scholarly journals Nitrogenase of Klebsiella pneumoniae. Interaction of the component proteins studied by ultracentrifugation

1973 ◽  
Vol 135 (3) ◽  
pp. 531-535 ◽  
Author(s):  
Robert R. Eady
Keyword(s):  
Klebsiella Pneumoniae ◽  
Sedimentation Coefficient ◽  
Sedimentation Velocity ◽  
Single Peak ◽  
Molar Ratio ◽  
Titration Curves ◽  
Additional Peak

Sedimentation-velocity analyses of mixtures of the component proteins of nitrogenase of Klebsiella pneumoniae at a 1:1 molar ratio, showed a single peak of sedimentation coefficient (12.4S) considerably greater than that obtained for the larger (Fe+Mo-containing) protein centrifuged alone (10.4S). When the ratio exceeded 1:1 (the smaller Fe-containing protein in excess) an additional peak corresponding in sedimentation coefficient (about 4.5S) to free Fe-containing protein appeared. When proteins, which had been inactivated by exposure to air were used, no interaction occurred. Na2S2O4 at 2mm both reversed and prevented interaction between the two proteins; sedimentation coefficients corresponded to those of the proteins when centrifuged alone. These results demonstrate the formation of a complex between the nitrogenase proteins, and, together with data of activity titration curves, are consistent with the formulation of the nitrogenase complex of K. pneumoniae as (Fe-containing protein)–(Fe+Mo-containing protein).

Binding of Al by protein in plasma of patients on maintenance hemodialysis.

1985 ◽  
Vol 31 (8) ◽  
pp. 1314-1316 ◽  
Author(s):  
M Cochran ◽  
D Patterson ◽  
S Neoh ◽  
B Stevens ◽  
R Mazzachi
Keyword(s):  
Molecular Mass ◽  
Binding Protein ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Human Albumin ◽  
Hemodialysis Patients ◽  
Single Peak ◽  
Molar Ratio ◽  
Maintenance Hemodialysis ◽  
Mass Fractions

Abstract Gel filtration of plasma from hemodialysis patients, with use of reagents and apparatus with carefully minimized background Al concentrations, reproducibly showed a single peak for Al, corresponding exactly to the elution position of transferrin. The Al/transferrin molar ratio in adjacent fractions was constant (mean 0.126, SE 0.006) in replicate experiments. In contrast, the association of Al with albumin varied. Using both equilibrium dialysis and gel-filtration techniques, in the presence and absence of calcium or phosphate, we could demonstrate no significant binding of Al by human albumin at Al concentrations of 1 to 12 mumol/L. We saw no Al peak in pooled, concentrated, low-molecular-mass fractions of plasma gel-filtered on Sephadex G-50. Evidently, transferrin is the sole Al-binding protein in plasma of hemodialysis patients.

scholarly journals Solution properties of polygalacturonic acid

1969 ◽  
Vol 114 (4) ◽  
pp. 863-870 ◽  
Author(s):  
R W Stoddart ◽  
I. P. C. Spires ◽  
K F Tipton
Keyword(s):  
Ph Dependence ◽  
Sedimentation Coefficient ◽  
Ruthenium Red ◽  
Optical Rotatory Dispersion ◽  
Neutral Sugar ◽  
Polygalacturonic Acid ◽  
Specific Viscosity ◽  
Ph Titration ◽  
Titration Curves ◽  
Low Concentrations

1. The specimen of polygalacturonic acid used in these studies was shown to contain very little neutral sugar, methyl ester groups or ash, and only residues of galacturonic acid. Its electrophoretic homogeneity was examined in pyridine–acetic acid buffer at pH6·5 and in borate buffer at pH9·2. The distribution of effective particle weights was shown to be fairly narrow. 2. The pH-titration curve of the polymer gave a pK value of 3·7. 3. The interaction of the polymer with Ruthenium Red was studied and titration curves were obtained for the spectral shifts associated with the formation of a complex. 4. Optical-rotatory-dispersion studies showed that the Drude constant, λc, was dependent on pH. 5. Polygalacturonic acid was shown to display non-Newtonian properties in solution and to have an anomalously high relative specific viscosity at low concentrations. 6. Studies were made of the pH-dependence of the sedimentation coefficient of the polymer. 7. These results are discussed in terms of the structure of the molecule and their relevance to the properties of pectic substances.

Amino-terminal proBNP in ovine plasma: evidence for enhanced secretion in response to cardiac overload

1998 ◽  
Vol 275 (4) ◽  
pp. H1200-H1208 ◽  
Author(s):  
C. J. Pemberton ◽  
T. G. Yandle ◽  
M. T. Rademaker ◽  
C. J. Charles ◽  
G. D. Aitken ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Natriuretic Peptide ◽  
Normal Control ◽  
Left Atrial ◽  
Left Ventricular ◽  
Size Exclusion ◽  
Single Peak ◽  
Molar Ratio ◽  
Coronary Artery Ligation ◽  
Amino Terminal

We have recently identified a novel amino-terminal fragment of pro-brain natriuretic peptide (NT-proBNP) in the circulation of humans, the concentration of which increases progressively as the left ventricle fails. To clarify the origins of NT-proBNP in experimental animals, we have developed an RIA for NT-proBNP based on residues 52–71 of ovine proBNP-(1—103) and used it to study cardiac processing, secretion, and metabolism of BNP in sheep with cardiac overload induced by coronary artery ligation (CAL) or rapid left ventricular pacing (rLVP). The concentration of NT-proBNP in left atrial plasma extracts drawn from normal control sheep was threefold that of mature BNP. Size-exclusion and reverse-phase HPLC analyses of plasma extracts coupled to RIA revealed a single peak of immunoreactive (ir) NT-proBNP [∼8,000 relative molecular weight ( M r)], quite distinct from a single peak of ir-mature BNP (∼3,000 M r). In contrast, ovine cardiac tissue contained only a single immunoreactive peak of high-molecular-weight BNP (∼11,000 M r), consistent in size with proBNP-(1—103). Sampling from the cardiac coronary sinus in normal control sheep ( n = 5) and sheep with CAL ( n = 5) revealed that the molar ratio of NT-proBNP to mature BNP was similar. There was a significant gradient of both mature and NT-proBNP across the heart in normal sheep, whereas after CAL the gradient was significant for mature BNP only. In both forms of cardiac overload (CAL and rLVP), left atrial plasma levels of NT-proBNP were significantly increased above normal levels, in contrast with mature BNP levels, which were raised only in the rLVP group of animals. Blockade of natriuretic peptide metabolism in sheep with heart failure (induced by rLVP) raised mature BNP levels threefold but did not affect levels of NT-proBNP. In conclusion, these studies show that NT-proBNP is formed from proBNP stores during secretion and, compared with mature BNP, accumulates in plasma because metabolism of NT-proBNP appears to differ from that of mature BNP. Although its function, if any, remains unclear, plasma NT-proBNP may prove to be a sensitive marker of cardiac overload and/or decompensation.

scholarly journals A T-lymphoma transmembrane glycoprotein (gp180) is linked to the cytoskeletal protein, fodrin.

1985 ◽  
Vol 101 (2) ◽  
pp. 477-487 ◽  
Author(s):  
L Y Bourguignon ◽  
S J Suchard ◽  
M L Nagpal ◽  
J R Glenney
Keyword(s):  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Membrane Extraction ◽  
Surface Glycoprotein ◽  
Molar Ratio ◽  
Surface Receptors ◽  
Transmembrane Glycoprotein ◽  
Parallel Increase ◽  
Double Label ◽  
T Lymphoma

A major mouse T-lymphoma surface glycoprotein (gp180) has been identified by labeling cells with 125I and [3H]glucosamine. After ligand-induced receptor patching and/or capping, the amount of gp 180 in the membrane-associated cytoskeleton fraction increases in direct proportion to the percentage of patched/capped cells. There is a parallel increase in the amount of fodrin in the membrane-associated cytoskeleton fraction. Evidence is presented that gp180 is the same as or very similar to the T-lymphocyte-specific glycoprotein T-200. An immunobinding assay of Nonidet P-40-solubilized plasma membrane selectively co-isolates gp180 and fodrin. After induction of receptor rearrangement, double-label immunofluorescence reveals that fodrin accumulated directly beneath gp180 patches and caps. Membrane extraction with Triton X-114 followed by sucrose gradient centrifugation permits isolation of a gp180-fodrin complex with a 1:1 molar ratio and sedimentation coefficient(s) of approximately 20. This complex remains stable during isoelectric focusing and exhibits a pl in the range of 5.2-5.7. On the basis of our results we conclude that gp180, an integral membrane glycoprotein, and fodrin, a component of the membrane-associated cytoskeleton, are closely associated into a complex. Furthermore, we contend that, through fodrin's association with actin, this complex is of functional significance in ligand-induced patching and capping of gp180. We also propose that, through lateral interactions in the plane of the membrane, the gp180-fodrin complex might be responsible for linking other surface receptors to the intracellular microfilament network during lymphocyte patching and capping.

scholarly journals Calcium-dependent stoichiometries of the KCa2.2 (SK) intracellular domain/calmodulin complex in solution

2014 ◽  
Vol 143 (2) ◽  
pp. 231-252 ◽  
Author(s):  
D. Brent Halling ◽  
Sophia A. Kenrick ◽  
Austen F. Riggs ◽  
Richard W. Aldrich
Keyword(s):  
Molar Mass ◽  
Analytical Ultracentrifugation ◽  
Protein Complexes ◽  
Sedimentation Coefficient ◽  
Molar Ratio ◽  
Composition Gradient ◽  
Sk Channels ◽  
Binding Studies ◽  
Calcium Dependent ◽  
Molar Excess

Ca2+ activates SK Ca2+-activated K+ channels through the protein Ca2+ sensor, calmodulin (CaM). To understand how SK channels operate, it is necessary to determine how Ca2+ regulates CaM binding to its target on SK. Tagless, recombinant SK peptide (SKp), was purified for binding studies with CaM at low and high Ca2+ concentrations. Composition gradient multi-angle light scattering accurately measures the molar mass, stoichiometry, and affinity of protein complexes. In 2 mM Ca2+, SKp and CaM bind with three different stoichiometries that depend on the molar ratio of SKp:CaM in solution. These complexes include 28 kD 1SKp/1CaM, 39 kD 2SKp/1CaM, and 44 kD 1SKp/2CaM. A 2SKp/2CaM complex, observed in prior crystallographic studies, is absent. At <5 nM Ca2+, 1SKp/1CaM and 2SKp/1CaM were observed; however, 1SKp/2CaM was absent. Analytical ultracentrifugation was used to characterize the physical properties of the three SKp/CaM stoichiometries. In high Ca2+, the sedimentation coefficient is smaller for a 1SKp:1CaM solution than it is for either 2SKp:1CaM or 1SKp:2CaM. At low Ca2+ and at >100 µM protein concentrations, a molar excess of SKp over CaM causes aggregation. Aggregation is not observed in Ca2+ or with CaM in molar excess. In low Ca2+ both 1SKp:1CaM and 1SKp:2CaM solutions have similar sedimentation coefficients, which is consistent with the absence of a 1SKp/2CaM complex in low Ca2+. These results suggest that complexes with stoichiometries other than 2SKp/2CaM are important in gating.

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