Excluded volume effects on the partition of macromolecules between two liquid phases

Partition parameters of several proteins and other macromolecules are measured in an aqueous two-phase liquid system containing polyethylene glycol and phosphate buffer. Distribution of macromolecules is a function of the relative volume excluded to the macromolecules in the two phases. A simple model with no adjustable parameters yields covolumes of the macromolecules with the polyethylene glycol. Covolumes are used to estimate effective molecular volumes and the magnitudes of excluded volume effects. The same approach is applied to mixtures of macromolecules.     

Effects of Hydration on Steric and Electric Charge-Induced Interstitial Volume Exclusion—a Model

The presence of collagen and charged macromolecules like glycosaminoglycans (GAGs) in the interstitial space limits the space available for plasma proteins and other macromolecules. This phenomenon, known as interstitial exclusion, is of importance for interstitial fluid volume regulation. Physical/mathematical models are presented for calculating the exclusion of electrically charged and neutral macromolecules that equilibrate in the interstitium under various degrees of hydration. Here, a central hypothesis is that the swelling of highly electrically charged GAGs with increased hydration shields parts of the neutral collagen of the interstitial matrix from interacting with electrically charged macromolecules, such that exclusion of charged macromolecules exhibits change due to steric and charge effects. GAGs are also thought to allow relatively small neutral, but also charged macromolecules neutralized by a very high ionic strength, diffuse into the interior of GAGs, whereas larger macromolecules may not. Thus, in the model, relatively small electrically charged macromolecules, such as human serum albumin, and larger neutral macromolecules such as IgG, will have quite similar total volume exclusion properties in the interstitium. Our results are in agreement with ex vivo and in vivo experiments, and suggest that the charge of GAGs or macromolecular drugs may be targeted to increase the tissue uptake of macromolecular therapeutic agents.     

Nuclear estradiol receptors in several "non-target" organs of rats]

Some properties of the macromolecules of the KCl-extracts of the nuclei of the uterus, kidney, liver, testis and prostate, specifically binding estradiol (E2), were studied. These macromolecules of the uterus and the liver were found to be maximally extracted from chromatin by the 0.6 M KCl concentration. The capacity of the macromolecules of the uterine, kidney and liver nuclear extracts to bind E2 specifically is destroyed completely by pronase, but not by RNA-ASe and DNA-ase, pointing to the protein nature of these macromolecules. Only estrogenic compounds, but not testosterone, 5alpha-dihydrotestosterone, progesterone or corticosterone were capable to compete with H3--E2 for the E2--binding sites of the macromolecules of the nuclear extracts of all the organs investigaeted. It is assumed that macromolecules of the nuclei of the investigated nontarget organs specifically binding E2 are estrogen receptors.     

Alignment of Synaptic Vesicle Macromolecules with the Macromolecules in Active Zone Material that Direct Vesicle Docking

Synaptic vesicles dock at active zones on the presynaptic plasma membrane of a neuron’s axon terminals as a precondition for fusing with the membrane and releasing their neurotransmitter to mediate synaptic impulse transmission. Typically, docked vesicles are next to aggregates of plasma membrane-bound macromolecules called active zone material (AZM). Electron tomography on tissue sections from fixed and stained axon terminals of active and resting frog neuromuscular junctions has led to the conclusion that undocked vesicles are directed to and held at the docking sites by the successive formation of stable connections between vesicle membrane proteins and proteins in different classes of AZM macromolecules. Using the same nanometer scale 3D imaging technology on appropriately stained frog neuromuscular junctions, we found that ∼10% of a vesicle’s luminal volume is occupied by a radial assembly of elongate macromolecules attached by narrow projections, nubs, to the vesicle membrane at ∼25 sites. The assembly’s chiral, bilateral shape is nearly the same vesicle to vesicle, and nubs, at their sites of connection to the vesicle membrane, are linked to macromolecules that span the membrane. For docked vesicles, the orientation of the assembly’s shape relative to the AZM and the presynaptic membrane is the same vesicle to vesicle, whereas for undocked vesicles it is not. The connection sites of most nubs on the membrane of docked vesicles are paired with the connection sites of the different classes of AZM macromolecules that regulate docking, and the membrane spanning macromolecules linked to these nubs are also attached to the AZM macromolecules. We conclude that the luminal assembly of macromolecules anchors in a particular arrangement vesicle membrane macromolecules, which contain the proteins that connect the vesicles to AZM macromolecules during docking. Undocked vesicles must move in a way that aligns this arrangement with the AZM macromolecules for docking to proceed.     

Excluded volume as a determinant of macromolecular structure and reactivity

The effect of excluded volume on the thermodynamic activity of globular macromolecules and macromolecular complexes in solution is studied in the hard-particle approximation. Activity coefficients are calculated as a function of the fraction of total volume occupied by macromolecules using relations obtained from scaled particle and lattice models. Significant and readily observable effects are predicted to occur as the fraction of volume occupied by globular macromolecules increases, including the following: (i) Compact quasi-spherical macromolecular conformations become increasingly energetically favored over extended anisometric conformations. (ii) Self- and heteroassociation processes are enhanced, particularly those leading to the formation of compact quasi-spherical aggregates. (iii) Depending upon the details of the reaction mechanism, the rate of an enzyme-catalyzed reaction may monotonically decrease, go through a maximum, or exhibit more complex behavior. A given degree of volume occupancy by larger macromolecules is predicted to have less effect on the structure and self-association of smaller macromolecules than the same degree of volume occupancy by smaller macromolecules has on the structure and self-association of larger macromolecules.     

用电导法研究悬浮介质中大分子物质对红血球细胞(RBC)聚集的一种抑制效应

通常认为悬浮介质中的大分子物质促进RBC聚集的桥联作用,使RBC聚集,但在高浓度时,大分子物质反而抑制RBC聚集。过去认为主要是高浓度大分子物质影响了RBC表面势能,但我们在研究中发现,在一定条件下低浓度的大分子物质也能抑制红细胞聚集。这说明:大分子物质对RBC聚集的抑制效应,不仅能通过改变RBC膜表面势能实观,还能通过其它途径实现,这种途径之一就是影响RBC膜的力学性质。     

Turnover of cell-surface macromolecules in cultured dog tracheal epithelial cells

We studied the metabolism of sulfated cell-surface macromolecules in dog tracheal epithelial cells in primary culture. To examine the time-course and rate of appearance of sulfated macromolecules at the cell surface, the cells were pulsed with 35SO4 for short periods (5-15 min), and the incubation medium was sampled for spontaneously released macromolecules (basal secretions) and for release induced by trypsin (trypsin-accessible secretions). Trypsin-accessible 35S-labeled macromolecules appeared on the cell surface within 5-10 min, increased linearly, and plateaued by 40 min; the median transit time for 35S-labeled macromolecules to reach the cell surface was 21 min. 35S-labeled macromolecules in basal secretions increased with a similar time-course, reaching a plateau by 40 min. Incorporation of [3H]serine into the protein moiety of trypsin-accessible macromolecules occurred more slowly; trypsin-accessible 3H-labeled macromolecules were barely detectable at 1 h and increased to a maximum after 2 h, suggesting the presence of a preformed pool of nonsulfated core protein. Pretreatment with cycloheximide, an inhibitor of protein synthesis, decreased trypsin-accessible 35S-labeled macromolecules log-linearly depending on the duration of pretreatment providing an estimate of the rate of depletion of the core protein pool (t1/2 = 32 min). During continuous exposure to 35SO4, 35S-labeled macromolecules accumulated on the cell surface (trypsin-accessible compartment) for 16 h, at which point the cell-surface pool was saturated (t1/2 = 7.5 h). After pulse-labeling the cells with 35SO4 for 15 min, the 35S-labeled macromolecules disappeared continuously from the cell surface (t1/2 = 4.6 h), and 79% of the radioactivity was recovered in the medium as nondialyzable macromolecules. Release of the 35S-labeled macromolecules from the cell surface was abolished at 4 degrees C, indicative of an energy-dependent process, but multiple proteinase inhibitors did not affect the release. We conclude that sulfate is metabolized rapidly into epithelial cell-surface macromolecules, which accumulate continuously into a relatively large cell-surface pool, before they are released by an undefined energy-dependent mechanism.     

生物大分子质谱电离技术的突破及核磁共振三维结构测定方法的建立——2002年诺贝尔化学奖简介

2002年诺贝尔化学奖授予了质谱和核磁共振领域的三位科学家以表彰他们对生物大分子鉴定及结构分析方法做出的贡献．其中两位科学家J．B．Fenn和K．Tanaka分别发展了生物大分子质谱分析的软解吸电离方法;另一科学家K．Wüthrich则将核磁共振技术成功地应用于生物大分子如蛋白质的溶液三维结构测定．他们的研究成果已使质谱和核磁共振技术成为生物大分子强有力的研究手段,极大地促进了生物大分子的研究进程,必将对整个生命科学研究产生深远的影响．     

Developments in describing equilibrium phase transitions of multivalent associative macromolecules

Biomolecular condensates are distinct cellular bodies that form and dissolve reversibly to organize cellular matter and biochemical reactions in space and time. Condensates are thought to form and dissolve under the influence of spontaneous and driven phase transitions of multivalent associative macromolecules. These include phase separation, which is defined by segregation of macromolecules from the solvent or from one another, and percolation or gelation, which is an inclusive networking transition driven by reversible associations among multivalent macromolecules. Considerable progress has been made to model sequence-specific phase transitions, especially for intrinsically disordered proteins. Here, we summarize the state-of-the-art of theories and computations aimed at understanding and modeling sequence-specific, thermodynamically controlled, coupled associative and segregative phase transitions of archetypal multivalent macromolecules.     

Regulation of synaptic vesicle docking by different classes of macromolecules in active zone material

The docking of synaptic vesicles at active zones on the presynaptic plasma membrane of axon terminals is essential for their fusion with the membrane and exocytosis of their neurotransmitter to mediate synaptic impulse transmission. Dense networks of macromolecules, called active zone material, (AZM) are attached to the presynaptic membrane next to docked vesicles. Electron tomography has shown that some AZM macromolecules are connected to docked vesicles, leading to the suggestion that AZM is somehow involved in the docking process. We used electron tomography on the simply arranged active zones at frog neuromuscular junctions to characterize the connections of AZM to docked synaptic vesicles and to search for the establishment of such connections during vesicle docking. We show that each docked vesicle is connected to 10-15 AZM macromolecules, which fall into four classes based on several criteria including their position relative to the presynaptic membrane. In activated axon terminals fixed during replacement of docked vesicles by previously undocked vesicles, undocked vesicles near vacated docking sites on the presynaptic membrane have connections to the same classes of AZM macromolecules that are connected to docked vesicles in resting terminals. The number of classes and the total number of macromolecules to which the undocked vesicles are connected are inversely proportional to the vesicles' distance from the presynaptic membrane. We conclude that vesicle movement toward and maintenance at docking sites on the presynaptic membrane are directed by an orderly succession of stable interactions between the vesicles and distinct classes of AZM macromolecules positioned at different distances from the membrane. Establishing the number, arrangement and sequence of association of AZM macromolecules involved in vesicle docking provides an anatomical basis for testing and extending concepts of docking mechanisms provided by biochemistry.     

Procedure for freeze-drying molecules adsorbed to mica flakes

The quick-freeze, deep-etch, rotary-replication technique is useful for visualizing cells and cell fractions but does not work with suspensions of macromolecules. These inevitably clump or collapse during deep-etching, presumably due to surface tension forces that develop during their transfer from ice to vacuum. Previous protocols have attempted to overcome such forces by attaching macromolecules to freshly cleaved mica before drying and replication. I describe here an adaptation of this procedure to the deep-etch technique as otherwise practiced. My innovation is to mix the molecules with an aqueous suspension of tiny flakes of mica and then to quick-freeze and freeze-fracture the suspension exactly as if one were dealing with cells. The fracture inevitably strikes the surfaces of many mica flakes and thereby cleaves the adsorbed macromolecules cleanly enough to reveal interesting substructure within them. The subsequent step of deep-etching exposes large expanses of unfractured mica and thus reveals intact macromolecules. These macromolecules are not obscured by salt deposits, even if they were frozen in hypertonic solutions, apparently because the fracturing step removes nearly all of the overlying electrolyte. Moreover, these macromolecules are minimally freeze-dried (since exposure is sufficient after only 3 min of etching at -102 degrees C) so they retain their three-dimensional topology. I show that molluscan hemocyanin is a good internal standard for this new technique. It is available commercially in stable solutions, mixes well with all sizes of macromolecules, and consists of particles that display distinct five-start surface helices, which have been measured carefully in the past and which possess a known handedness, useful for determining the orientation of micrographs when examining the various helical patterns possessed by most types of extended macromolecules. The fractured hemocyanin particles also display characteristic internal structures, which permit determination of the elevation of the "molecular cleavage" described above. Finally, molluscan hemocyanin is delicate enough to reflect bad freezing or poor replication, if these steps become a problem. A survey of several macromolecules is presented, including soluble enzymes, antibodies, filamentous proteins and nucleoproteins. These images, for the most part, correspond to those previously obtained by negative staining. New details of their structures are noted, and the images are used to illustrate both the advantages and drawbacks of the new procedure.     

The Isolation and Characterization of the Two Macromolecular Antitumor Agents from Streptomyces

Purification of the two antitumor macromolecules, A216 and A280 substances, from culture filtrates of Streptomyces is achieved by chromatography using ion-exchanged celluloses. The purified macromolecules appeared homogeneous are characterized as a protein from the chemical and biological properties by paper electrophoresis, paper chromatography and ultracentrifuge.

The simple method for approximation of molecular weight of a protein from distribution coefficient on gel filtration is proposed. The molecular weights of both macromolecules given by gel filtration are near to those given by ultracentrifugal analysis.     

荧光能量转移技术在生物学研究中的应用

荧光能量转移(FRET)是指两个携带不同荧光基团的大分子在相互间距离足够近时(10～100A)所发生的能量非放射性地由一个荧光基团向另一个荧光基团转移的现象。结合绿色荧光蛋白的发现,FRET技术可用于检测生物大分子中不同亚基的位置和生物大分子间的相互作用。近年来,FRET技术在生物学研究中的突破性进展是在活体细胞中实时监测生物大分子之间的相互作用。本文就绿色荧光蛋白的发现,FRET技术的原理、研究进展和应用前景作简要综述。     

Organic matrixlike macromolecules associated with the mineral phase of sea urchin skeletal plates and teeth

The skeletal plates and teeth of the echinoid Paracentrotus lividus contain a heterogeneous assemblage of macromolecules that are not part of the connective tissue, but are presumably intimately associated with the mineral phase. Upon dissolution of the Mg-calcite mineral phase, some of these molecules are insoluble. The insoluble fractions of the teeth and skeletal plates are quite different, the former being predominantly protein and the latter, primarily some unknown nonproteinaceous material. The soluble constituents are similar in both tissues. These hydrophilic macromolecules have been partially separated and characterized. In both hard parts, two distinct classes of macromolecules are present, as indicated by the amino acid compositions of their protein constituents. These two classes of macromolecules are also present in the shells of a foraminifer and in various mollusks, both of which are formed by the "organic matrix-mediated" biomineralization process. The locations of these macromolecules in the teeth and skeletal plates are not known, nor whether they form coherent structures. It is therefore premature to conclude that these macromolecules do function as an organic matrix, although the results presented are in agreement with such an interpretation.     

Plant suspension culture media macromolecules-pectic substances, protein, and peroxidase

Macromolecules secreted into the media by a nondifferentiating suspension culture of tobacco cells were found to be composed of protein and polysaccharide, and to account for the viscosity of the media. The concentration, composition, and viscosity of these macromolecules changed significantly with the age of the culture and growth temperature. The concentration changed from 0.02 mg/ml in newly inoculated cultures to over 1 mg/ml in older cultures. The macromolecules contained from 6 to 18% protein and 3 to 4 μmoles hydroxy-proline/mg nitrogen, more than 20 times the level found in whole cells. The macromolecules contained 5 to 25% pectic substances whose carboxyl groups were either methyl esterified or combined with calcium. Arabinose, xylose, glucose, galactose, and mannose were identified in acid hydrolysates of the macromolecules. Peroxidase activity of the macromolecules increased as cultures became older. Peroxidase isoenzyme patterns changed with culture age and growth temperature. The relation of the macromolecules to cell walls and intercellular substances is discussed.     

Macromolecules and the origin of life

From our knonwledge of present day organisms, it is hard to imagine a living assembly, even at its most primitive stage, without macromolecules.In order to look for the macromolecules which possibly participated in the assembly of the primitive organisms, the reaction and formation of polymers in HCN under irradiation of ultraviolet ray of 184.9 nm. was studied.As a example of a simple way of producing an assembly of macromolecules, the mechanism of coacervation was studied by using gelatin as the material.     

Boundary element solution of macromolecular electrostatics: interaction energy between two proteins.

The boundary element technique is implemented to solve for the electrostatic potential of macromolecules in an ionic solution. This technique entails solving surface integral equations that are equivalent to the Poisson and the Poisson-Boltzmann equations governing the electrostatic potential inside the macromolecules and and in the solvent. A simple but robust method is described for discretizing the macromolecular surfaces in order to approximate the integral equations by linear algebraic equations. Particular attention is paid to the interaction energy between two macromolecules, and an iterative procedure is devised to make the calculation more efficient. This iterative procedure is illustrated in the electron transfer system of cytochrome c and cytochrome c peroxidase.     

Fibronectin in bovine vitreous. An immunochemical study

The vitreous body is a transparent gel essentially composed of hyaluronan, collagen and proteoglycans. These components are assembled in a three-dimensional structure that is maintained by self-aggregation of macromolecules and the interactions between these different macromolecules. We confirmed the presence of fibronectin in vitreous body using immunochemical methods and by indirect immunofluorescence on cryostat sections. We also determined its distribution in vitreous extracts as compared to those of collagen, proteoglycans and hyaluronan. Because of its high affinity to these macromolecules fibronectin in the vitreous appears to play an important role in strengthening and stabilizing the gel structure.     

Glomerular permselection: shape and flow

The selective permeability of the glomerular basement membrane to macromolecules is a function of the size and charge of the macromolecule. Evidence suggests that shape may also be a factor. The orientation of macromolecules in solution is dependent on their size, shape, and frictional interactions with moving solvent molecules. The spaces between the glomerular visceral epithelial cells (slit pores) may produce a non-uniform distribution of fluid flow within the basement membrane, and this non-uniformity may increase during disease. This report is of a model that relates the filtration of rigid prolate ellipsoidal (cigar) shaped macromolecules to the size and shape of the filter and to the velocity of solvent flow. The calculations, using published macromolecular and glomerular parameters correspond well to published data. The glomerular visceral epithelial cell, by altering the number, size and distribution of the intercellular spaces, may regulate the passage of ellipsoidal shaped macromolecules, such as albumin and IgG, into and through glomerular structures.     

Modeling biological macromolecules in solution: 3. The Λ-R intersection method for triaxial ellipsoids

The general triaxial ellipsoid model for the gross conformation of macromolecules in solution represents a signficiant advance over the previously, almost ubiquitously used ellipsoid of revolution model. A new method is presented which involves the graphical intersection of two triaxial hydrodynamic functions (Λ and R) involving viscosity, sedimentation and fluorescence depolarization. The method is restricted to macromolecules asymmetric enough for the functions to be sufficiently sensitive but not so asymmetric for there to be problems of internal rotations between parts of the macromolecules. The method is illustrated by application to data for neurophysin II monomers and dimers.