scholarly journals Isolation of a sixth dynein subunit adenosine triphosphatase of Chlamydomonas axonemes.

1988 ◽  
Vol 106 (1) ◽  
pp. 133-140 ◽  
Author(s):  
G Piperno
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Adenosine Triphosphatase ◽  
Sucrose Gradient ◽  
Soluble Fraction ◽  
Sedimentation Coefficient ◽  
Apparent Molecular Weight ◽  
A Subunit ◽  
Dynein Arms ◽  
Different Levels

This study of the axoneme led to the identification of a previously unknown adenosine triphosphatase (ATPase), which is likely a major component of inner dynein arms. The ATPase was isolated from a soluble fraction of axonemes obtained from pf 28, a Chlamydomonas mutant lacking the outer dynein arms. The activity hydrolyzed up to 2.3 mumol of ATP.min-1.mg-1 of protein (at pH 7.2, in the presence of both Ca++ and Mg++), had a sedimentation coefficient of 11S in sucrose gradient, and cosedimented with four polypeptides of apparent molecular weight 325,000, 315,000 140,000, and 42,000. Several arguments indicate that the new ATPase is a component of the inner dynein arms. Three or four polypeptides cosedimenting with the activity belong to a group of axonemal components that are deficient in the axonemes of pf 23 and pf 30, two mutants that display different levels of inner dynein arm deficiency. The 42,000 component is axonemal actin, a subunit of two other inner dynein ATPases. The two polypeptides of molecular weight greater than 300,000 have electrophoretic mobility similar to that of high molecular weight components of outer and inner dynein arms. In spite of some similarities each ATPase isolated from inner or outer arms is composed of a different set of polypeptides. Different ATPases may be required for the modulation of localized sliding of adjacent outer double microtubules in the axoneme.

Subcellular Distribution of Low- and High Molecular-Weight Renin and its Relation to a Renin Inhibitor in Pig Renal Cortex

1980 ◽  
Vol 59 (5) ◽  
pp. 337-345 ◽  
Author(s):  
G. A. Sagnella ◽  
P. R. B. Caldwell ◽  
W. S. Peart
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Subcellular Distribution ◽  
Renal Cortex ◽  
Soluble Fraction ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Main Peak ◽  
Low Molecular Weight ◽  
Molecular Weight Form

1. The subcellular distribution of low-molecular-weight and high-molecular weight forms of pig renin has been investigated. 2. Renin, in aqueous extracts of a ‘renin granular fraction’ prepared by differential centrifugation, after gel filtration on Sephadex G-100 displayed an apparent molecular weight of 40 000 and was not activated by acidification to pH 2.8. 3. Renin in the soluble fraction separated on Sephadex G-100 at neutral pH displayed a main peak of activity with an apparent molecular weight of 40000. When eluates were acidified to pH 2.8 (2°C, 60 min) a marked increase in renin activity was observed in the region corresponding to an apparent molecular weight of 50 000. 4. A renin inhibitory material was isolated from the soluble fraction by DEAE chromatography. This material displayed an apparent molecular weight of 50000 and it was destroyed by acidification to pH 2.8. 5. The presence of the proteolytic inhibitor N-ethylmaleimide yielded an apparently high-molecular-weight form of renin (60000–70000) from the soluble fraction, but this was not found in the granular fraction. 6. We conclude that pig renal renin is stored within membrane-bounded subcellular organelles as the low-molecular-weight form. High-molecular-weight renin and renin inhibitory activity are localized to the cortical soluble fraction. In addition, the soluble fraction contains a material which in the presence of N-ethylmaleimide results in the formation of an apparently high-molecular-weight renin.

A PLASMINOGEN ACTIVATOR INHIBITOR (PAI-2) CIRCULATES IN TWO HIGH MOLECULAR WEIGHT FORMS IN PREGNANCY

1987 ◽  
Author(s):  
N A Booth ◽  
A Reith ◽  
B Bennett
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Plasminogen Activator ◽  
Apparent Molecular Weight ◽  
Late Pregnancy ◽  
Plasminogen Activator Inhibitor ◽  
Molecular Weights ◽  
Histiocytic Cell ◽  
Sds Page ◽  
Pai 1

Normal vascular endothelium and platelet α-granules contain an inhibitor of plasminogen activator (PAI-1) of about 48000 molecular weight, which is released by stimuli such as thrombin. An immunologically distinct inhibitor (PAI-2) of about 47000 molecular weight has been purified from placenta and from a histiocytic cell line U-937. The level of PA-inhibition in plasma is raised in late pregnancy and this may be due to increases in PAI-1 or in PAI-2 or in both.Using SDS-PAGE and zymography on fibrin/plasminogen /u-PA detector gels, we have found that normal plasma contains a band of inhibition of apparent molecular weight 40000, which can be neutralised by antiserum raised against PAI-1. Pregnancy plasma contained this band as well as additional inhibitor bands of apparent molecular weights 75000 and 130000. The novel high molecular weight PA-inhibitors were detectable by zymography at about 12 weeks gestation. They were specific for plasminogen activator and did not inhibit plasmin. They were inhibited by antiserum raised against PAI-2 from U-937 cells (a gift from Dr EKO Kruithof) and thus are immunologically related to PAI-2. They may represent circulating complexes of PAI-2 with another protein or aggregates of PAI-2, which retain inhibitory activity after SDS-PAGE. PAI-2 appears to represent a pregnancy associated protein that circulates in a number of different molecular weight forms.

scholarly journals A high molecular weight soluble fraction of tempeh protects against fluid losses in Escherichia coli-infected piglet small intestine

2007 ◽  
Vol 98 (2) ◽  
pp. 320-325 ◽  
Author(s):  
Jeroen L. Kiers ◽  
M. J. Rob Nout ◽  
Frans M Rombouts ◽  
Marius J. A. Nabuurs ◽  
Jan van der Meulen
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Small Intestine ◽  
High Molecular Weight ◽  
Internal Control ◽  
Soluble Fraction ◽  
Fluid Secretion ◽  
Fluid Absorption ◽  
Fluid Losses ◽  
Etec Infection

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhoea in children and piglets. Infection of ETEC results in fluid secretion and electrolyte losses in the small intestine. In this study the effects of tempeh, a traditional fungal fermented soyabean product, on fluid losses induced by ETEC infection in piglets was investigated. Pairs of ETEC-infected and non-infected small intestinal segments of piglets were perfused simultaneously for 8 h with pre-digested tempeh, its supernatant and saline as an internal control. In saline perfused segments, ETEC infection reduced net fluid absorption by more than 500 μl/cm2, whereas this reduction was significantly less for pre-digested tempeh and its supernatant (75 and 282 μl/cm2, respectively). The supernatant of pre-digested tempeh was also compared with its permeate and retentate fractions. These fractions were created by ultra-filtration and contained respectively low and high molecular weight (>5 kDa) compounds. Again ETEC infection caused a significant reduction of net fluid absorption when perfused with saline (386 μl/cm2) and also with the permeate fraction (300 μl/cm2), but much less with the supernatant and the retentate fraction (125 and 140 μl/cm2, respectively). The reduction in net fluid absorption upon ETEC infection when perfused with supernatant of either undigested or pre-digested tempeh was not different. Therefore from this study it can be concluded that a high molecular weight soluble fraction of tempeh is able to protect against fluid losses induced by ETEC, suggesting that this could play a potential role in controlling ETEC-induced diarrhoea.

Sulphydryl Oxidation in the Mechanism of Molecular-Weight Conversion of Renin in Dog Kidney

1982 ◽  
Vol 62 (2) ◽  
pp. 157-162 ◽  
Author(s):  
Fumihiko Ikemoto ◽  
Kazuo Takaori ◽  
Hiroshi Iwao ◽  
Kenjiro Yamamoto
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Renal Cortex ◽  
Soluble Fraction ◽  
Low Molecular Weight ◽  
Oxidizing Agent ◽  
Nitrobenzoic Acid ◽  
Binding Substance ◽  
Sh Group ◽  
Dog Kidney

1. A high-molecular-weight renin (Mr 60 000) was formed by the reaction of a low-molecular-weight renin (Mr 40 000) with a renin-binding substance in canine renal cortical extract in the presence of the sulphydryl (SH) group oxidizing agent potassium tetrathionate; thus the reaction required SH oxidation. 2. Renin extracted from isolated renin granules was adsorbed on to thiopropyl Sepharose 6B, and then liberated with dithiothreitol (50 mmol/l), indicating that it possessed on SH moiety(s). 3. However, the renin was capable of reaction with the renin-binding substance even after its SH moiety (or moieties) was protected with 5,5′-dithiobis-(2-nitrobenzoic acid). 4. The high-molecular-weight renin was converted into the low-molecular-weight renin by incubation (37°C, 15 min) with cytosol (soluble fraction) of renal cortex and liver. Such converting ability was diminished after the cytosol was treated with perchloric acid or potassium tetrathionate. 5. These results suggest that the reaction of renin with the renin-binding substance does not require disulphide bond(s) and that an enzymelike substance which is sensitive to SH oxidation is involved in the conversion from the high-molecular-weight renin into the low-molecular weight renin.

END GROUP AND SEDIMENTATION DATA ON FRAGMENTED HIGH MOLECULAR WEIGHT RIBONUCLEATES

1963 ◽  
Vol 41 (1) ◽  
pp. 1927-1941 ◽  
Author(s):  
B. G. Lane ◽  
J. Diemer ◽  
C. A. Blashko
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Snake Venom ◽  
Group Analysis ◽  
Sedimentation Coefficient ◽  
Snake Venom Phosphodiesterase ◽  
Ehrlich Ascites ◽  
Spontaneous Hydrolysis ◽  
Hydrolysis Of ◽  
End Group

A method for end group analysis of ribonucleate preparations using purified snake venom phosphodiesterase is described. Unusual difficulties encountered with the method are discussed. The technique is useful for detection of end groups resulting from enzymic and chemical fragmentation of high molecular weight ribonucleates. Preliminary studies indicate that the method has limited usefulness because of a spontaneous hydrolysis of ribonucleates which occurs under the conditions which are optimal for hydrolysis with snake venom phosphodiesterase (pH 9, in the presence of magnesium). Physicochemical studies have shown that the pronounced dependence of sedimentation coefficient on ionic strength which has been reported by other investigators is also observed with fragmented high molecular weight ribonucleates and with 16S + 24S ribonucleates of Ehrlich ascites cells. The changes of sedimentation rate are associated with configurational and aggregation effects.

CA2+-INDUCED STRUCTURAL TRANSITIONS OF THE PLATELET GP IIb-IIIa COMPLEX

1987 ◽  
Author(s):  
B Steiner ◽  
D R Phillips
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
High Molecular Weight ◽  
Functional Activity ◽  
Sucrose Gradient ◽  
Membrane Glycoprotein ◽  
Structural Transitions ◽  
Heterodimeric Complex ◽  
Triton X ◽  
Active Subunits

Previous studies have shown that the membrane glycoprotein (GP) IIb-IIIa complex can be reversibly dissociated by incubating platelets for 5 min at 37°C in an EDTA-containing buffer. Prolonged incubations (30 min) with EDTA, however, result in the formation of high molecular weight aggregates of GP IIb and GP IIIa. These aggregates of individual GP's neither bind fibrinogen nor support platelet aggregation, indicating that chelation of Ca2+ can affect the functional activity of GP IIb-IIIa. The present study was designed to identify conditions for the generation of functionally active GP IIb and GP IIIa. Functionally active subunits were defined as those which reformed GP IIb-IIIa complexes. The complexes were quantified by sucrose gradient sedimentation (complexed, dissociated and aggregated GP’s have different sedimentation coefficients) and thrombin hydrolysis (dissociated and aggregated GP lib are susceptible to hydrolysis by thrombin while GP lib in the GP IIb-IIIa complex is thrombin resistant). Purified GP IIb-IIIa could be dissociated by a 5 min incubation at 37°C with ≤ 10−5 M Ca2+. When the complexes were dissociated in the presence of Ca2+ concentrations below 10−6 m, the monomeric GP IIIa was converted to a slower sedimenting form; this change in structure caused it to become functionally inactive. In the presence of very low Ca2+ concentrations 10−6 M) both dissociated subunits subsequently formed high molecular weight aggregates. However, these changes in structure and loss in function could be prevented by dissociating the complexes in 10−6 M Ca2+ and immediately readding raM Ca2+ at 4°C. When this solution was warmed to 20°C, almost 70% of the dissociated subunits reformed heterodimeric complexes. Storage at 4°C for as long as 6 h did not alter the functional activity of these subunits. Octylglucoside, but not Triton X-100, completely inhibited reassociation. Experiments performed in the presence of various H+ and salt concentrations showed that the interactive forces between GP IIb and GP IIIa are both electrostatic and hydrophobic. Thus, conditions have been obtained for the preparation of functionally active GP IIb and GP IIIa which can reform the native heterodimeric complex. Various Ca2+ concentrations can have multiple effects on the structure of the dissociated subunits.

MOLECULAR WEIGHT AND PHYSICAL PROPERTIES OF A LIPOPROTEIN FROM THE FLOATING FRACTION OF EGG YOLK

1958 ◽  
Vol 36 (1) ◽  
pp. 937-949 ◽  
Author(s):  
K. J. Turner ◽  
W. H. Cook
Keyword(s):  
Molecular Weight ◽  
Sodium Chloride ◽  
Lipid Content ◽  
Soluble Fraction ◽  
Egg Yolk ◽  
Low Molecular Weight ◽  
Molecular Weights ◽  
Ether Extraction ◽  
Soluble Material ◽  
Different Levels

Ether extraction removes 80% of the floating fraction of egg yolk from its solution in 2.5% sodium chloride. On removal of the ether and dialysis against specific solvents two thirds of the remaining lipoprotein (40% lipid) precipitates, leaving a soluble fraction containing 50% lipid. The soluble and insoluble portions probably represent different levels of degradation rather than different lipoproteins since both materials are apparently derived by ether extraction from a natural entity of much higher lipid content. The soluble fraction is heterogeneous and has mean molecular weights of M w = 5.4 × 105by light-scattering methods, and M n = 3.4 × 105by osmotic pressure. A "light" fraction obtained by centrifuging and representing 38% of the soluble material had M w = 3.4 to 3.5 × 105. Heterogeneity in lipid content appears to be responsible for the low molecular weight (M w = 2.4 × 105) obtained by sedimentation.

Expression of high molecular weight tau in the central and peripheral nervous systems

1993 ◽  
Vol 105 (3) ◽  
pp. 729-737
Author(s):  
I.S. Georgieff ◽  
R.K. Liem ◽  
D. Couchie ◽  
C. Mavilia ◽  
J. Nunez ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Molecular Weight ◽  
Nervous System ◽  
High Molecular Weight ◽  
Tumor Cells ◽  
Apparent Molecular Weight ◽  
In Vitro Transcription ◽  
The Central Nervous System ◽  
Immunohistochemical Studies

Using a novel PCR approach, we have cloned a cDNA encoding the entire high molecular weight tau molecule from rat dorsal root ganglia. The resulting 2080 bp cDNA differs from low molecular weight rat brain tau by the insertion of a novel 762 bp region (exon 4a) between exons 4 and 5. This cDNA clone is identical in sequence with a high molecular weight tau (HMW) cDNA from rat PC12 tumor cells and is closely related to a HMW tau cDNA from mouse N115 tumor cells. In vitro transcription/translation produces a protein that migrates on SDS-PAGE with the same apparent molecular weight as HMW tau purified from rat sciatic nerve. The HMW tau protein is generated from an 8 kb mRNA, which can be detected by northern blots in peripheral ganglia, but not in brain. A more sensitive assay using PCR and Southern blot analysis demonstrates the presence of exon 4a in spinal cord and in retina. In combination with immunohistochemical studies of spinal cord, these data suggest that HMW tau, though primarily in the peripheral nervous system, is also expressed in limited areas of the central nervous system, although its presence cannot be detected in the cerebral cortices.

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