Purification and properties of guinea-pig submandibular-gland kallikrein.
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ABSTRACT:
SUBMITTER: Fiedler F
PROVIDER: S-EPMC1154063 | biostudies-other | 1983 Jan
REPOSITORIES: biostudies-other
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Project description:The kallikrein from pig submandibular glands was highly purified, with an overall yield of 31%. Affinity chromatography on bovine basic pancreatic trypsin inhibitor linked to Sepharose 4B was an especially effective step in the purification procedure, giving a purification factor of 80. The enzyme is a single-chain molecule, occurring, as does pig urinary kallikrein, as a major B-form of apparent mol.wt. 39600 and minor amounts of an A-form of apparent mol.wt. 35900; the two forms can be separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The amino acid composition of pig submandibular kallikrein is very similar to, but not quite identical with, that of the two-chain beta-kallikrein isolated from pig pancreatic autolysates. Submandibular kallikrein contains notably more glucosamine and hexoses than does pancreatic beta-kallikrein. Submandibular kallikrein, and also urinary kallikrein, exhibit an unusual biphasic hydrolysis of substrate esters that is not shared by pancreatic beta-kallikrein. For the submandibular enzyme, the K(m) for the initial reaction phase of the hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester is 0.15+/-0.01mm (mean+/-s.e.m.), but rises to 0.69+/-0.04mm (mean+/-s.e.m.) in the stationary reaction phase; the V(max.) does not differ significantly between the two phases. The esterolytic activities of submandibular and urinary kallikreins on a number of esters of different amino acids resemble each other much more closely than those of pancreatic beta-kallikrein.
| S-EPMC1186352 | biostudies-other
Project description:1. Methods for the purification of dog submandibular-gland hyaluronidase from sedimentable and non-sedimentable portions of a homogenate and from the whole homogenate are presented. The method consists of three main steps: removal of mucin by acid precipitation or gel filtration on Sephadex G-200, ammonium sulphate precipitation and CM-cellulose chromatography. By this method specific activities of up to 1.28 and 0.78mumoles of N-acetylglucosamine/min./mg. of protein were obtained for the purified freeze-dried non-sedimentable hyaluronidase and for the sedimentable hyaluronidase respectively. 2. A comparison of some of the properties of the non-sedimentable and the sedimentable hyaluronidase preparation indicated that there was little difference between the two and that they both resembled lysosomal hyaluronidase from rat liver.
| S-EPMC1187104 | biostudies-other
Project description:1. Polynucleotide phosphorylase has been isolated and partially purified from crude preparations of guinea-pig liver nuclei. 2. The enzyme is particulate and associated with RNA and lipids characteristic of membranes. 3. It has phosphorolysis and exchange activities, but the latter may be due to a contaminating enzyme. 4. The phosphorolysis activity is dependent on bivalent cations, preferably Mg(2+), has a pH optimum between 8.6 and 9.2 and is inhibited by potassium chloride and sodium chloride. 5. The enzyme catalyses phosphorolysis of poly A, poly C, poly U, rRNA and tRNA. Poly G is only phosphorolysed to a very small extent and DNA is not a substrate. 6. The enzyme appears to lack nucleoside diphosphate polymerization activity.
| S-EPMC1179382 | biostudies-other
Project description:The submandibular gland of the rat contains several enzymes belonging to the kallikrein family. These include tissue kallikrein, antigen gamma (T-kininogenase), esterase B and tonin. In the present study, a new member of this family, which we have named KLP-S3, was identified and purified from the submandibular gland. KLP-S3 was classified as a kallikrein-like enzyme on the basis of its immunological similarity to other kallikrein-like enzymes and its showing 70% and 73% identity in partial amino acid sequence with tissue kallikrein and tonin respectively. Furthermore, the 44 sequenced amino acid residues showed complete correspondence to the mRNA S3 of the kallikrein gene family, which was the rationale for the name kallikrein-like protein (KLP) S3. KLP-S3 consisted of three isoenzymes with pI 6.75, 6.90 and 6.95, which significantly differed from those of other kallikrein-like enzymes. In conjunction with its immunological relationship to kallikrein, this parameter (pI) was considered robust enough to identify the enzyme during purification, since a specific physiological substrate for KLP-S3 has yet to be identified. In SDS/PAGE the three isoenzymes ran as one band with a molecular mass of 25,800 Da, which after reduction with 2-mercaptoethanol was split into two chains with molecular masses of 16,500 and 13,300 Da. In common with other kallikrein-like enzymes, KLP-S3 was inhibited by phenylmethanesulphonyl fluoride, and was thus classified as a serine protease. It was also inhibited by soya-bean trypsin inhibitor but not by aprotinin. It showed weak reactivity against the chromogenic substrates S2288, S2266, S2366 and S2302 (D-Ile-Pro-Arg 4-nitroanilide, D-Val-Leu-Arg 4-nitroanilide, Glu-Pro-Arg 4-nitroanilide and D-Pro-Phe-Arg 4-nitroanilide respectively) and did not cleave rat T-kininogen or dog high-molecular-mass/low-molecular-mass kininogen. Its specific angiotensin II-generating activity (angiotensin I as substrate) was 0.04% of that of rat tonin. KLP-S3 (1-100 nM) induced a statistically significant angiotensin-independent contraction of isolated rat aorta rings. The maximum contraction was 15% of the response to the alpha-adrenoceptor agonist phenylephrine (1 microM). The concentration of KLP-S3 in the rat submandibular gland was by single radial immunodiffusion estimated to be 47 +/- 3 micrograms/mg of protein.
| S-EPMC1130763 | biostudies-other
Project description:A method has been developed for the purification to homogeneity of guinea-pig complement component C2. Contrary to previous reports, guinea-pig C2 is a single polypeptide chain with apparent mol.wt. of 102000, the same as human C2. It is cleaved by C1s to yield fragments C2a (apparent molwt. 74000) and C2b (apparent mol.wt. 34000). The amino acid composition and N-terminal sequences of these fragments are similar to those of human C2a and C2b. Human and guinea-pig C2 show more extensive sequence homology to Factor B than previously identified. The known homology around the sites of cleavage by C1s and Factor D has now been extended by a stretch of ten identical or conservatively substituted residues. Sequence homology has now been identified at the N-terminal of C2b and Factor Ba. The properties of the classical-pathway C3 convertases assembled from human C4b, C1s and human or guinea-pig C2 have been compared. The rates of cleavage of human and guinea-pig C2 by C1s (and therefore the rates of assembly of the C3 convertases) are similar. The rate of decay of the activity of the C3 convertase formed from guinea-pig C2 is 10-fold lower than for human C2. This greater stability reflects a higher affinity of guinea-pig C2a for human C4b. The presence of C2b is not necessary for C3 convertase activity.
| S-EPMC1158446 | biostudies-other
Project description:Biomechanical changes in the sclera likely underlie the excessive eye elongation of axial myopia. We studied the biomechanical characteristics of myopic sclera at the microscopic level using scanning acoustic microscopy (SAM) with 7-μm in-plane resolution. Guinea pigs underwent form-deprivation (FD) in one eye from 4 to 12 days of age to induce myopia, and 12-μm-thick scleral cryosections were scanned using a custom-made SAM. Two-dimensional maps of the bulk modulus (K) and mass density (ρ) were derived from the SAM data using a frequency-domain approach. We assessed the effect on K and ρ exerted by: 1) level of induced myopia, 2) region (superior, inferior, nasal or temporal) and 3) eccentricity from the nerve using univariate and multivariate regression analyses. Induced myopia ranged between -3D and -9.3D (Mean intraocular difference of -6.2 ± 1.7D, N = 11). K decreased by 0.036 GPa for every 1.0 D increase in induced myopia across vertical sections (p < 0.001). Among induced myopia right eyes, K values in the inherently more myopic superior region were 0.088 GPa less than the inferior region (p = 0.002) and K in the proximal nasal region containing the central axis were 0.10 GPa less than temporal K (p = 0.036). K also increased 0.12 GPa for every 1 mm increase in superior vertical distance (p < 0.001), an effect that was blunted after 1 week of FD. Overall, trends for ρ were less apparent than for K. ρ values increased by 20.7 mg/cm3 for every 1.00 D increase in induced myopia across horizontal sections (p < 0.001), and were greatest in the region containing the central posterior pole. ρ values in the inherently more myopic superior region were 13.1 mg/cm3 greater than that found in inferior regions among control eyes (p = 0.002), and increased by 11.2 mg/cm3 for every 1 mm increase in vertical distance (p = 0.001). This peripheral increase in ρ was blunted after 1 week of FD. Scleral material properties vary depending on the location in the sclera and the level of induced myopia. Bulk modulus was most reduced in the most myopic regions (both induced myopia and inherent regional myopia), and suggests that FD causes microscopic local decreases in sclera stiffness, while scleral mass density was most increased in the most myopic regions.
| S-EPMC6712578 | biostudies-literature
Project description:The sequence of guinea pig cytomegalovirus (GPCMV) was determined by direct sequencing of salivary gland homogenates obtained following sustained, serial in vivo passage of pathogenic virus in guinea pigs. The 233,501-nucleotide salivary gland (SG) genome was noted to have 11 differences compared to the tissue culture-passaged virus, although no variations were noted in putative protein coding sequences.
| S-EPMC3622957 | biostudies-literature
Project description:The muscularis mucosae, a type of smooth muscle located between the urothelium and the urinary bladder detrusor, has been described, although its properties and role in bladder function have not been characterized. Here, using mucosal tissue strips isolated from guinea pig urinary bladders, we identified spontaneous phasic contractions (SPCs) that appear to originate in the muscularis mucosae. This smooth muscle layer exhibited Ca(2+) waves and flashes, but localized Ca(2+) events (Ca(2+) sparks, purinergic receptor-mediated transients) were not detected. Ca(2+) flashes, often in bursts, occurred with a frequency (?5.7/min) similar to that of SPCs (?4/min), suggesting that SPCs are triggered by bursts of Ca(2+) flashes. The force generated by a single mucosal SPC represented the maximal force of the strip, whereas a single detrusor SPC was ?3% of maximal force of the detrusor strip. Electrical field stimulation (0.5-50 Hz) evoked force transients in isolated detrusor and mucosal strips. Inhibition of cholinergic receptors significantly decreased force in detrusor and mucosal strips (at higher frequencies). Concurrent inhibition of purinergic and cholinergic receptors nearly abolished evoked responses in detrusor and mucosae. Mucosal SPCs were unaffected by blocking small-conductance Ca(2+)-activated K(+) (SK) channels with apamin and were unchanged by blocking large-conductance Ca(2+)-activated K(+) (BK) channels with iberiotoxin (IbTX), indicating that SK and BK channels play a much smaller role in regulating muscularis mucosae SPCs than they do in regulating detrusor SPCs. Consistent with this, BK channel current density in myocytes from muscularis mucosae was ?20% of that in detrusor myocytes. These findings indicate that the muscularis mucosae in guinea pig represents a second smooth muscle compartment that is physiologically and pharmacologically distinct from the detrusor and may contribute to the overall contractile properties of the urinary bladder.
| S-EPMC3154705 | biostudies-literature
Project description:As a result of studies of guinea-pig live testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), a new testosterone 17beta-dehydrogenase was discovered. The new enzyme was purified to a single homogeneous protein from the 105 000 g-supernatant fraction of guinea-pig liver by (NH4)2SO4 fractional precipitation and two gel-filtration stages, DEAE-cellulose column chromatography and hydroxyapatite column chromatography. It was characterized by many properties. The enzyme has almost the same properties as the classical testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), with respect to cofactor requirement, pH optima for dehydrogenation, effect of phosphate ion on the NAD+-dependent reaction and molecular weight, but characteristic differences were observed in substrate-specificity between the two dehydrogenases. With various androstane derivatives, the configuration of the A/B-ring junction was closely connected with enzyme activity. 5alpha-Androstanes, such as 5alpha-androstane-3alpha,17beta-diol, 5alpha-androstane-3beta,17beta-diol and 17beta-hydroxy-5alpha-androstan-3-one, and 5beta-congeners, such as 5beta-androstane-3alpha,17beta-diol, 5beta-androstane-3beta,17beta-diol and 17beta-hydroxy-5beta-androstan-3-one, served as substrates for both the EC 1.1.1.64 enzyme and the new enzyme. The EC 1.1.1.64 enzyme oxidized testosterone more rapidly than did the new enzyme. These comparisons were based on the relative activities, apparent Km values and apparent Vmax values.
| S-EPMC1164717 | biostudies-other
Project description:Background and purposeTo investigate the role of connexin 43 in the maintenance of spontaneous activity in prostate tissue from young and old guinea pigs.Experimental approachConventional intracellular microelectrode and tension recording techniques, coupled with Western blot analysis and immunohistochemistry for connexin 43 (CX43) were used. The effects of three gap junction uncouplers, 18? glycyrrhetinic acid (10?µM, 40?µM), carbenoxolone (10?µM, 50?µM) and octanol (0.5?mM, 1?mM), were studied in cells displaying slow wave activity and on spontaneously contracting tissue from prostate glands of young (2-5 months) and old (9-16 months) guinea pigs.Key results18? Glycyrrhetinic acid (40?µM), carbenoxolone (50?µM) or octanol (0.5?mM) abolished slow wave activity in prostate tissue from young and old guinea pigs and depolarized membrane potential by approximately 5?mV. These treatments also abolished all contractions in both sets of prostate tissue. These effects were reversed upon washout. Western blot analysis and CX43 immunohistochemistry showed that there was no age-related difference in the expression and distribution of CX43 in prostate tissues.Conclusion and implicationsWhen gap junctional communication via CX43 was disrupted, spontaneous activity was abolished at a cellular and whole tissue level; CX43 is therefore essential for the maintenance of spontaneous slow wave activity and subsequent contractile activity in the guinea pig prostate gland.
| S-EPMC3010576 | biostudies-literature