapidly labeled in vivo by a single intraperitoneal injection of [3H]proline with maximum incorporation occurring at about 3. More than 90% of the [3h]proline found in parotid gland homogenates is incorporated into PRPs with less than 1% of the radioactivity in alpha-amylase. Tritium incorporated into PRPs was isolated as [3H]proline after acid hydrolysis. One acidic and six basic 3H-labeled PRPs were isolated from the 100,000 x g supernatant fraction of parotid gland homogenates by Sephadex G-100 and ion exchange chromatography. The six basic proteins accounted for about 90% of the total PRPs isolated.     

Purification and characterization of a low molecular weight cysteine proteinase inhibitor from bovine muscle

A low-M_r tight binding proteinase inhibitor was purified from bovine muscle by alkaline denaturation of cysteine proteinases, gel filtration on Sexphadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. Chromatofocusing separated three isoforms which are similar in their M_r of about 14 000, their stability with heating at 80°C and their inhibitory activity towards cathepsin H, cathepsin B and papain. The equilibrium constants (K_i) were determined for these three cysteine proteinases but for cathepsin H, association (k_ass) and dissociation (k_diss) rate constants were also evaluated. K_i values of 56 nM and 8.4 nM were found for cathepsin B and cathepsin H, respectively. For papain, K_i was in the range of 0.1–1 nM. The kinetic features of enzyme-inhibitor binding suggest a possible role for this low-M_r protein inhibitor in controlling ‘in vivo’ cathepsin H proteolytic activity. With regard to cathepsin B, such a physiological role was less evident.     

Purification and characterization of a low molecular weight cysteine proteinase inhibitor from bovine muscle

A low-Mr tight binding proteinase inhibitor was purified from bovine muscle by alkaline denaturation of cysteine proteinases, gel filtration on Sephadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. Chromatofocusing separated three isoforms which are similar in their Mr of about 14 000, their stability with heating at 80 degrees C and their inhibitory activity towards cathepsin H, cathepsin B and papain. The equilibrium constants (Ki) were determined for these three cysteine proteinases but for cathepsin H, association (kass) and dissociation (kdiss) rate constants were also evaluated. Ki values of 56 nM and 8.4 nM were found for cathepsin B and cathepsin H, respectively. For papain, Ki was in the range of 0.1-1 nM. The kinetic features of enzyme-inhibitor binding suggest a possible role for this low-Mr protein inhibitor in controlling 'in vivo' cathepsin H proteolytic activity. With regard to cathepsin B, such a physiological role was less evident.     

Purification and characterization of inhibitor against the cysteine proteinase of Rana catesbeiana

1. Inhibitors of cysteine proteinase were found in tadpole tail of metamorphosing bullfrog. 2. One of the inhibitors was purified by affinity chromatography with CM-papain agarose, gel filtration with Superose 12 and ion exchange chromatography with Mono S. 3. The molecular weight of the inhibitor was 130,000-140,000 and the isoelectric point was pH 9.6. 4. The inhibitor had inhibitory effects on ficin, papain and tadpole tail cysteine proteinase. 5. The inhibitor is possibly involved in the regulation of muscle degradation in tail regression of metamorphosing tadpole.     

Purification and characterization of a low molecular mass cysteine proteinase inhibitor from human amniotic fluid

We have purified the human low molecular mass cysteine proteinase inhibitor in good yield from amniotic fluid, using ultrafiltration through 100-kDa and 1-kDa cut-off filters, chromatography on Ultrogel AcA 54, and affinity chromatography on alkylated papain-agarose. Approximately 1-4 mg/l of this inhibitor are present in amniotic fluid. The purified inhibitor had an apparent molecular mass of 10.5-12 kDa, as judged by its electrophoretic behavior. Amino acid analysis showed it to be rich in acidic and aliphatic residues and in cysteine. No carbohydrate side-chains could be demonstrated. The purified inhibitor inhibited papain, ficin, cathepsins B, C, and H, the cathepsin B-like enzyme from B16 melanoma cells, and a bovine chromaffin granule enkephalin-converting activity. No inhibition of Ca2-dependent neutral cysteine proteinase, serine- or metallo-proteinases was seen. Analysis of the purified inhibitor by isoelectric focusing revealed 7 major bands with pI values of 7.95, 7.0, 6.7, 6.55, 6.25, 5.5, and 5.2, all of which inhibited papain.     

Purification and characterization of a cysteine proteinase from silkworm eggs

Eggs of the silkworm, Bombyx mori, contain a high level of a proteinase which is most active in acidic pH region. The proteinase was purified from an extract of eggs by a six-step procedure which included conventional chromatographic fractionations. The molecular mass of the proteinase was estimated to be 350 kDa by gel filtration and 47 kDa by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels, suggesting an octameric structure. The amino acid composition was found to resemble that of mammalian lysosomal cysteine proteinases, in particular cathepsin L. The NH2-terminal 10-residue sequence is Val-Gln-Phe-Phe-Asp-Leu-Val-Lys-Glu-Glu-. The enzyme appears to be a member of the class of cysteine proteinases since it was strongly inhibited by sulfhydryl-reactive compounds and N-[N-(1,3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). The enzyme hydrolyzed various protein substrates, such as hemoglobin, vitellogenin, vitellin, and lipophorin, with maximal activity around pH 3-3.5. The specificity of the cleavage sites in the oxidized B chain of insulin was rather well defined and there was high affinity for hydrophobic residues at the P2 and P3 positions. The cysteine proteinase is thought to be involved in protein degradation during embryonic development of silkworm eggs.     

Purification and characterization of a bovine colostrum low molecular weight cysteine proteinase inhibitor

Large amounts of cysteine proteinase inhibitors were found in bovine colostrum. One had a molecular weight of 90,000, and the other a molecular weight of 10,500. The concentrations of both these inhibitors were highest the day after parturition, and were about one-tenth as much on day 7. The lower molecular weight inhibitor was purified by acid treatment, ammonium sulfate fractionation, gel filtration on Sephadex G-50, CM-Sephadex chromatography and rechromatography on Sephadex G-50. The purified preparation gave a single band on SDS-polyacrylamide gel electrophoresis. This inhibitor contained one tryptophanyl residue and one cystinyl residue, and did not contain a free thiol group. Values obtained for its isoelectric point (pI) were 10.0 and 10.3. This material strongly inhibited cathepsin B, cathepsin H, and papain. the higher molecular weight inhibitor was partially purified. It had a pI of 4.2 and inhibited papain, cathepsin H, and cathepsin B.     

Purification and partial characterization of a cysteine proteinase from Trypanosoma rangeli

Abstract Epimastigotes of the American Trypanosome Trypanosoma rangeli contain a very low cysteine proteinase (CP) activity. The enzyme was purified to homogeneity by affinity chromatography on ConA-Sepharose and Cystatin-Sepharose. This CP had a similar apparent molecular mass and an identical N-terminal sequence (15 amino acids) as compared with cruzipain from Trypanosoma cruzi ; cross-reacted immunologically with the latter enzyme, it was inhibited by E-64 and TLCK, but not by PMSF, o-phenanthroline or Pepstatin, and was able to use the same substrates, although with different order of effectiveness and optimum pH.     

Partial purification and characterization of cysteine proteinase inhibitor from chicken plasma

A high-molecular-weight cysteine proteinase inhibitor (CPI) was purified from chicken (Gallus gallus) plasma using polyethylene glycol (PEG) fractionation and affinity chromatography on carboxymethyl–papain–Sepharose-4B. The CPI was purified 96.8-fold with a yield of 28.9%. Based on inhibitory activity staining for papain, CPI was shown to have an apparent molecular mass of 122 kDa. No inhibitory activity was obtained under reducing condition, indicating that CPI from chicken plasma was stabilized by disulfide bonds. CPI was stable in temperature ranges from 40 to 70 °C for 10 min; however, more than 50% of the inhibitory activity towards papain was lost within 30 min of heating at 90 °C. CPI was stable in the presence of salt up to 3%. The purified CPI exhibited the inhibitory activity toward autolysis of arrowtooth flounder (Atheresthes stomias) and Pacific whiting (Merluccius productus) natural actomyosin (NAM) in a concentration-dependent manner.     

Purification and characterization of CMP-N-acetylneuraminic acid hydroxylase from pig submandibular glands

N-Glycoloylneuraminic acid (Neu5Gc) is synthesized as its CMP-giycosideby the action of CMPN-acetylneuramlnic acid (CMP-Neu5Ac) hydroxylase.This enzyme is a soluble cytochrome bs-dependent monooxygenaseand has been purified to apparent homogeneity from pig submandibularglands by precipitation with N-cetyN,N,N-trimethylam-moniumbromide and fractionation on Q-Sepharose, Cibacron Blue 3GA-Agarose,Reactive Brown 10-Agarose, Hexyl-Agarose and Superose S.12.This procedure resulted in an 8960-fold purification of thehydroxylase with a recovery of 0.8%. The molecular mass of thisprotein was shown to be 65 kDa on SDS-PAGE and 60 kDa as determinedby gel filtration on Superose S.12, which suggests that theenzyme is a monomer. The purified CMP-Neu5Ac hydroxylase isactivated by FeSO₄ and inhibited by iron-binding reagents suchas o-phenanthroline, KCN, Tiron and ferro-zine. An apparentKm of 11 µM was determined for the substrate CMP-Neu5Acusing purified hydroxylase in the presence of Triton X-100-solubilizedmicrosomes. In a reconstituted system consisting of purifiedhydroxylase, cytochrome b₅, cytochrome b₅ reductase and catalase,an apparent K_m of 3 µM was measured. The apparent K_mforcytochrome b₅ in this system was 0.24 µM. Immunizationof a rabbit with enriched and purified hydroxylase led to anantiserum that inhibited CMP-Neu5Ac hydroxylase activity andreacted with the purified 65 kDa protein on a Western blot afterSDS-PAGE. Antibodies specific for this 65 kDa protein were isolatedand showed a strong reaction with the purified CMP-Neu5Ac hydroxylasefrom mouse liver after immunoblotting. Initial experiments withthis monospecific antibody suggest that the activity of thehydroxylase in a particular tissue correlates with the amountof immuno-reactive protein. cytochrome b₅ N-glcoloylneuraminic acid hydroxylase pig submandibular gland mucin sialic acid     

Purification and characterization of thiol proteinase inhibitor from rat liver

A thiol proteinase inhibitor was purified from rat liver by essentially the same procedure as reported previously (Kominami, E., Wakamatsu, N., and Katunuma, N. (1981) Biochem. Biophys. Res. Commun. 99, 568-575), but without heat treatment. The purified inhibitor appears homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate and displayed no multiple forms. The inhibitor has Mr = 12,500 and contains 50.5% of polar amino acid residues, 9.3% aromatic amino acids, and no tryptophan. The presence of 2 half-cystines/molecule and the absence of free thiol groups indicate that the inhibitor possesses one disulfide bridges. The inhibitor inhibits cathepsin H by forming an enzyme-inhibitor complex in a molar ratio of 1:1. It inhibits most thiol proteinases such as cathepsin H, L, B, and C, papain, and ficin, but not calcium-activated neutral proteinase or serine proteinases or carboxyl proteinases. The inhibitor was found in various rat tissues. Immunological diffusion analysis with anti-liver thiol proteinase inhibitor serum indicated that the rat liver inhibitor is immunologically identical with the inhibitors from other rat tissues. On subcellular fractionation of rat liver, the thiol proteinase inhibitor was recovered in the cytosol fraction.     

Studies on chromatin-associated protein phosphokinase of submandibular gland from isoproterenol-treated rats

Water-soluble chromatin from rat submandibular gland nuclei was isolated, and had a DNA: RNA:protein ratio of 8:1:20. The spectral properties of this preparation were similar to those described for chromatins from other tissues. The rat submandibular gland chromatin possessed protein phosphokinase activity. It was able to incorporate ³²P from [γ-³²P]-ATP into chromatin proteins, and into dephospho-phosvitin. The chromatin-associated protein phosphokinase activity (measured with dephospho-phosvitin as substrate) required Mg²⁺, Na⁺ or K⁺ and dithiothreitol for optimal activity. A single injection of isoproterenol influenced the activity of this enzyme system, so that it was decreased at 2 h, showed a transient increase at 4 h, and a large increase at 10–16 h after the injection. This event appears to precede the increase in ribosomal RNA induced by Ipr [13]. By 48 h the chromatin-associated protein kinase returned to the normal control levels. These changes appeared to be commensurate with the corresponding alterations in the non-histone acidic protein complement of these chromatins. Actinomycin D or cycloheximide, when administered 30 min prior to isoproterenol, blocked the increase in chromatin-associated protein kinase at 4 as well as 10 h after the injection of isoproterenol. Injection of pilocarpine did not influence the chromatin-associated protein phosphokinase activity. Dichloroisoproterenol appeared to be antagonistic to the influence of isoproterenol in mediating changes in chromatin-associated protein kinase. The results suggest that the isoproterenol-induced increase in chromatin-bound protein phosphokinase which precedes the increase in RNA synthesis is related to the eventual onset of DNA synthesis in rat submandibular gland stimulated by isoproterenol. The chromatin-bound protein phosphokinase activity (or activities) may have a regulatory role on gene action, mediated through the control of phosphorylation of nuclear non-histone acidic proteins [26].     

Purification and characterization of kallikrein-like serine proteases from rat submandibular glands.

The purification and characterization of kallikrein-like proteases from rat submandibular glands is described. The proteolytic activity of each fraction during purification was monitored on the synthetic substrate N-alpha-tosyl-L-arginine methyl ester (TAME). The purification scheme involved ammonium sulfate precipitation, chromatography on columns of DEAE-Sepharose and Sephadex G-100 and chromatofocusing. Three TAME-hydrolytic activity peaks were eluted from DEAE-Sepharose as unbound fraction (Pool 1), at 125 mM NaCl (Pool 2) and at 250 mM NaCl concentration (Pool 4). Pool 1 further resolved into two protease fractions (1A1 and 1A2), pool 2 into three protease fractions (2A1, 2A2 and 2A3) and pool 4 gave a single major protease peak (4A1) by chromatofocusing on PBE-94. Protease pools 2A2, 2A3, and 4A1 each gave a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular weight of 34 kDa, 46 kDa and 46 kDa respectively. Pools 1A1, 1A2, 2A1 and 2a2 gave a single precipitin line with anti-rat glandular kallikrein antibodies. 2A3 and 4A1 did not react with these antibodies. Synthetic substrates DL-val-leu-arg-pNA and Bz-pro-phe-arg-pNA, specific for kallikrein-like proteases, were hydrolyzed preferentially by 2A3 and 4A1 but were poor substrates for 1A1, 1A2, 2A1 and 2A2.     

Purification and characterization of human bronchial proteinase inhibitor

We describe a purification procedure for the human bronchial proteinase inhibitor which involves trichloroacetic acid precipitation of sputum followed by ion-exchange and gel filtration chromatography. The inhibitor shows a major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but exhibits microheterogeneity on high-resolution chromatography. It has a molecular mass of 15.5-16 kDa as determined by electrophoresis and gel filtration and is 90% active against leukocyte elastase. The amino acid sequence of the N-terminal portion of the inhibitor was determined and was found to be identical (through 29 amino acids) to that recently reported for the human seminal plasma proteinase inhibitor I (Seemuller et al. (1986) FEBS Lett. 199, 43-48).     

Studies on phosphoproteins of submandibular gland nuclei isolated from isoproterenol-treated rats

Rat submandibular gland nuclei incubated with γ-³²P-ATP incorporated the label into histone and non-histone phosphoproteins. The latter was the predominantly radioactive fraction. After a single injection of isoproterenol (Ipr), the incorporation of ³²P into non-histone phosphoproteins decreased during the first few hours, followed by an increase at 4 h which reached its peak at 24 h at a higher level compared with normal controls. The values returned to the control level at 40 h after the injection. The changes were reflected in the initial rates as well as the total level of incorporation of ³²P into the phosphoproteins. Temporally, the onset of increase in the phosphorylation of non-histone phosphoproteins appeared to precede that in RNA synthesis, although peak activity of the phosphorylation coincided with the peak of RNA synthesis. The non-histone phosphoproteins which depicted maximal changes in response to Ipr were further characterized as phenol-soluble acidic phosphoproteins. The phosphorylation of histone phosphoproteins also declined after the injection of Ipr, but the recovery of the rate of phosphorylation was not observed until 16 h after the injection, reaching the control levels at 24 h. Treatment of rats with actinomycin D or cycloheximide, prior to Ipr, abolished the increase in phosphorylation of non-histone phosphoproteins observed at 24 h after Ipr. Further, the changes in the phosphorylation of nuclear phosphoproteins induced by Ipr were blocked by prior treatment of the animals with dichloroisoproterenol. The results suggest that the phosphorylation of the non-histone phosphoproteins plays an important role in the events controlling the synthesis of RNA which precedes the replication of DNA and cell. In addition, the regulation of the metabolism of nuclear phosphoproteins may be controlled through a function of the cytoplasmic membrane.     

Purification and characterization of a serine proteinase inhibitor from human articular cartilage

An inhibitor of serine proteinases from human articular cartilage was purified to homogeneity by sequential ultrafiltration and ion exchange chromatography on CM-Sephadex C-50. The apparent molecular weight of the cationic glycoprotein (pI > 10) was determined to be 16.5 · 10³ by SDS gel electrohoresis. The inhibitor blocked the activity of leukocyte elastase, cathepsin G and trypsin but not leukocyte collagenase. In kinetic studies for the interactions with leukocyte elastase a firm enzyme-inhibitor binding was obtained. Amino acid analyses did not reveal homologies with other serine proteinase inhibitors already purified from human tissues.     

Purification and characterization of an extracellular cysteine proteinase produced by Micrococcus sp. INIA 528

Micrococcus sp. INIA 528, a micro-organism isolated from raw ewe's milk Manchego cheese, produced an extracellular proteinase. This enzyme was purified to homogeneity from culture supernatant fluid in two chromatographic steps, with a 29-fold increase of specific activity and a 28% recovery of proteinase activity. The homogeneous protein was characterized biochemically. The molecular weight of the enzyme was determined to be 19.4 kDa by mass spectrometry. The purified enzyme was inhibited by E-64, PMSF and iodoacetamide and activated by cysteine, glutathione, dithiothreitol and β-mercaptoethanol. These results suggest that the enzyme is a cysteine proteinase. Optimal conditions for activity on azocasein were 34°C and a pH of 7.0. The proteinase preferentially degraded β-casein, while after a longer incubation period α^s1-casein was also extensively hydrolysed. The proteinase had a K _m value of 6.12 g 1⁻¹ for casein and 2.20 g 1⁻¹ for azocasein.