Rate Of Protein Transport Research Articles

Charge-Gated Transport of Proteins in Nanostructured Optical Films of Mesoporous Silica

The admission into and diffusion through nanoscale pores by molecules is a fundamental process of great importance to biology and separations science. Systems (e.g., chromatography, electrophoresis) designed to harness such processes tend to remove the separation process from the detection event, both spatially and temporally. Here, we describe the preparation and characterization of thin optical Fabry-Pérot films of mesoporous silica (pSiO(2)) that can detect protein infiltration by optical interferometry, which probes the separation process in real time and in the same ultrasmall physical volume (5 nL). Admission of a protein into the pores is controlled by the diameter (∼50 nm) and the surface charge of the pores, and both the rate and the degree of protein infiltration are a function of solution pH. Test proteins bovine serum albumin (BSA, 66 kDa), bovine hemoglobin (BHb, 65 kDa), and equine myoglobin (EMb, 18 kDa) are admitted to or excluded from the nanophase pores of this material based on their size and charge. The rate of protein transport within the pores of the pSiO(2) film is slowed by 3 orders of magnitude relative to the free-solution diffusion values, and it is maximized when pH = pI.

PKA‐Mediated Golgi Remodeling During cAMP Signal Transmission

Cyclic AMP (cAMP)-dependent protein kinase A (PKA) is part of the set of signaling proteins that are stably associated to the cytosolic surface of Golgi membranes in mammalian cells. In principle, Golgi-associated PKA could participate in either signal transduction events and/or the coordination of Golgi transport activities. Here, we show data indicating that although Golgi-associated PKA is activated fast and efficiently during cell stimulation by an extracellular ligand it does not contribute significantly to cAMP signal transmission to the nucleus. Instead, most of the PKA catalytic subunits Calphaderived from the Golgi complex remain localized in the perinuclear cytoplasm where they induce changes in Golgi structural organization. Thus, in stimulated cells the Golgi complex appears collapsed, showing increased colocalization of previously segregated markers and exhibiting merging of different proximal cisternae within a single stack. In contrast, the trans-Golgi network remains as a separate compartment. Consequently, the rate of protein transport is increased whereas glycan processing is not severely affected. This remodeling process requires the presence of PKA activity associated to the Golgi membranes. Together these data indicate that Golgi-associated PKA activity is involved in the adaptation of Golgi dynamic organization to extracellular signaling events.

Golgi Cisternal Unstacking Stimulates COPI Vesicle Budding and Protein Transport

The Golgi apparatus in mammalian cells is composed of flattened cisternae that are densely packed to form stacks. We have used the Golgi stacking protein GRASP65 as a tool to modify the stacking state of Golgi cisternae. We established an assay to measure protein transport to the cell surface in post-mitotic cells in which the Golgi was unstacked. Cells with an unstacked Golgi showed a higher transport rate compared to cells with stacked Golgi membranes. Vesicle budding from unstacked cisternae in vitro was significantly increased compared to stacked membranes. These results suggest that Golgi cisternal stacking can directly regulate vesicle formation and thus the rate of protein transport through the Golgi. The results further suggest that at the onset of mitosis, unstacking of cisternae allows extensive and rapid vesiculation of the Golgi in preparation for its subsequent partitioning.

Cilostazol prevents impairment of slow axonal transport in streptozotocin-diabetic rats

We studied the effects of cilostazol, an antiplatelet and vasodilating agent, on axonal transport patterns of cytoskeletal proteins in the motor fibers of sciatic nerve of streptozotocin-induced diabetic rats. Proteins labeled with l-[ 35S]methionine in 6-mm consecutive segments of the nerve were analyzed electrophoretically following fractionation into Triton-soluble and-insoluble subpopulations. Transport rates of proteins (particularly neurofilaments) in slow component a were reduced by 50% 2 weeks after labeling (4 weeks after streptozotocin). An apparent reduction of tubulin and actin was observed at later intervals after induction of diabetes. Actin transported in slow component b was also impaired, though to a lesser extent than in component a. Cilostazol prevented transport impairment of both slow components a and b without affecting hyperglycemia or reduction in body weight gain. These results suggest that in sciatic motor fibers early defects in slowly transported proteins are more marked in slow component a, and that impairment may be caused primarily by hemodynamic abnormalities.

Mechanisms controlling diffusion and release of model proteins through and from partially esterified hyaluronic acid membranes

The effects of polymer percent esterification and protein molecular weight on the diffusion of two model proteins, deoxyribonuclease (DNase) and ribonuclease A (RNase A), through and from partially esterified hyaluronic acid membranes were compared. The permeability of the polymer membranes was inversely related to the degree of polymer esterification and the molecular weight of the protein. Transport rates of proteins through the membranes decreased dramatically over narrow ranges of polymer esterification. As expected, the apparent diffusivity of the larger protein in the polymer matrix was more sensitive to changes in membrane hydration than that of the smaller protein. These observations demonstrated the dependence of the mobility of large molecular weight proteins on polymer hydration and chain relaxation. The relationship between protein diffusion through and release from the modified hyaluronate matrices was also investigated using RNase A as a model. The release profiles from fully esterified membranes showed lag behavior and varied with protein load and hyaluronate hydrolysis rates, while release from less esterified membranes was rapid and independent of polymer esterification or hydrolysis. Potential applications of modified hyaluronate matrices in the controlled delivery of proteins are discussed.

Rates of Protein Transport Across Rat Alveolar Epithelial Cell Monolayers

The transport of model proteins, ranging from 12, 300 to 150, 000 Da, across tight rat alveolar epithelial cell monolayers (>2000 cm2) grown on polycarbonate filters, was studied. Model proteins were 14C-cytochromec, 14C-ovalbumin, granulocyte-colony stimulating factor (G-CSF), 14C-bovine serum albumin (BSA), 125I-transferrin, and 14C-immunoglobulin G. Cytochrome c was extensively metabolized, as indicated by < 10% of the dose being translocated in intact form. This contrasts with 20–80% for the other model proteins studied. The flux of cytochrome c and G-CSF was symmetric in the apical-to-basolateral (ab) and basolateral-to-apical (ba) directions. By contrast, the flux of intact ovalbumin, BSA, transferrin and immunoglobulin G showed asymmetry, with the ab flux being higher by 2-5 times. There was no relationship between ab or ba fluxes and the molecular weights of these four model proteins. Since some of the proteins were translocated at much greater rates than are consistent with restricted diffusion or pinocytosis, receptor-mediated or adsorptive transcytosis may be involved.

Transport rates of proteins in porous materials with known microgeometry

Many biological and biotechnological systems involve the diffusion of macromolecules through complicated macroporous (pore size on the order of 10-100 microns) environments. In this report, we present and evaluate an experimental system for measuring the rate of protein transport in an inert, macroporous membrane. For this particular membrane system, the microgeometry was characterized in terms of distribution of pore size, position, and orientation. Although the rate of protein desorption was much less than expected based on continuum diffusion models, we demonstrate that the measured transport rates are consistent with diffusion of protein in a complex, interconnected network of water-filled pores. The porous systems exhibit transitional behavior in quantitative agreement with the behavior of percolation lattices (mean square error 7%, n = 29). Predictive mathematical models of the diffusion process were developed: these models used percolation concepts to describe pore topology, continuum models of diffusion/dissolution to describe protein movement at each single pore, and measured pore size distributions. Effective diffusion coefficients for protein transport in aqueous, constricted macropores were predicted by this technique. Predicted diffusion coefficients, based on measured and derived microstructural parameters, agree with experimentally measured diffusion coefficients within a factor of 2. This approach may be useful in the design of porous polymer systems for biological applications and for evaluating other biological systems where conduction of mass, heat, momentum, or charge occurs in a heterogeneous environment.

Quantitative determination of macromolecular transport rate across intestinal Peyer's patches.

We used horseradish peroxidase (HRP) (mol wt, 40,000) to compare in vitro, in Ussing chambers, the rates of protein transport across segments of piglet jejunum with and without Peyer's patches. The mean HRP transport rate across intestinal segments with a patch, 25.2 +/- 4.2 SE ng . min-1 . cm-2 (22 animals), was increased threefold (P less than 0.0005) compared with control (no patch) tissue, 7.9 +/- 1.0 ng . min-1 . cm-2 (n = 29). Neither rate showed saturation with increasing concentrations of HRP; both were inhibited 75-95% by a temperature drop from 37 to 15 degrees C. Transport across patch-containing tissue was inhibited 48 +/- 6% (n = 5, P less than 0.0025) by 1 mM NaF, but NaF had no consistent effect on the transport across tissue without Peyer's patches. We conclude that HRP transport is increased across Peyer's patches. This transport is dependent on metabolism and does not involve specific receptors. These findings support the concept that the Peyer's patch serves an antigen-sampling function in the gut.