The occurrence of pituitary prolactin depletion–transformation in lactating rats: dependence on strain of rats, homogenization conditions and method of assay

1987 ◽  
Vol 113 (1) ◽  
pp. 71-80 ◽  
Author(s):  
D. M. Lawson ◽  
D. J. Haisenleder ◽  
R. R. Gala ◽  
J. A. Moy
Keyword(s):  
Molecular Form ◽  
Gel Filtration ◽  
Molecular Forms ◽  
Sprague Dawley ◽  
Rapid Changes ◽  
Prolactin Content ◽  
Pituitary Prolactin ◽  
Triton X ◽  
Benzamidine Hydrochloride ◽  
Molecular Nature

ABSTRACT The objectives of this study were to determine (1) whether pre-release transformation (depletion) of pituitary prolactin occurs as the result of suckling to the same extent in several strains of lactating rats, (2) the molecular nature of the transformed hormone, (3) whether the quantity of transformed (depleted) prolactin recovered is dependent upon the type of homogenization buffer used and (4) whether the method of assay influences the extent to which transformed prolactin is detected. During the course of these experiments other factors such as the methods of handling and storing pituitaries and homogenates were also found to influence the amount of prolactin recovered. The results indicated that transformation of prolactin is a very labile event which is affected by many factors. Strain and supplier of rats was critical to the observation of suckling-induced depletion of prolactin, with Spartan- and Holtzman-derived Sprague–Dawley strains exhibiting the most consistent responses. When transformation was observed, it mattered little which buffer was used for homogenization; however, alkaline or acidic buffers extracted more prolactin than did neutral buffers. Triton X-100 also significantly enhanced the efficiency of extraction by neutral buffers. Maintaining pituitaries on dry ice immediately upon removal from the animal increased the amount of prolactin recovered, as did freezing the homogenate for 1–5 weeks before assay. The addition of the protease inhibitor, benzamidine hydrochloride, did not affect the pituitary content of prolactin. Assay of prolactin by polyacrylamide electrophoresis and densitometry yielded more prolactin than either radioimmunoassay or the Nb2 lymphoma bioassay. The molecular nature of pituitary prolactin, extracted at neutral pH, as judged by gel filtration was altered slightly but consistently by suckling, such that large molecular forms increased at the expense of the smallest molecular form. We conclude from these studies that great care must be exercised when attempting to characterize dynamic changes in pituitary prolactin content in lactating rats. Strain and supplier of rats, methods of handling and storing pituitaries, types of buffers used for homogenization and methods of assay all influence the amount of prolactin recovered and can influence the extent to which rapid changes in pituitary prolactin are detected. J. Endocr. (1987) 113, 71–80

Molecular Forms of Purified Cytoplasmatic and Membrane Bound Bovine-Brain-Acetylcholinesterase Solubilized by Different Methods

1981 ◽  
Vol 36 (11-12) ◽  
pp. 968-972 ◽  
Author(s):  
K.-P. Rueß ◽  
M. Liefländer
Keyword(s):  
Molecular Form ◽  
Bovine Brain ◽  
Water Soluble ◽  
Molecular Forms ◽  
Enzyme Complex ◽  
Detergent Enzyme ◽  
Neuronal Tissues ◽  
Membrane Bound ◽  
Triton X ◽  
Brain Acetylcholinesterase

Abstract Membrane bound, Triton X-100 solubilized bovine nucleus caudatus acetylcholinesterase is sedimenting in presence of Triton X-100 concentrations higher than the CMC as a 10.5 S-detergent-enzyme complex. There is evidence that this complex does neither represent the molecular enzyme arrangement present in the membrane, nor the molecular form originally released from the membrane. The purified, cytoplasmatic acetylcholinesterase is sedimenting as a 10.5 S-form too. This form is clearly to be distinguished from the detergent enzyme complex, for it is obviously not capable of aggregating, whereas the 10.5 S-detergent-enzyme complex aggregates on detergent removal to defined water soluble oligomers with sedimentation coefficients of 16 S (700000 ± 10000), 20.6 S (960000 ± 60000) and 23.3 S (~ 1200000). In contrast to acetylcholinesterase from erythrocytes this aggregation is not easily reversibly by incubation with Triton X-100, reflecting differences in the hydrophobic part of the enzymes. Purified acetylcholinesterase solubilized without detergent under autolytic or tryptic conditions is mainly sedimenting as a 4.5 S-form. Such slow sedimenting forms detected in crude solubilisates of neuronal tissues, may originate at least partially form autolytic solubilization.

Effects of ethanol and inhibitors of its metabolism on the circulating levels of Met-enkephalin in greyhounds

1989 ◽  
Vol 120 (3) ◽  
pp. 473-480 ◽  
Author(s):  
S. Medbak ◽  
D. F. J. Mason ◽  
L. H. Rees
Keyword(s):  
Oral Administration ◽  
Active Component ◽  
Gel Filtration ◽  
Bilateral Adrenalectomy ◽  
Molecular Forms ◽  
Gel Filtration Chromatography ◽  
Conscious Animals ◽  
Circulating Levels ◽  
Molecular Nature ◽  
Different Doses

ABSTRACT The mechanisms involved in the release of Metenkephalin-like immunoreactivity (MLI) into the circulation following oral administration of ethanol and chlorpropamide were investigated in dogs. The origin of plasma MLI and the sites where it may be metabolized were also studied. Moreover, the molecular nature of circulating MLI was characterized. In conscious animals oral administration of ethanol (0·15 ml/kg) led to a significant (P<0·01) rise in plasma MLI concentrations in chlorpropamidepretreated animals from a basal level of 43 ± 6 (mean ± s.e.m.) to a peak of 66 ± 8 ng/l. Similar rises in MLI concentrations were observed following administration of ethanol with disulfiram and ethanol with chlorpropamide and captopril. In contrast, the administration of ethanol alone or ethanol with 4-methylpyrazole resulted in a decrease in plasma MLI concentrations. Comparisons of two different doses of i.v. acetaldehyde, the first metabolite of ethanol, showed that plasma MLI concentrations rose significantly (P<0·05) only after the larger dose (8 mg/kg), rising from 45±7 to 81 ± 18 ng/l. These results suggest that acetaldehyde is the active component in the chlorpropamide+ ethanol-induced MLI secretion. Plasma MLI was also measured following acetaldehyde infusion in adrenalectomized dogs with and without hexamethonium treatment. Acute bilateral adrenalectomy resulted in a decrease (P<0·05) in plasma MLI concentrations, but the levels remained detectable. Moreover, subsequent acetaldehyde infusion led to rises in plasma MLI similar to those observed in animals with intact adrenals. These MLI responses were not altered by the concurrent i.v. administration of hexamethonium. Gel filtration chromatography revealed that Met-enkephalin exists in the circulation predominantly in larger molecular forms with approximate sizes of 18 000 and 8000 Da in the basal state, after stimulation and following adrenalectomy. Journal of Endocrinology (1989) 120, 473–480

scholarly journals Physicochemical and Biochemical Properties of Commercially Available Urokinase Preparations

1977 ◽  
Author(s):  
M. Nishida ◽  
H. Nishimaki ◽  
S. Miyake ◽  
T. Suyama ◽  
S. Morisue
Keyword(s):  
Gel Electrophoresis ◽  
Molecular Form ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Biochemical Properties ◽  
Molecular Forms ◽  
Starch Gel Electrophoresis ◽  
Starch Gel ◽  
The Difference ◽  
Fresh Urine

Comparative studies were made on eleven urokinase preparations commercially available. Analysis by both gel filtration and sodium dodecyl gel electrophoresis revealed the variety of molecular forms of the activator in the preparations showing molecular weight approximately 54,000 (A), 47,000 (B) and 34,000 (C). Starch gel electrophoresis at pH 8.8 indicated that (B) and (C) moved to anode whereas (A) and the active component of fresh urine slightly moved to the cathod. Proteolytic digestion of (A) produced the same component as shown by (B) and (C) on starch gel electrophoresis. Plasminogen activating activity of (B) and (C) was found to be less than that of (A) when measured by the procedure of CTA fibrinolytic method with the physiological blood level of plasminogen. The present data suggest that variety of molecular form in the preparation may be due to the difference of purification procedure in view of proteolytic degradation of the enzyme, and (A) seems to be the naturally occuring type of urokinase in urine.

RECOVERY OF PITUITARY SECRETION OF GONADOTROPHINS AND PROLACTIN DURING RE-FEEDING AFTER CHRONIC RESTRICTED FEEDING IN FEMALE RATS

1976 ◽  
Vol 69 (3) ◽  
pp. 329-339 ◽  
Author(s):  
YOSHIHIKO NAKANISHI ◽  
JUNICHI MORI ◽  
HIROSHI NAGASAWA
Keyword(s):  
Reproductive Function ◽  
Serum Levels ◽  
The Other ◽  
Female Rats ◽  
Restricted Feeding ◽  
Sprague Dawley ◽  
Serum Lh ◽  
Prolactin Content ◽  
Almost All ◽  
Pituitary Prolactin

SUMMARY The effects on reproductive function of restriction to one half of the normal food intake for 30 or 60 days and subsequent unrestricted feeding were investigated in adult female Sprague–Dawley rats. Restricted feeding resulted in the cessation of oestrous cycles within 9 to 28 days, associated with decreased pituitary, ovarian and uterine weights. Pituitary content and concentration of FSH were increased by restricted feeding and the levels of FSH in 60 day underfed rats were about twice as high as those of the controls at dioestrus. There was little difference in pituitary LH content between the underfed groups and the controls at the end of restricted feeding. Pituitary LH concentration was significantly higher in rats underfed for 60 days than in the control rats. Pituitary prolactin content was one half and one third of that in control rats in rats underfed for 30 and 60 days respectively. Pituitary prolactin concentration was also decreased by restricted feeding. At the end of restricted feeding, no differences in serum LH and prolactin levels were found between the groups, whereas the serum FSH level in 60 day underfed rats was higher than that in the controls at dioestrus. After re-feeding, normal oestrous cycles returned within 3–5 days in almost all rats, regardless of the length of the previous cessation of oestrous cycles. Pituitary contents of FSH and LH in underfed rats decreased after re-feeding following the return of oestrous cycles. The rate of decrease was much greater in 60 day underfed rats than that in 30 day underfed rats. On the other hand, serum levels of FSH, LH and prolactin in these underfed rats were increased by re-feeding and the levels in the evening of the first pro-oestrus were higher than those in the morning of dioestrus and pro-oestrus. Serum levels of these hormones increased more in 60 day underfed rats than in the other groups at any stage. After re-feeding, pituitary, ovarian and uterine weights increased and the uterine epithelial layer was clearly repaired in both underfed groups, although not always to the control levels by the first oestrus. The end-bud system of the mammary gland which degenerated during restricted feeding was comparable to that of the controls at the first oestrus after re-feeding in 30 day underfed rats, but not in 60 day underfed rats.

scholarly journals Molecular forms of phosphatase and ribonuclease in phosphate deficient and N,N-dimethylmorpholinium chloride treated Spirodela oligorrhiza (Lemnaceae)

2015 ◽  
Vol 48 (1) ◽  
pp. 65-85 ◽  
Author(s):  
J. S. Knypl
Keyword(s):  
Gel Filtration ◽  
Molecular Forms ◽  
Membrane Material ◽  
Alkaline Phosphatases ◽  
Ph Optimum ◽  
Membrane Bound ◽  
Soluble Acid ◽  
Spirodela Oligorrhiza ◽  
Triton X ◽  
Membrane Bound Enzymes

Soluble, membrane bound, and extracellular phosphatases (EC 3.1.3.2 and 3.1.3.1) of control, N,N-dimethylmorphołinłum chloride (DMMC) treated, and phosphate deficient (-P) axenic <i>Spirodela oligorrhiza</i> plants were analysed by Sephadex G-150 gel filtration. Soluble, acid enzymes of control plants were separated into two molecular forms with apparent MW ≥ 400 000 and 85 000. Phosphatase with MW 34 000 replaced the latter isoenzyme in the presence of DMMC. Two alkaline enzymes with apparent MW 210 000 and 36 000 were detected in -P plants. Triton X-100 solubilized a number of acid and alkaline phosphatases from membrane material. DMMC caused the appearance of two membrane bound enzymes (MW 48 000 and 14 000) which were not detected in the control. Senescimg control and DMMC treated plants released an acid phosphatase (MW 48 000; pH optimum 5.2) into the nutrient medium. -P plants released, in addition ,an alkaline phosphatase (MW 170,000; pH optimum 7.8-8.2). Ribonucleases (EC 2.7.7.17.) with apparent MW 31000 and 28 000 daltons were induced by DMMC and -P, respectively.

Release of cholecystokinin and secretin by sodium oleate in dogs: molecular form and bioactivity

1992 ◽  
Vol 262 (1) ◽  
pp. G35-G43 ◽  
Author(s):  
G. Sun ◽  
T. M. Chang ◽  
W. J. Xue ◽  
J. F. Wey ◽  
K. Y. Lee ◽  
...  
Keyword(s):  
Pancreatic Juice ◽  
Sodium Oleate ◽  
Molecular Form ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Conscious Dogs ◽  
Oral Ingestion ◽  
Intraduodenal Infusion ◽  
Predominant Form ◽  
Duodenal Lumen

The release of cholecystokinin (CCK) and secretin into both circulation and duodenal lumen, after intraduodenal perfusion with sodium oleate or oral ingestion of fat, was studied in anesthetized and conscious dogs, respectively. Intraduodenal infusion with sodium oleate (4 mmol.kg.-1.h-1, pH 9.5) in anesthetized dogs with diversion of bile and pancreatic juice stimulated the release of both CCK and secretin not only into the circulation but also into the duodenal lumen. The concentration of CCK and secretin in the luminal perfusate increased from 0.2 +/- 0.1 to 2.1 +/- 0.4 nM and 0.34 +/- 0.16 to 2.59 +/- 0.63 nM, respectively. Intraduodenal infusion of NaHCO3 solution at pH 9.5 did not result in release of either hormone. Luminal release of both hormones was also observed by intraduodenal infusion of sodium oleate in the dogs without diversion of bile and pancreatic juice, albeit at lower concentrations than those released in the dogs with diversion. Analysis of the molecular form of luminal secretin by gel filtration, ion-exchange chromatography, and high-performance liquid chromatography showed only a single form of secretin with molecular size, hydrophobicity, and charge similar to those of natural porcine secretin. In contrast, multiple forms of CCK were released into both circulation and duodenal lumen with CCK-58 as the predominant form. In conscious dogs, CCK-58 was also found to be the predominant form of CCK released into the circulation after oral ingestion of fat.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. II. Lectin binding to the isolated glycoproteins of normal and malignant gastric mucosa.

1984 ◽  
Vol 32 (7) ◽  
pp. 690-696 ◽  
Author(s):  
J Fischer ◽  
G Uhlenbruck ◽  
P J Klein ◽  
M Vierbuchen ◽  
R Fischer
Keyword(s):  
Molecular Weight ◽  
Gastric Mucosa ◽  
High Molecular Weight ◽  
Lectin Binding ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Gastric Cancer Cells ◽  
Tissue Sections ◽  
Gradient Gel Electrophoresis ◽  
Triton X

Using affinity chromatography on HPA-, PNA-, Con A, and WGA-agarose columns only a part (10-30%) of the high molecular weight mucous glycoproteins could be isolated from the Triton X-100 solubilized components of normal as well as carcinomatous gastric mucosa. The main part of the mucus was not bound by the lectins, which corresponds to our earlier lectin histochemical observations on paraffin-embedded tissue sections. The lectin-bound mucous glycoproteins had a relatively lower molecular weight, ranging from about 250-1,000 kilodaltons, as indicated by polyacrylamide gradient gel electrophoresis and by gel filtration on Biogel A 1.5 m column. In gas chromatographic analysis the molar ratio of aminohexoses to galactose was found to be much higher (3:1) in the lectin-bound mucous substances than in the whole high molecular weight mucus (1:1). This finding indicates that lectins have a higher affinity to the hexosamine rich components of mucus, which may be special forms of mucous glycoprotein molecules or the incompletely glycosylated core and backbone regions of the oligosaccharide chains of mucus. Extremely high hexosamine values (10:1) were found in the PNA isolated mucus of gastric adenocarcinoma. Since it is known that PNA binds to the terminal disaccharide, beta-galactose-(1-3)-N-acetylgalactosamine, which is localized at the reducing end of the oligosaccharide chains of mucus, it is highly probable that the elongation of the oligosaccharide side chains is disturbed in gastric cancer cells.

scholarly journals Globular and asymmetric acetylcholinesterase in frog muscle basal lamina sheaths.

1986 ◽  
Vol 102 (3) ◽  
pp. 762-768 ◽  
Author(s):  
M Nicolet ◽  
M Pinçon-Raymond ◽  
F Rieger
Keyword(s):  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Muscle Fibers ◽  
Frog Muscle ◽  
Motor Endplate ◽  
Molecular Forms ◽  
Nonionic Detergent ◽  
Plasmic Membrane ◽  
Triton X

After denervation in vivo, the frog cutaneus pectoris muscle can be led to degenerate by sectioning the muscle fibers on both sides of the region rich in motor endplate, leaving, 2 wk later, a muscle bridge containing the basal lamina (BL) sheaths of the muscle fibers (28). This preparation still contains various tissue remnants and some acetylcholine receptor-containing membranes. A further mild extraction by Triton X-100, a nonionic detergent, gives a pure BL sheath preparation, devoid of acetylcholine receptors. At the electron microscope level, this latter preparation is essentially composed of the muscle BL with no attached plasmic membrane and cellular component originating from Schwann cells or macrophages. Acetylcholinesterase is still present in high amounts in this BL sheath preparation. In both preparations, five major molecular forms (18, 14, 11, 6, and 3.5 S) can be identified that have either an asymmetric or a globular character. Their relative amount is found to be very similar in the BL and in the motor endplate-rich region of control muscle. Thus, observations show that all acetylcholinesterase forms can be accumulated in frog muscle BL.

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

1990 ◽  
Vol 10 (2) ◽  
pp. 131-139
Author(s):  
Oyewole Adeyemo ◽  
E. O. Okegbile ◽  
O. O. Olorunsogo
Keyword(s):  
Molecular Weight ◽  
Membrane Protein ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Boar Sperm ◽  
Sperm Membrane ◽  
Triton X

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.

scholarly journals CCK-8 and CCK-58 differ in their effects on nocturnal solid meal pattern in undisturbed rats

2012 ◽  
Vol 303 (8) ◽  
pp. R850-R860 ◽  
Author(s):  
Miriam Goebel-Stengel ◽  
Andreas Stengel ◽  
Lixin Wang ◽  
Gordon Ohning ◽  
Yvette Taché ◽  
...  
Keyword(s):  
Locomotor Activity ◽  
Food Intake ◽  
Molecular Forms ◽  
Reduce Food Intake ◽  
Dark Phase ◽  
Sprague Dawley ◽  
Solid Meal ◽  
Sprague Dawley Rats ◽  
And Behavior

Various molecular forms of CCK reduce food intake in rats. Although CCK-8 is the most studied form, we reported that CCK-58 is the only detectable endocrine peptide form in rats. We investigated the dark-phase rat chow intake pattern following injection of CCK-8 and CCK-58. Ad libitum-fed male Sprague-Dawley rats were intraperitoneally injected with CCK-8, CCK-58 (0.6, 1.8, and 5.2 nmol/kg), or vehicle. Food intake pattern was assessed during the dark phase using an automated weighing system that allowed continuous undisturbed monitoring of physiological eating behavior. Both CCK-8 and CCK-58 dose dependently reduced 1-h, dark-phase food intake, with an equimolar dose of 1.8 nmol being similarly effective (−49% and −44%). CCK-58 increased the latency to the first meal, whereas CCK-8 did not. The intermeal interval was reduced after CCK-8 (1.8 nmol/kg, −41%) but not after CCK-58. At this dose, CCK-8 increased the satiety ratio by 80% and CCK-58 by 160%, respectively, compared with vehicle. When behavior was assessed manually, CCK-8 reduced locomotor activity (−31%), whereas grooming behavior was increased (+59%). CCK-58 affected neither grooming nor locomotor activity. In conclusion, reduction of food intake by CCK-8 and CCK-58 is achieved by differential modulation of food intake microstructure and behavior. These data highlight the importance of studying the molecular forms of peptides that exist in vivo in tissue and circulation of the animal being studied.

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