Differences between the Sugar Moieties of Liver- and Bone-Type Alkaline Phosphatases: A Re-Evaluation

1994 ◽  
Vol 31 (1) ◽  
pp. 25-30 ◽  
Author(s):  
Masakazu Miura ◽  
Yoshikatsu Sakagishi ◽  
Keishi Hata ◽  
Tsugikazu Komoda
Keyword(s):  
Sialic Acid ◽  
Heat Exposure ◽  
Molecular Size ◽  
Heat Stability ◽  
Additional Treatment ◽  
Alkaline Phosphatases ◽  
Sugar Moiety ◽  
Heat Stable ◽  
The Difference ◽  
Sialic Acid Linkage

We re-evaluated the differences between the sugar moieties of liver and bone alkaline phosphatases (ALPs). Sialic acid was added to ALP sugar moieties by α2,3- or 2,6-sialyltransferase treatment of the asialo-form ALP (neuraminidase-treated ALP). Asialo-bone ALP was converted to a liver-like ALP by the 2,6-sialyltransferase treatment. The resulting liver-like ALP was less susceptible to neuraminidase than non-treated bone ALP, but was still labile to heat exposure at 56°C like non-treated bone ALP. However, after the O-linked sugar moiety had been released by additional treatment with O-glycanase the liver-like ALP became more heat stable at 56°C, like non-treated liver ALP. Non-treated liver ALP reacted specifically with anti-liver ALP monoclonal antibody, and non-treated bone ALP reacted with both anti-liver and anti-bone ALP antibodies. The asialo-bone ALP still reacted with anti-bone ALP antibody, whereas the asialo-form liver ALP showed little, if any, reaction with anti-liver and anti-bone ALP antibodies. Neuraminidase and O-glycanase-treated bone ALP reacted less with anti-bone ALP antibody. After O-glycanase treatment, bone ALP molecules deprived of an O-linked sugar moiety had a molecular size and heat stability similar to liver ALP. The difference between liver and bone ALP molecules may be due not only to their manner of sialic acid linkage but also to the attachment of the O-linked sugar moiety.

Stability of buffalo casein micelles

1979 ◽  
Vol 46 (2) ◽  
pp. 401-405 ◽  
Author(s):  
Nripendra C. Ganguli
Keyword(s):  
Sialic Acid ◽  
Gel Filtration ◽  
Skim Milk ◽  
Heat Stability ◽  
Casein Micelles ◽  
Heat Stable ◽  
Micellar Casein ◽  
Acid Release ◽  
The Stability ◽  
Better Than

SUMMARYBuffalo skim-milk is less heat stable than cow skim-milk. Interchanging ultracentrifugal whey (UCW) and milk diffusate with micellar casein caused significant changes in the heat stability of buffalo casein micelles (BCM) and cow casein micelles (CCM). Buffalo UCW dramatically destabilized COM, whereas buffalo diffu-sate with CCM exhibited the highest heat stability.Cow κ-casein stabilizes αs-casein against precipitation by Ca better than buffalo º-casein. About 90% of αs-casein could be stabilized by κ: αs ratios of 0·20 and 0·231 for cow and buffalo, respectively.Sialic acid release from micellar κ-casein by rennet was higher than from acid κ-casein in both buffalo and cow caseins, the release being slower in buffalo. The released macropeptide from buffalo κ-casein was smaller than that from cow κ-casein as revealed by Sephadex gel filtration.Sub-units of BCM have less sialic acid (1·57mg/g) than whole micelles (2·70mg/g). On rennet action, 47% of bound sialic acid was released from sub-units as against 85% from whole micelles. The sub-micelles are less heat stable than whole micelles. Among ions tested, added Ca reduced heat stability more dramatically in whole micelles, whereas added phosphate improved the stability of micelles and, more strikingly, of sub-micelles. Citrate also improved the heat stability of sub-micelles but not of whole micelles.

scholarly journals Postnatal changes in sialylation of glycoproteins in rat liver

1991 ◽  
Vol 280 (1) ◽  
pp. 179-185 ◽  
Author(s):  
S Oda-Tamai ◽  
S Kato ◽  
N Akamatsu
Keyword(s):  
Sialic Acid ◽  
Liver Microsomes ◽  
Heat Stability ◽  
Postnatal Period ◽  
Sialic Acid Residue ◽  
Membrane Glycoproteins ◽  
Sialic Acid Content ◽  
Liver Growth ◽  
Neuraminidase Treatment ◽  
The Difference

Glycoproteins containing N-linked oligosaccharides were prepared from plasma and liver microsomes of rats aged 0-5 weeks, and galactose and sialic acid content were determined. The sialic acid/galactose ratios in plasma membrane N-glycans remained at about 1 throughout the postnatal period, suggesting that most of the galactose residues are sialylated. In the same way, it was suggested that most of the galactose residues of microsomal N-glycans were sialylated at 0, 4 and 5 weeks of age, but that the degree of sialylation was lower at the other ages, with a minimum at 2 weeks. When the activities of sialyltransferase and galactosyltransferase in liver Golgi membranes were determined, age-dependent changes were found, not only in the specific activities of the enzymes, but also in the Golgi membrane content per g of liver. The activity of galactosyltransferase per g of liver increased immediately after birth, whereas that of sialyltransferase remained at a low level for 2 weeks and then increased to a constant level at 4 weeks. It is probable that this delayed increase in the activity of sialyltransferase results in the decreased sialylation of microsomal N-glycans at 1, 2 and 3 weeks. Sialyltransferase was solubilized from the liver microsomes of rats aged 2, 3 and 4 weeks and characterized. Phosphocellulose column chromatography separated the activity into two subfractions, designated transferase I and transferase II in the order of elution. The increase in total sialyltransferase activity during this period was caused mainly by an increase in transferase I. Rechromatography of each transferase from 3-week-old rats after neuraminidase treatment showed that transferase I but not transferase II contained sialic acid residue(s) and that desialylated transferase I was eluted in a similar way as transferase II. Although the apparent Km value for CMP-N-acetylneuraminic acid and the heat stability of transferase I were different from those of transferase II, the difference was abolished by treating transferase I with neuraminidase, suggesting that transferase II may be a desialylated form of transferase I. These changes in the sialylation of membrane glycoproteins, including sialyltransferase, may be related to the control of liver growth during postnatal development.

scholarly journals Adeno-Associated Virus Serotype 4 (AAV4) and AAV5 Both Require Sialic Acid Binding for Hemagglutination and Efficient Transduction but Differ in Sialic Acid Linkage Specificity

2001 ◽  
Vol 75 (15) ◽  
pp. 6884-6893 ◽  
Author(s):  
Nikola Kaludov ◽  
Kevin E. Brown ◽  
Robert W. Walters ◽  
Joseph Zabner ◽  
John A. Chiorini
Keyword(s):  
Sialic Acid ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Target Cells ◽  
Adeno Associated Virus ◽  
Virus Serotype ◽  
Sialic Acid Binding ◽  
The Difference ◽  
Protein Treatment ◽  
Sialic Acid Linkage

ABSTRACT Adeno-associated virus serotype 4 (AAV4) and AAV5 have different tropisms compared to AAV2 and to each other. We recently reported that α2-3 sialic acid is required for AAV5 binding and transduction. In this study, we characterized AAV4 binding and transduction and found it also binds sialic acid, but the specificity is significantly different from AAV5. AAV4 can hemagglutinate red blood cells from several species, whereas AAV5 hemagglutinates only rhesus monkey red blood cells. Treatment of red blood cells with trypsin inhibited hemagglutination for both AAV4 and AAV5, suggesting that the agglutinin is a protein. Treatment of Cos and red blood cells with neuraminidases also indicated that AAV4 bound α2-3 sialic acid. However, resialylation experiments with neuraminidase-treated red blood cells demonstrated that AAV4 binding required α2–3 O-linked sialic acid, whereas AAV5 required N-linked sialic acid. Similarly, resialylation of sialic acid-deficient CHO cells supported this same conclusion. The difference in linkage specificity for AAV4 and AAV5 was confirmed by binding and transduction experiments with cells incubated with either N-linked or O-linked inhibitors of glycosylation. Furthermore, AAV4 transduction was only blocked with soluble α2-3 sialic acid, whereas AAV5 could be blocked with either α2–3 or α2-6 sialic acid. These results suggest that AAV4 and AAV5 require different sialic acid-containing glycoproteins for binding and transduction of target cells and they further explain the different tropism of AAV4 and AAV5.

scholarly journals A GENETICALLY DISTINCT FORM OF CYCLIC AMP PHOSPHODIESTERASE ASSOCIATED WITH CHROMOMERE 3D4 IN DROSOPHILA MELANOGASTER

Genetics ◽  
1979 ◽  
Vol 91 (3) ◽  
pp. 521-535
Author(s):  
John A Kiger ◽  
Eric Golanty
Keyword(s):  
Drosophila Melanogaster ◽  
Cyclic Amp ◽  
Structural Gene ◽  
Regulatory Gene ◽  
Heat Stability ◽  
Normal Female ◽  
Dependent Manner ◽  
Cyclic Amp Phosphodiesterase ◽  
Heat Stable ◽  
Camp Levels

ABSTRACT Two cyclic AMP phosphodiesterase enzymes (E.C.3.1.4.17) are present in homogenates of adult Drosophila melanogaster. The two enzymes differ from one another in heat stability, affinity for Mg++, Ca++ activation and molecular weight. They do not differ markedly in their affinities for cyclic AMP, and both exhibit anomalous Michaelis-Menten kinetics. The more heatlabile enzyme is controlled in a dosage-dependent manner by chromomere 3D4 of the X chromosome and is absent in flies that are deficient for chromomere 3D4. Chromomere 3D4 is also necessary for the maintenance of normal cAMP levels, for male fertility, and for normal female fertility and oogenesis. The structural gene(s) for the more heat-stable enzyme is located outside of chromomeres 3C12-3D4. Whether 3D4 contains a structural gene, or a regulatory gene necessary for the presence of the labile enzyme, remains to be determined.

Systematic Optimisation of Microtiter Plate Lectin Assay to Improve Sialic Acid Linkage Detection

2021 ◽  
Vol 24 ◽  
Author(s):  
Nur Hanina Izzati Khairol Mokhtar ◽  
Ainulkhir Hussin ◽  
Aidil Abdul Hamid ◽  
Shahrul Hisham Zainal Ariffin ◽  
Muhammad Ashraf Shahidan
Keyword(s):  
Sialic Acid ◽  
Specific Binding ◽  
Specific Antigen ◽  
Blocking Agent ◽  
Microtiter Plate ◽  
Tween 20 ◽  
Nonspecific Binding ◽  
Binding Assays ◽  
High Background ◽  
Sialic Acid Linkage

Aims: We aimed to develop a high-throughput lectin assay with minimized background signals to investigate the interactions of lectins and sialic acid glycans, focusing on prostate-specific antigen (PSA). Background: High background signals resulting from nonspecific binding are a significant concern for microtiter plate-based enzyme-linked lectin sorbent assays (ELLSAs), as they can mask specific binding signals and cause false-positive results. Methods: In this study, we constructed an ELLSA based on different washing step parameters, including the number of washing cycles, NaCl and Tween-20 concentrations, and the type of blocking agent and evaluated the effects on both specific and nonspecific binding signals. Furthermore, we performed a PSA binding assay using the optimized ELLSA. Results: The optimal washing parameters based on the highest specific binding signal proposed four cycles of washing steps using a washing buffer containing a high salt concentration (0.5 M NaCl) and mild detergent (0.05% Tween-20). The utilization of the optimized washing parameters in this assay was shown to be sufficient to obtain the optimal binding signals without the use of any blocking agent. Binding assays performed using the optimized ELLSA revealed that the glycan of the PSA sample used in this study mainly consists of terminal α2,6-linked sialic acid, as strongly recognized by Sambucus nigra agglutinin (SNA) with a KD value of 12.38 nM. Conclusion: The ELLSA reported in this study provides a simple yet sensitive assay for sialic acid linkage recognition.

scholarly journals The control of diuresis in the tsetse fly Glossina austeni: a preliminary investigation of the diuretic hormone

1975 ◽  
Vol 63 (2) ◽  
pp. 391-401
Author(s):  
J. D. Gee
Keyword(s):  
Sialic Acid ◽  
Nervous Tissue ◽  
Preliminary Investigation ◽  
Tsetse Fly ◽  
Malpighian Tubules ◽  
Diuretic Hormone ◽  
Glossina Austeni ◽  
Heat Stable ◽  
Soluble Molecule ◽  
Degradative Enzyme

The rate of secretion of the Malpighian tubules of Glossina austeni is controlled by a diuretic hormone. This hormone is present in the nervous tissue of the fly together with a degradative enzyme that can be activated by boiling. It is demonstrated that the Malpighian tubules are able to destroy the diuretic hormone; they may therefore participate in the control of diuresis. The diuretic hormone appears to be a heat-stable, non-dialysable, alcohol-soluble molecule, containing amino acid, glucose and sialic acid residues.

Abstract 90: Inimitable Ouabain: The Endogenous Circulating Cardiotonic Steroid that Singularly Stimulates Sodium Potassium Pump Activity at Low Doses

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Yeon Jae Kim ◽  
Elisha Hamilton ◽  
William Hannam ◽  
Chia-Chi Liu ◽  
Rachel Teh ◽  
...  
Keyword(s):  
Low Dose ◽  
Lactone Ring ◽  
Cardiac Contractility ◽  
Cancer Cell Line ◽  
Structural Components ◽  
Sodium Potassium ◽  
Sugar Moiety ◽  
Unsaturated Lactone ◽  
Sodium Potassium Pump ◽  
The Difference

Rationale: Cardiotonic steroids (CTS), such as digoxin, have been used to treat heart failure (HF) for over 200 years. They inhibit the sodium-potassium pump (NKP), and increase cardiac contractility by inhibiting efflux of sodium through the pump (“digitalis hypothesis”). CTS possess three structural components: a saturated/unsaturated lactone ring, steroid core, and sugar moiety, each of which may be involved in NKP inhibition/stimulation. It is now known that inhibition of the NKP in patients with HF increases mortality, and all major beneficial treatments increase its activity. Endogenous circulating CTS such as ouabain are generally thought to inhibit the NKP, despite studies sporadically reporting ouabain-induced pump stimulation. This study aims to identify whether ouabain-induced pump stimulation occurs, and if so, which structural components are involved in causing pump stimulation. Methods & Results: Cardiac myocytes were isolated from male New Zealand White rabbits, placed in a Tyrode’s solution, and whole-cell patch clamped. They were exposed to 0-30nM ouabain, 0-50nM dihydroouabain (ouabain with a saturated lactone ring) or 0-500nM ouabagenin (ouabain lacking a sugar moiety) for 1 min, followed by a potassium-free solution, with the difference in current yielding the NKP current. Compared to the 0.47±0.05 pA/pF Tyrode’s solution control (n=11), 5nM ouabain significantly increased NKP current to 0.69±0.09 pA/pF ( P <0.05, n=6). Exposure to dihydroouabain or ouabagenin did not significantly change NKP current in the studied concentration range. Cell viability assays carried out on the breast cancer cell line MCF7, which have an NKP structure extremely similar to that of cardiomyocytes, showed significantly elevated viability above control values (n=2) following 24h treatment with 0-9nM ouabain; maximum viability was 116±5% at 0.28nM ( P <0.05, n=4). A significant change in viability was not observed for ouabagenin or digoxin in the same concentration range. Conclusion: Low-dose ouabain uniquely stimulates NKP activity. Low-dose dihydroouabain and ouabagenin do not, suggesting that a sugar moiety and unsaturated lactone ring are required for pump stimulation. Ouabain in its unaltered form may be a potential treatment for HF.

Response of alveolar epithelial solute permeability to changes in lung inflation

1980 ◽  
Vol 49 (6) ◽  
pp. 1032-1036 ◽  
Author(s):  
E. A. Egan
Keyword(s):  
Pore Radius ◽  
Alveolar Epithelium ◽  
Molecular Size ◽  
High Volume ◽  
Lung Inflation ◽  
Solute Permeability ◽  
Alveolar Epithelial ◽  
The Difference ◽  
Total Capacity ◽  
Inflation Volume

The relation between the solute permeability of th alveolar epithelium, characterized as a pore radius, and lung inflation was studied in anesthetized dogs. Pore radius was calculated from measurements of the rate of efflux of several radiolabeled solutes of known molecular size from alveolar saline. Individual animals were studied at two or more separate inflation volumes. The pore radius during the first volume studied averaged 20 A in high-volume animals (mean inflation 82% of capacity) and 15 A at lower volume (mean inflation, 47% of capacity). The difference was significantly P < 0.05. Lungs inflated to total capacity showed free solute movement across the lung epithelium. Increasing inflation volume in an animal always produced a larger pore radius. Decreasing the inflation volume did not produce a smaller pore radius; it remained the same or became larger. Volume induced increases in lung epithelial solute permeability do not reverse immediately at lower volumes, suggesting this phenomenon represents lung injury.

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