QUANTITATIVE STUDIES ON ISOLATED PANCREATIC ISLETS OF MAMMALS

1963 ◽  
Vol 42 (4) ◽  
pp. 615-624 ◽  
Author(s):  
Claes Hellerström ◽  
Bo Hellman
Keyword(s):  
Enzyme Activity ◽  
B Cells ◽  
Enzyme Level ◽  
Enzyme Reaction ◽  
Histochemical Staining ◽  
Pancreatic Tissue ◽  
Free Access ◽  
Isolated Pancreatic Islets ◽  
Insulin Synthesis ◽  
Dipeptidase Activity

ABSTRACT Microtitrimetric assays of dipeptidase activity were performed in isolated pancreatic islet tissue from mice. Considerable enzyme activity was found in both the endocrine and exocrine pancreas of normal mice, the enzyme level of the exocrine parenchyma being significantly higher. In obesehyperglycaemic mice with free access to food, isolated islets of Langerhans had a much higher enzyme activity than in normal animals. The increased islet dipeptidase activity in the obese-hyperglycaemic animals may, at least in part, be accounted for by their higher proportion of B cells. The intense insulin synthesis and renewal of B cells in these animals have been considered as alternative explanations. Histochemical staining for leucine aminopeptidase revealed a moderate enzyme reaction in both the endocrine and exocrine pancreatic tissue of normal and obese-hyperglycaemic mice.

scholarly journals QUANTITATIVE STUDIES ON ISOLATED PANCREATIC ISLETS OF MAMMALS: ADENOSINE TRIPHOSPHATASE ACTIVITY IN NORMAL AND OBESE-HYPERGLYCEMIC MICE

1964 ◽  
Vol 12 (7) ◽  
pp. 491-497 ◽  
Author(s):  
INGE-BERT TÄLJEDAL ◽  
BO HELLMAN ◽  
CLAES HELLERSTRÖM
Keyword(s):  
Enzyme Activity ◽  
Adenosine Triphosphatase ◽  
Endocrine Pancreas ◽  
Islet Cells ◽  
Adenosine Diphosphate ◽  
Histochemical Staining ◽  
Isolated Pancreatic Islets ◽  
Quantitative Studies ◽  
Substrate Cleavage ◽  
Substrate Medium

Microchemical and histochemical methods were used for the characterization, localization and assay of adenosine triphosphate (ATP) splitting enzymes in homogenates and sections of the endocrine pancreas from obese-hyperglycemic mice and their lean litter mates. The following observations were made: 1. Dephosphorylation of ATP was maximal at pH 9.1. It was strongly stimulated by magnesium ions at an optimal concentration of 1 mM. ATP cleavage was inhibited by adenosine diphosphate, sodium azide and p-chloromercuribenzoic acid. The addition of l-cysteine, sodium cyanide or sodium fluoride to the substrate medium had no effect on the enzyme activity. Substitution of ATP by equimolar amounts of other phosphate esters in the medium considerably reduced the substrate cleavage. These results are taken as evidence for the presence of sulfhydryl-dependent adenosine triphosphatase (ATPase) in the islet tissue. 2. Histochemical staining revealed a strong ATP splitting enzyme activity in the capillaries and walls of larger blood vessels throughout the pancreas; a rather weak and diffuse cytoplasmic reaction being found in the islet cells. 3. Microchemical assays revealed a lower cleavage of ATP in the islets as compared with the exocrine pancreas and the liver. The cleavage of ATP was more intense in the islets of the obese-hyperglycemic mice than in those of the lean litter mates. 4. Starvation for 7 days induced no significant changes in the enzyme activity of the endocrine pancreas.

Ultrastructural Cytochemistry of Acetylcholinesterase Enzyme Activity in Pancreatic Tissue Transplants in Rats

1994 ◽  
Vol 3 (2) ◽  
pp. 171-177 ◽  
Author(s):  
Ernest Adeghate ◽  
Tibor Donáth
Keyword(s):  
Enzyme Activity ◽  
Enzyme Reaction ◽  
Ultrastructural Level ◽  
Pancreatic Tissue ◽  
Reaction Products ◽  
Nerve Fibres ◽  
Tissue Transplants ◽  
Acetylcholinesterase Reaction ◽  
Acetylcholinesterase Enzyme ◽  
Product Forms

The distribution of acetylcholinesterase (AChE) at the ultrastructural level was investigated in normal and in pancreatic fragments transplanted for 56 days into the anterior eye chamber of heterologous rats using enzyme cytochemical methods. Acetylcholinesterase reaction products were seen on the basal surface of the acinar cells in normal pancreas. Acetylcholinesterase enzyme activity was also detected on the axolemma of the surviving nerve fibres. This enzyme reaction product forms alternating thick and thin bands on the axolemma. Some of these AChE-positive nerve fibres accompany blood vessels that also survive after transplantation. AChE were seen in cytoplasm adjacent to the surviving alpha and pancreatic polypeptide cells. We conclude that the ability of some neurons and cells to produce and or store acetylcholinesterase is still retained after transplantation of pancreatic tissue into the anterior eye chamber of rats.

QUANTITATIVE STUDIES ON ISOLATED PANCREATIC ISLETS OF MAMMALS: ENZYMIC HYDROLYSIS OF NUCLEOSIDE DIPHOSPHATES AND p-NITROPHENYL PHOSPHATE IN NORMAL AND CORTISONE-TREATED RATS

1966 ◽  
Vol 36 (2) ◽  
pp. 115-NP ◽  
Author(s):  
I.-B. TÄLJEDAL ◽  
B. HELLMAN ◽  
C. HELLERSTRÖM
Keyword(s):  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Sodium Fluoride ◽  
Acid Phosphatase Activity ◽  
Staining Intensity ◽  
Histochemical Staining ◽  
Enzymic Hydrolysis ◽  
Isolated Pancreatic Islets ◽  
Hydrolysis Of

SUMMARY Chemical micromethods and histochemical staining were employed for studies of the enzymic hydrolysis of inosine diphosphate (IDP) and adenosine diphosphate (ADP) and the non-specific acid phosphatase activity of the endocrine pancreas from normal and cortisone-treated rats. The following observations were made: 1. Enzymic dephosphorylation of IDP and ADP was maximal at about pH 8·0. Magnesium and manganese ions enhanced the phosphate liberation, the hydrolysis of ADP being more activated than that of IDP. A marked inhibition of enzyme activity towards either substrate was produced by sodium fluoride, sodium cyanide and ethylene-diaminotetraacetate. Acid phosphatase activity was maximal at about pH 5·5, a tendency for a second activity optimum was noted at about pH 4·0. Acid phosphatase activity was markedly inhibited by sodium fluoride, tartaric acid and formaldehyde. 2. Histochemical staining revealed marked enzyme activity towards IDP and ADP in the capillaries and walls of the large blood vessels throughout the pancreas, whereas the islet cells displayed a moderate reaction. The staining intensity was the same with IDP as with ADP. 3. Cortisone administration reduced the rate of cleavage of both IDP and ADP in both the endocrine and the exocrine pancreas, but the enzymic splitting of these substrates remained unchanged in the liver. Acid phosphatase activity was not influenced in any of these tissues by the steroid treatment.

scholarly journals Expression of Glucokinase in Glucose-Unresponsive Human Fetal Pancreatic Islet-Like Cell Clusters1

1997 ◽  
Vol 82 (3) ◽  
pp. 943-948
Author(s):  
Jian Tu ◽  
Bernard E. Tuch
Keyword(s):  
Glucose Utilization ◽  
Glucose Sensor ◽  
Genetic Disorder ◽  
Fold Increase ◽  
Pancreatic Tissue ◽  
Maximal Velocity ◽  
Β Cell ◽  
Western Blots ◽  
Insulin Synthesis ◽  
Glucose Challenge

Abstract Glucokinase (GK) is the glucose sensor in the adult β-cell, resulting in fuel for insulin synthesis and secretion. Defects in this enzyme in the β-cell are responsible for the genetic disorder maturity-onset diabetes of the young, with the β-cell being unable to secrete insulin appropriately when challenged with glucose. The human fetalβ -cell is also unable to secrete insulin when exposed to glucose, but whether GK is present and functional in this developing cell is unknown. To determine the expression of GK in human fetal pancreatic tissue, cytosolic protein was extracted from human fetal islet-like cell clusters (ICCs) at 17–19 weeks gestation and examined for protein content and enzyme activity. On Western blots, a single band corresponding to GK was seen at 52 kDa, and this was similar to that obtained from human adult islets. The maximal velocity (Vmax) of GK was less in fetal ICCs than that in adult islets (8.7 vs. 20.7 nmol/mg protein·h); similar Km values were found in both ICCs and islets. No attempt was made to determine which cells in an ICC contained GK. Glucose utilization was determined radiometrically; the Vmax of the high Km component was less in ICCs than in islets (31.3 pmol/ICC·h vs. 101.4 pmol/islet·h). Culture of ICCs for 3–7 days in medium containing 11.2 mmol/L glucose resulted in a 3.7-fold increase in the Vmax of GK and a 1.8-fold increase in glucose utilization. These enhanced activities of glucose phosphorylation and glycolysis, however, did not lead to the β-cell being able to secrete insulin when exposed to glucose. In conclusion, glucokinase is present and functional in human fetal ICCs, but the inability of the human fetal β-cell to secrete insulin in response to an acute glucose challenge is not due to immaturity of this enzyme.

scholarly journals Decreased rate of ketone-body oxidation and decreased activity of d-3-hydroxybutyrate dehydrogenase and succinyl-CoA:3-oxo-acid CoA-transferase in heart mitochondria of diabetic rats

1986 ◽  
Vol 240 (1) ◽  
pp. 49-56 ◽  
Author(s):  
L Grinblat ◽  
L F Pacheco Bolaños ◽  
A O Stoppani
Keyword(s):  
Enzyme Activity ◽  
Enzyme Reaction ◽  
Diabetic Rats ◽  
Heart Mitochondria ◽  
Maximal Velocity ◽  
Coa Transferase ◽  
Slow Adaptation ◽  
Significant Difference ◽  
Metabolic Conditions ◽  
Onset Of Diabetes

Heart mitochondria from chronically diabetic rats (‘diabetic mitochondria’), in metabolic State 3, oxidized 3-hydroxybutyrate and acetoacetate at a relatively slow rate, as compared with mitochondria from normal rats (‘normal mitochondria’). No significant differences were observed, however, with pyruvate or L-glutamate plus L-malate as substrates. Diabetic mitochondria also showed decreased 3-hydroxybutyrate dehydrogenase and succinyl-CoA: 3-oxoacid CoA-transferase activities, but cytochrome content and NADH-dehydrogenase, succinate dehydrogenase, cytochrome oxidase and acetoacetyl-CoA thiolase activities proved normal. The decrease of 3-hydroxybutyrate dehydrogenase activity was observed in diabetic mitochondria subjected to different disruption procedures, namely freeze-thawing, sonication or hypoosmotic treatment, between pH 7.5 and 8.5, at temperatures in the range 6-36 degrees C, and in the presence of L-cysteine. Determination of the kinetic parameters of the enzyme reaction in diabetic mitochondria revealed diminution of maximal velocity (Vmax) as its outstanding feature. The decrease in 3-hydroxybutyrate dehydrogenase in diabetic mitochondria was a slow-developing effect, which reached full expression 2-3 months after the onset of diabetes; 1 week after onset, no significant difference between enzyme activity in diabetic and normal mitochondria could be established. Insulin administration to chronically diabetic rats for 2 weeks resulted in limited recovery of enzyme activity. G.l.c. analysis of fatty acid composition and measurement of diphenylhexatriene fluorescence anisotropy failed to reveal significant differences between diabetic and normal mitochondria. The Arrhenius-plot characteristics for 3-hydroxybutyrate dehydrogenase in membranes of diabetic and normal mitochondria were similar. It is assumed that the variation of the assayed enzymes in diabetic mitochondria results from a slow adaptation to the metabolic conditions resulting from diabetes, rather than to insulin deficiency itself.

INFLUENCE OF THE ISLET A AND B CELLS ON THE EXOCRINE PANCREATIC TISSUE IN THE DUCK

1962 ◽  
Vol 48 (1-2) ◽  
pp. 137-141 ◽  
Author(s):  
Arne Wallgren ◽  
Bo Hellman
Keyword(s):  
B Cells ◽  
Pancreatic Tissue ◽  
A And B Cells

scholarly journals QUANTITATIVE AND HISTOCHEMICAL STUDIES ON THE DESORPTION AND READSORPTION OF ALDOLASE IN CROSS-STRIATED MUSCLE

1969 ◽  
Vol 17 (5) ◽  
pp. 314-320 ◽  
Author(s):  
H. ARNOLD ◽  
J. NOLTE ◽  
D. PETTE
Keyword(s):  
Enzyme Activity ◽  
Actin Filaments ◽  
Phosphate Buffer ◽  
Striated Muscle ◽  
Psoas Muscle ◽  
Histochemical Staining ◽  
Intracellular Location ◽  
Successive Extraction ◽  
Complete Extraction

Complete extraction of aldolase from minced rabbit psoas muscle was achieved by successive extraction steps in 0.1 M phosphate buffer. Aldolase was then readsorbed quantitatively to the depleted myofibrils. Extraction, readsorption and a final redsorption of the enzyme were followed quantitatively by enzyme activity determinations and qualitatively by histochemical staining of aldolase. The intracellular location of the readsorbed enzyme was found to be identical with that of aldolase in native muscle. In both cases, aldolase was localized within the isotropic bands. These results as well as the previously demonstrated binding of the enzyme to F-actin suggest that aldolase is located within the interfilamentary sarcoplasm of the isotropic bands and is probably also bound in vivo to the actin filaments.

scholarly journals The possible mechanisms by which phanoside stimulates insulin secretion from rat islets

2007 ◽  
Vol 192 (2) ◽  
pp. 389-394 ◽  
Author(s):  
Nguyen Khanh Hoa ◽  
Åke Norberg ◽  
Rannar Sillard ◽  
Dao Van Phan ◽  
Nguyen Duy Thuan ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
B Cells ◽  
Pancreatic Islets ◽  
Insulin Release ◽  
Pertussis Toxin ◽  
Insulin Response ◽  
Gk Rat ◽  
Isolated Pancreatic Islets ◽  
Gk Rats ◽  
Rat Islets

We recently showed that phanoside, a gypenoside isolated from the plant Gynostemma pentaphyllum, stimulates insulin secretion from rat pancreatic islets. To study the mechanisms by which phanoside stimulates insulin secretion. Isolated pancreatic islets of normal Wistar (W) rats and spontaneously diabetic Goto-Kakizaki (GK) rats were batch incubated or perifused. At both 3.3 and 16.7 mM glucose, phanoside stimulated insulin secretion several fold in both W and diabetic GK rat islets. In perifusion of W islets, phanoside (75 and 150 μM) dose dependently increased insulin secretion that returned to basal levels when phanoside was omitted. When W rat islets were incubated at 3.3 mM glucose with 150 μM phanoside and 0.25 mM diazoxide to keep K-ATP channels open, insulin secretion was similar to that in islets incubated in 150 μM phanoside alone. At 16.7 mM glucose, phanoside-stimulated insulin secretion was reduced in the presence of 0.25 mM diazoxide (P<0.01). In W islets depolarized by 50 mM KCl and with diazoxide, phanoside stimulated insulin release twofold at 3.3 mM glucose but did not further increase the release at 16.7 mM glucose. When using nimodipine to block L-type Ca2+ channels in B-cells, phanoside-induced insulin secretion was unaffected at 3.3 mM glucose but decreased at 16.7 mM glucose (P<0.01). Pretreatment of islets with pertussis toxin to inhibit exocytotic Ge-protein did not affect insulin response to 150 μM phanoside. Phanoside stimulated insulin secretion from Wand GK rat islets. This effect seems to be exerted distal to K-ATP channels and L-type Ca2+ channels, which is on the exocytotic machinery of the B-cells.

scholarly journals ELECTRON MICROSCOPE OBSERVATIONS ON THE SURFACE ADENOSINE TRIPHOSPHATASE-LIKE ENZYMES OF HELA CELLS INFECTED WITH HERPES VIRUS

1963 ◽  
Vol 19 (2) ◽  
pp. 337-347 ◽  
Author(s):  
M. A. Epstein ◽  
S. J. Holt
Keyword(s):  
Enzyme Activity ◽  
Hela Cells ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Enzyme Reaction ◽  
Thin Sections ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Simplex Virus ◽  
Vacuolar Membranes

HeLa cells infected with herpes simplex virus have been examined in thin sections by electron microscopy after cytochemical staining for the presence of surface enzymes splitting adenosine triphosphate. As with uninfected HeLa cultures (18), the opaque enzyme reaction product was localized at the plasma membranes of about half the cells, tending to be present where there were microvilli and absent on smooth surfaces. Where mature extracellular herpes particles were found in association with cell membranes showing the enzyme activity, they were invariably likewise stained, and conversely, those mature particles which lay close against cells without reaction product at the surface were themselves free of it. Particles found budding into cytoplasmic vacuoles were also always without opaque deposit since this was never seen at vacuolar membranes, even in cells having the activity at the surface. The enzyme reaction product thus provided a marker indicating the manner in which the particles escape from cells and mature by budding out through cellular membranes, carrying, in the process, a portion of the latter on to themselves to form the outer viral limiting membrane. In some instances, virus particles were observed with more opaque material covering them than was present at the cell membrane with which they were associated. This finding has been taken as evidence for a physiological waxing and waning of surface enzyme activity of adenosine triphosphatase type. The fine structure of the mature extracellular virus as prepared here, using glutaraldehyde fixation, is also recorded. The observations and interpretations are discussed in full.

Influence of carcinogens and group-specific compounds on DNAse II

1969 ◽  
Vol 47 (10) ◽  
pp. 987-989 ◽  
Author(s):  
Marvin S. Melzer
Keyword(s):  
Enzyme Activity ◽  
Enzyme Level ◽  
Iodoacetic Acid ◽  
Dnase Ii ◽  
Histidine Residues ◽  
Carcinogenic Process

A number of cancer-causing and group-specific compounds were tested for their effects on the activity of DNAse II. The following were almost completely inhibitory: iodoacetic acid, N-bromosuccinimide (at an N-bromosuccinimide/enzyme level ≥ 24), and H2O2 (at an H2O2/enzyme level > 10 000). Either noninhibitory or less than 30% inhibitory were iodoacetamide and diisopropylfluorophosphate. Noninhibitory were such carcinogens as beta-butyrolactone, diepoxybutane, 3-hydroxyxanthine, and ascaridole. Also noninhibitory was malonaldehyde.From these results (and others in the literature), it was concluded that (1) the carcinogens tested (at least in their unmetabolized forms) might not act directly on DNAse II in the critical reaction of the carcinogenic process, and (2) tryptophan, methionine, and/or histidine residues play important roles in the enzyme activity.

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