scholarly journals Association of newly synthesized islet prohormones with intracellular membranes.

1984 ◽  
Vol 99 (2) ◽  
pp. 418-424 ◽  
Author(s):  
B D Noe ◽  
M N Moran
Keyword(s):  
Secretory Granules ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Proteolytic Processing ◽  
Membrane Association ◽  
Freezing And Thawing ◽  
Ph Optimum ◽  
Intracellular Membranes ◽  
Membrane Lysis ◽  
Repeated Freezing

Results from recent studies have indicated that pancreatic islet prohormone converting enzymes are membrane-associated in islet microsomes and secretory granules. This observation, along with the demonstration that proglucagon is topologically segregated to the periphery within alpha cell secretory granules in several species, led us to investigate the possibility that newly synthesized islet prohormones might be associated with intracellular membranes. Anglerfish islets were incubated with [3H]tryptophan and [14C]isoleucine for 3 h, then fractionated by differential and density gradient centrifugation. Microsome (M) and secretory granule (SG) fractions were halved, sedimented, and resuspended in the presence or absence of dissociative reagents. After membrane lysis by repeated freezing and thawing, the membranous and soluble components were separated by centrifugation. Extracts of supernatants and pellets were chromatographed by gel filtration; fractions were collected and counted. A high proportion (77-79%) of the newly synthesized proinsulin and insulin was associated with both M and SG membranes. Most of the newly synthesized proglucagons and prosomatostatins (12,000-mol-wt precursors) were also membrane-associated (86-88%) in M and SG. In contrast, glucagon- and somatostatin-related peptides exhibited much less membrane-association in SG (24-31%). Bacitracin, bovine serum albumin EDTA, RNAse, alpha-methylmannoside, N-acetylglucosamine, and dithiodipyridine had no effect on prohormone association with membranes. However, high salt (1 M KCl) significantly reduced membrane-association of prohormones. Binding of labeled prohormones to SG membranes from unlabeled tissue increased with incubation time and was inhibited by unlabeled prohormones. The pH optimum for prohormone binding to both M and SG membranes was 5.2. It is suggested that association of newly synthesized prohormones with intracellular membranes could be related to the facilitation of proteolytic processing of prohormones and/or transport from their site of synthesis to the secretory granules.

THE MECHANISM OF THE ACID ACTIVATION OF RABBIT UTERINE RENIN

1979 ◽  
Vol 92 (4) ◽  
pp. 720-730 ◽  
Author(s):  
Jørgen Jørgensen
Keyword(s):  
Proteolytic Enzymes ◽  
Pressor Response ◽  
Gel Filtration ◽  
Acid Activation ◽  
Freezing And Thawing ◽  
Ph Optimum ◽  
Active Renin ◽  
Inactive Form ◽  
Repeated Freezing ◽  
Rate Of Activation

ABSTRACT Besides active renin an inactive form of renin could be demonstrated in uterine tissue. On gel filtration it was eluted as a molecule of slightly higher molecular weight than active renin, and it could be irreversibly activated by acidification at 37°C. The activation had a pH optimum between pH 3.8 and pH 5.3. Acid activated uterine renin was found identical to active uterine renin by 1) the formation of angiotensin I with time after addition of rat substrate, 2) the pressor response in the rat, 3) neutralization by antirenin and 4) similar Michaelian constants. Repeated freezing and thawing, acidification at 4°C and dialysis against 4 mol/l NaCl did not give any activation. A lower rate of activation of diluted samples and activation by trypsin at pH 7.4 suggest that proteolytic enzymes are involved in the activation.

scholarly journals ISOZYMES OF ACID PHOSPHATASE IN NORMAL AND CALMETTE-GUÉRIN BACILLUS-INDUCED RABBIT ALVEOLAR MACROPHAGES

1968 ◽  
Vol 128 (5) ◽  
pp. 1031-1048 ◽  
Author(s):  
S. G. Axline
Keyword(s):  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Alveolar Macrophages ◽  
Acid Phosphatase Activity ◽  
Enzyme Inhibitors ◽  
Freezing And Thawing ◽  
Ph Optimum ◽  
Lysosomal Fraction ◽  
Repeated Freezing ◽  
Major Acid

The acid phosphatase activity of normal alveolar and BCG-induced alveolar macrophages has been examined. Five electrophoretically distinct forms of acid phosphatase have been identified in both normal and BCG-induced macrophages. The acid phosphatases can be divided into two major categories. One category, containing four distinct forms, is readily solubilized after repeated freezing and thawing or mechanical disruption The second category, containing one form, is firmly bound to the lysosomal membrane and can be solubilized by treatment of the lysosomal fraction with Triton X-100. The Triton-extractable acid phosphatase and the predominant aqueous soluble acid phosphatase have been shown to differ in the degree of membrane binding, in solubility, in net charge, and in molecular weight. The two pre-dominant phosphatases possess identical pH optimum and do not differ in response to enzyme inhibitors. BCG stimulation has been shown to result in a nearly twofold increase in acid phosphatase activity. A nearly proportionate increase in the major acid phosphatase forms has been observed.

scholarly journals Factor V activity of platelets: evidence for an activated factor V molecule and for a platelet activator

Blood ◽  
1977 ◽  
Vol 49 (5) ◽  
pp. 819-834 ◽  
Author(s):  
B Osterud ◽  
SI Rapaport ◽  
KK Lavine
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Factor V ◽  
Freezing And Thawing ◽  
Early Step ◽  
Platelet Factor ◽  
Plasma Factor ◽  
Native Factor

Abstract This study was prompted by the observation that fresh platelet suspensions--prepared by gel filtration or albumin density gradient centrifugation--possessed only minimal factor V activity, whereas frozen-and-thawed platelet suspensions possessed striking factor V activity. Results of experiments with fresh suspensions suggested that unaltered platelets did not bind plasma factor V. The factor V activity of frozen-and-thawed platelet suspensions was markedly diminished after exposure to a factor V antibody, was not activated by thrombin, and was not associated with an increase in factor V antigen over that found in fresh platelet suspensions. Consequently, disruption by freezing and thawing must have resulted in the appearance of small amounts of an activated factor V molecule in platelet suspensions. Disrupted platelets were shown to activate native factor V, but an interaction between a platelet activator and traces of native factor V in fresh suspensions could not be demonstrated to account for the full activity of frozen-and-thawed suspensions. Apparently, therefore, platelets also contained an activated factor V molecule. Adding collagen, but not adenosine 5′-diphosphate to fresh platelet suspensions increased their factor V activity. Release of an activated platelet factor V molecule after exposure to collagen could represent a physiologically significant early step in hemostasis.

Interference in a radioimmunoassay for human thyrotropin.

1977 ◽  
Vol 23 (3) ◽  
pp. 584-588 ◽  
Author(s):  
C A Spencer ◽  
G S Challand
Keyword(s):  
Clinical Evidence ◽  
Chemical Nature ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
High Plasma ◽  
Normal Serum ◽  
Freezing And Thawing ◽  
Separation Stage ◽  
Molecular Weights ◽  
Repeated Freezing

Abstract Abnormally high plasma thyrotropin values were found by radioimmunoassay in some patients when an antiserum to porcine thyrotropin was used, normal results being obtained with an antiserum raised to human thyrotropin. These discrepancies were found in some subjects with no biochemical or clinical evidence of hypothyroidism and occasionally in sera from patients with unequivocal hyperthyroidism. We found a substance in serum that cross reacts with the anti-porcine antiserum, is stable on repeated freezing and thawing, and is independent of the 125I-labeled tracer preparation. It is unlikely that this substance is a separation-stage artefact related to immunoglobulins. Its apparent molecular weight (gel filtration) is 114 000, as compared with apparent molecular weights for standard thyrotropin and endogenous thyrotropin (as found in idiopathic hypothyroidism) of 34 700 and 38 000, respectively. We believe the substance is a normal serum constituent that is present in large quantities in a minority of subjects. Apparently unrelated to TSH, its exact chemical nature remains unidentified.

scholarly journals Purification, characterization and identification of rat liver histidine-pyruvate aminotransferase isoenzymes

1976 ◽  
Vol 155 (1) ◽  
pp. 107-115 ◽  
Author(s):  
T Noguchi ◽  
E Okuno ◽  
Y Minatogawa ◽  
R Kido
Keyword(s):  
Density Gradient ◽  
Rat Liver ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Aminotransferase Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum

1. Histidine-pyruvate aminotransferase (isoenzyme 1) was purified to homogeneity from the mitochondrial and supernatant fractions of rat liver, as judged by polyacrylamide-gel electrophoresis and isolectric focusing. Both enzyme preparations were remarkably similar in physical and enzymic properties. Isoenzyme 1 had pI8.0 and a pH optimum of 9.0. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors in the following order of activity: phenylalanine greater than tyrosine greater than histidine. Very little activity was found with tryptophan and 5-hydroxytryptophan. The apparent Km values were about 2.6mM for histidine and 2.7 mM for phenylalanine. Km values for pyruvate were about 5.2mM with phenylalanine as amino donor and 1.1mM with histidine. The aminotransferase activity of the enzyme towards phenylalanine was inhibited by the addition of histidine. The mol.wt. determined by gel filtration and sucrose-density-gradient centrifugation was approx. 70000. The mitochondrial and supernatant isoenzyme 1 activities increased approximately 25-fold and 3.2-fold respectively in rats repeatedly injected with glucagon for 2 days. 2. An additional histidine-pyruvate aminotransferase (isoenzyme 2) was partially purified from both the mitochondrial and supernatant fractions of rat liver. Nearly identical properties were observed with both preparations. Isoenzyme 2 had pI5.2 and a pH optimum of 9.3. The enzyme was specific for pyruvate and did not function with 2-oxoglutarate. The order of effectiveness of amino donors was tyrosine = phenylalanine greater than histidine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values for histidine and phenylalanine were about 0.51 and 1.8 mM respectively. Km values for pyruvate were about 3.5mM with phenylalanine and 4.7mM with histidine as amino donors. Histidine inhibited phenylalanine aminotransferase activity of the enzyme. Gel filtration and sucrose-density-gradient centrifugation yielded a mol.wt. of approx. 90000. Neither the mitochondrial nor the supernatant isoenzyme 2 activity was elevated by glucagon injection.

scholarly journals The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing

1987 ◽  
Vol 245 (2) ◽  
pp. 575-582 ◽  
Author(s):  
H W Davidson ◽  
J C Hutton
Keyword(s):  
Secretory Granule ◽  
Gel Electrophoresis ◽  
Secretory Granules ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Carboxypeptidase B ◽  
Carboxypeptidase H ◽  
Insulin Secretory Granule

A carboxypeptidase B-like enzyme was detected in the soluble fraction of purified insulin secretory granules, and implicated in insulin biosynthesis. To investigate the role of this activity further, we purified the enzyme from rat insulinoma tissue by gel-filtration chromatography and affinity elution from p-aminobenzoyl-arginine. A yield of 42%, with a purification factor of 674 over the homogenate, was achieved. Analysis of the purified carboxypeptidase by SDS/polyacrylamide-gel electrophoresis under either reducing or non-reducing conditions showed it to be a monomeric protein of apparent Mr 55,000. The preparation was also homogeneous by high-performance gel-filtration chromatography. The enzyme bound to concanavalin A, showing it to be a glycoprotein. Amino acid analysis or chemical deglycosylation and SDS/polyacrylamide-gel electrophoresis indicated a protein Mr of 50,000, suggesting a carbohydrate content of approx. 9% by weight. The purified enzyme was able to remove basic amino acids from the C-terminus of proinsulin tryptic peptides to generate insulin, but did not further degrade the mature hormone. It was inhibited by EDTA, 1,10-phenanthroline and guanidinoethylmercaptosuccinic acid, and stimulated 5-fold by CoCl2. The pH optimum of the conversion of diarginyl-insulin into insulin was in the range 5-6, with little activity above pH 6.5. Activity was also expressed towards a dansylated tripeptide substrate (dansyl-phenylalanyl-leucyl-arginine; Km = 17.5 microM), and had a pH optimum of 5.5. These properties are indistinguishable from those of the activity located in secretory granules, and are compatible with the intragranular environment. The insulin-secretory-granule carboxypeptidase shared several properties of carboxypeptidase H from bovine adrenal medulla and pituitary. We propose that the carboxypeptidase that we purified is the pancreatic isoenzyme of carboxypeptidase H (crino carboxypeptidase B; EC 3.4.17.10), and is involved in the biosynthesis of insulin in the pancreatic beta-cell.

scholarly journals Effect of cations on structure-linked sedimentability of lysosomal hydrolases

1968 ◽  
Vol 109 (1) ◽  
pp. 149-154 ◽  
Author(s):  
M. Anthony Verity ◽  
R. Caper ◽  
W. Jann Brown
Keyword(s):  
High Speed ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Lysosomal Hydrolases ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
Bivalent Cation ◽  
Repeated Freezing ◽  
Triton X ◽  
Total Enzyme

1. A partially purified lysosomal preparation was obtained from mouse liver sucrose homogenates by differential and discontinuous gradient centrifugation. 2. Triton X-100 or repeated freezing and thawing of the lysosomal suspension (subfraction B) allowed comparison of free and activated values for acid phosphohydrolase, β-glucuronidase and N-acetylglucosaminidase in the presence and absence of ascorbate. 3. The distribution of hydrolase activities between supernatant and pellet after high-speed centrifugation was measured and the percentages of total enzyme found in the supernatant were: acid phosphohydrolase, 40·7; β-glucuronidase, 51; N-acetylglucosaminidase, 39·4. 4. Differential rates of elution of the three hydrolases from the membrane fraction occurred with increasing Na+ and K+ concentrations, whereas complex biphasic elution curves were obtained as a function of bivalent cation concentration with Ca2+ and Mg2+. 5. Sucrose-density-gradient centrifugation of frozen-and-thawed subfraction B demonstrated highly significant changes in the protein gradient profile in the presence of a low concentration of bivalent cation, indicating membrane aggregation and enzyme–membrane association. 6. The data provide further evidence for the nature of lysosomal enzyme binding and indicate the presence of different enzyme–membrane bonds conferring structure-linked latency upon individual lysosomal enzymes.

scholarly journals Proteolytic conversion of proinsulin into insulin. Identification of a Ca2+-dependent acidic endopeptidase in isolated insulin-secretory granules

1987 ◽  
Vol 246 (2) ◽  
pp. 279-286 ◽  
Author(s):  
H W Davidson ◽  
M Peshavaria ◽  
J C Hutton
Keyword(s):  
Secretory Granules ◽  
Subcellular Fractionation ◽  
Proteolytic Processing ◽  
Intermediate Form ◽  
Maximal Effect ◽  
Ph Optimum ◽  
Physiological Importance ◽  
Endogenous Insulin ◽  
Carboxypeptidase H

The nature of the endoproteolytic activity involved in the post-translational processing of proinsulin has been investigated in rat insulinoma tissue. 125I-proinsulin was converted by lysed insulin-secretory granules into insulin via an intermediate form identified as des-dibasic-proinsulin. This activity co-localized with immunoreactive (endogenous) insulin and carboxypeptidase H upon subcellular fractionation of the tissue, indicating a secretory-granular location. Under optimized conditions, conversion was quantitative. Inhibitor studies demonstrated that processing occurred by a reaction sequence involving cleavage on the C-terminal side of the pairs of basic amino acids, with subsequent removal of the newly exposed basic residues by carboxypeptidase H. Endoproteolytic activity was abolished by EDTA and CDTA (1,2-cyclohexanediaminetetra-acetic acid), but not by 1,10-phenanthroline or by group-specific inhibitors of serine, thiol or acidic proteinases. Inhibition by EDTA and CDTA could be reversed by both Ca2+ and Zn2+, although the former appeared to be the ion of physiological importance. Addition of Ca2+ in the absence of chelators stimulated endoproteinase activity, with a maximal effect at 5 mM, a concentration consistent with the intragranular environment. Similarly the pH optimum of 5.5 coincides with the prevailing intragranular pH. Together these properties suggest that the Ca2+-dependent endopeptidase described here is involved in vivo in the proteolytic processing of proinsulin.

scholarly journals Isolation and some molecular parameters of elastase-like normal proteinases from horse blood leucocytes

1976 ◽  
Vol 153 (2) ◽  
pp. 389-396 ◽  
Author(s):  
A Dubin ◽  
A Koj ◽  
J Chudzik
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polymorphonuclear Leucocytes ◽  
Molecular Parameters ◽  
Apparent Homogeneity ◽  
Repeated Freezing ◽  
Horse Blood ◽  
Nitrophenyl Ester

Cytoplasmic granules were isolated from horse blood polymorphonuclear leucocytes by the heparin method and extracted with 0.9% NaCl by repeated freezing. Soluble proteins were separated on a column of Sephadex G-75 followed by chromatography on a column of CM-Sephadex with a NaCl gradient. Gel filtration, density-gradient centrifugation, isoelectric focusing and 0.1% sodium dodecyl sulphate/polyacrylamide-gel electrophoresis at pH 7.0 and at pH 4.5 were used to determine molecular parameters of proteinases. Three enzymes hydrolysing both casein and N-benzyloxycarbonyl-L-alanine nitrophenyl ester were found in the granule extract: proteinase 1, mol.wt. 38000, pI5.3; proteinase 2A, mol.wt. 24500, pI8.8; and proteinase 2B, mol.wt. 20500, pI above 10. The latter two elastase-like proteinases were purified to apparent homogeneity.

Purification and Properties of Acid Phosphatase I From the Cellular Slime Mold Polysphondylium pallidum

1974 ◽  
Vol 52 (2) ◽  
pp. 126-136 ◽  
Author(s):  
Ilona A. Horgen ◽  
Paul A. Horgen ◽  
Danton H. O'Day
Keyword(s):  
Acid Phosphatase ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Neutral Hydrogen ◽  
Slime Mold ◽  
Cellular Slime Mold ◽  
Ph Optimum ◽  
Purification Method ◽  
Polysphondylium Pallidum ◽  
Cellular Slime

A procedure for the purification of a phosphomonoesterase, designated as acid phosphatase I, from the cellular slime mold Polysphondylium pallidum is described. Ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography are utilized in this purification method. The enzyme was judged to be homogeneous by gel filtration and by acylamide gel electrophoresis. The molecular weight of the enzyme was estimated by gel filtration and density gradient centrifugation to be 150 000 daltons. Acid phosphatase I was shown to be relatively heat stable, and it lost no activity when kept at 4 °C, pH 7.35, for over 30 days. The pH optimum was 3.5, but the enzyme was found to be more stable when kept near neutral hydrogen ion concentrations. P. pallidum acid phosphatase I was most effective using the natural substrates, fructose-1,6-pbosphate, β-glycerolphosphate, and 5′-mononucleotides. Various compounds including known phosphatase inhibitors were tested as to their effect on the activity of the enzyme. The slime-mold acid phosphatase appears in many ways to be a typical acid phosphomonoesterase.

Sign in / Sign up

Export Citation Format

Share Document