Verne N. Schumaker

[6] Sequential flotation ultracentrifugation

Publisher Summary Plasma lipoproteins have lower hydrated densities relative to the other plasma proteins; therefore sequential flotation ultracentrifugation has been the principal method used for their isolation and classification. This chapter describes the sequential flotation ultracentrifugation method and some important features for the proper use of sequential flotation ultracentrifugation, such as it is important to calculate the effect of differences in path length or it is virtually impossible to reduce the level of contaminating proteins, such as albumin, below detection limits with a single spin. The ability to process simultaneously large volumes or many small samples is one of the principal advantages of sequential flotation ultracentrifugation. However, the technique suffers from certain limitations, such as during the course of long centrifugation, extensive lipid peroxidation can occur. This problem may be minimized by the inclusion of appropriate chelating agents and antioxidants. Full fractionation and purification by ultracentrifugal methods are both lengthy and costly. Also sequential flotation ultracentrifugation separates lipoproteins according to density only, and the investigator must be aware that particles differing in stage of metabolism, composition, charge, and size may be combined in any of the fractions obtained with this method.

Apolipoprotein B and Low-Density Lipoprotein Structure: Implications for Biosynthesis of Triglyceride-Rich Lipoproteins

ApoB100 is a very large glycoprotein essential for triglyceride transport in vertebrates. It plays functional roles in lipoprotein biosynthesis in liver and intestine, and is the ligand recognized by the LDL receptor during receptor-mediated endocytosis. ApoB100 is encoded by a single gene on chromosome 2, and the message undergoes a unique processing event to form apoB48 message in the human intestine, and, in some species, in liver as well. The primary sequence is relatively unique and appears unrelated to the sequences of other serum apolipoproteins, except for some possible homology with the receptor recognition sequence of apolipoprotein E. From its sequence, structure prediction shows the presence of both sheet and helix scattered along its length, but no transmembrane domains apart from the signal sequence. The multiple carbohydrate attachment sites have been identified, as well as the locations of most of its disulfides. ApoB is the single protein found on LDL. These lipoproteins are emulsion particles, containing a core of nonpolar cholesteryl ester and triglyceride oil, surrounded by an emulsifying agent, a monolayer of phospholipid, cholesterol, and a single molecule of apoB100. An emulsion particle model is developed to predict accurately the physical and compositional properties of an LDL of any given size. A variety of techniques have been employed to map apoB100 on the surface of the LDL, and all yield a model in which apoB surrounds the LDL like a belt. Moreover, it is concluded that apoB100 folds into a long, flexible structure with a cross-section of about 20 x 54 A2 and a length of about 585 A. This structure is embedded in the surface coat of the LDL and makes contact with the core. During lipoprotein biosynthesis in tissue culture, truncated fragments of apoB100 are secreted on lipoproteins. Here, it was found that the lipoprotein core circumference was directly proportional to the apoB fragment size. A cotranslational model has been porposed for the lipoprotein assembly, which includes these structural features, and it is concluded that in permanent hepatocyte cell lines, apoB size determines lipoprotein core circumference.

Two DNA restriction fragment length polymorphisms associated with Ag(t/z) and Ag(g/c) antigenic sites of human apolipoprotein B.

Several high frequency restriction fragment length polymorphisms (RFLPs) associated with the human gene for apolipoprotein B have been previously reported by Priestly et al.1 The EcoRI RFLP here was shown to be very strongly associated with the Ag(t/z) Immunochemlcal polymorphism of human low density llpoprotelns, allowing correct Ag(t/z) phenotyplng of 17 (out of 17 tested) unrelated individuals. The Xbal RFLP was associated with the Ag(g/c) Immunochemlcal polymorphism, permitting correct phenotyplng of 14 (out of 17 tested) unrelated Individuals. Its close association with an RFLP permitted localization of the Ag(t/z) polymorphism to the C-termlnal end of the apolipoprotein B peptlde, and allowed detailed discussion of Its probable molecular basis.

Semi-flexible joint in the C1q subunit of the first component of human complement

Abstract C1q appears in electron micrographs in two different projections: lateral projections, in which the molecules resemble bunches of tulips; and top projections, in which the molecules are seen as six terminal subunits connected to a central portion. We have measured 39 particularly well-formed top views to determine the distribution of distances of terminal subunits from the central portion, from which may be calculated the distribution of the angles made by the connecting strands with an axis through the central portion. This distribution peaks sharply at a preferred angle of 50 °. A limited degree of flexibility must exist, however, for a few molecules are found with angles ranging from 20 ° to 80 °. Therefore, we suggest the existence of a semi-flexible joint at the point of interruption of the collagen-like amino acid sequence where the connecting strands join to the central portion of the C1q molecule.

Conformation and restricted segmental flexibility of C1, the first component of human complement

Seventy selected images of chemically crosslinked C1 are analyzed to illustrate structural details of the C1qC1r2C1s2 complex. From inspection of these images, the C1r2C1s2 tetramer can be seen to be located in the region of the C1q arms, cleanly separated from the C1q heads and from at least 90%, if not all, of the C1q stem. From measurements made upon 65 images, the semicone angles formed between the spreading arms and the symmetry axis passing through the stem of C1 may be calculated. Unlike C1q, for which a wide variety of angles is found, the C1 complex appears to possess a restricted range of angular flexibility with an average value of about 50 degrees. The volume inside the cone formed by the spreading arms of C1q is too small to contain the entire C1r2C1s2 tetramer; at least some of the tetramer must lie outside the cone when it is bound to C1q to form C1. From our knowledge of the sizes and structures of its subunits, and from symmetry considerations, a model is proposed for the configuration of the C1 complex in which the middle portion of the C1r2C1s2 tetramer is centrally located among the arms close to the stem of the C1q and with the two protruding ends of the tetramer wrapped around the outside of the cone. Functional implications of this more rigid structure are discussed with relevance to C1q-induced aggregation of latex beads and C1-induced disaggregation.

A theory of bivalent antibody-bivalent hapten interactions☆

Abstract A theory is developed to describe the concentrations of the differently-sized complexes, both circular and linear, which are formed between bivalent hapten and bivalent antibody. The theory is based upon the mass law and rigorously applies only to homogeneous antibodies. The equilibrium constants which are used in the theory include terms to account for statistical factors, binding energies, and steric restrictions. A computer program based upon the equations is described, and the results of computations discussed in terms of published studies of the interaction between bivalent haptens and bivalent antibodies.

POLYDISPERSITY OF HUMAN LOW‐DENSITY LIPOPROTEINS*

Human lipoproteins are often divided into three main groups: the high-density lipoproteins, the low-density lipoproteins and the chylomicra. The high-density lipoproteins are further subdivided into two classes, HDLz and HDL3. The lowdensity lipoporteins are also subdivided into two classes, the low-density, Sr 0-20, and the very-low-density, Sf 20-400, lipoproteins (Scanu, 1965). Where are these lipoproteins manufactured and what physiological roles do they play? The chylomicra are formed in the intestine, and they function in transporting alimentary lipids from the intestines to the tissues. The very-low-density lipoproteins are manufactured principally in the liver, and apparently supply neutral fats synthesized by the liver to other tissues and storage depots. The highdensity lipoproteins also are manufactured by the liver. Some evidence indicates that these may serve to facilitate the exodus of lipids from the plasma and to keep the tissues free from unwanted lipid deposits (Fredrickson, Levy, and Lees, 1967). Lipoproteins are unique among biological macromolecules in that they are not polymers, for the most part. By and large, the lipid moiety is held in place by noncovalent bonds. And the lipoproteins may grow or shrink depending upon the availability and chemical potential of environmental lipids. Thus, a heterogeneity in size, density, and molecular weight is expected to exist among these molecules, and it is found (Nichols, 1967). The analytical ultracentrifuge has been employed as a primary tool for the study of this heterogeneity. The value of this technique becomes clear when writing the equation relating the sedimentation coefficient to other macromolecular parameters:

Human/mouse chimeric monoclonal antibodies with human IgG1, IgG2, IgG3 and IgG4 constant domains: Electron microscopic and hydrodynamic characterization

The unique structure of the human IgG3 constant region with its greatly extended hinge can clearly be seen in electron micrographs, which compare a series of recombinant proteins with the same murine anti-dansyl variable domain but constant domains from human IgG1, IgG2, IgG3 and IgG4. The hinge region of IgG3 was found to be very long, with some measurements extending to 100 A. It exhibited considerable flexibility allowing the Fc to be displaced far toward either side. Upon addition of bivalent hapten, all of the monoclonal antibodies formed complexes. IgG1, IgG3 and IgG4 formed circular dimers, composed of two antibodies forming a ring-shaped complex, presumably through the binding of two bivalent haptens. IgG2, on the other hand, showed a distribution of complexes which was noticeably different from the other subclasses. Some circular dimers, some linear dimers and a large amount of monomer were seen. This was interpreted in terms of an energy barrier to ring closure arising from the orientation of the Fab arms of IgG2 probably leading to linear dimers as the predominate complex seen with the analytical ultracentrifuge. A substantial number of these dimers probably dissociated upon dilution for examination in the electron microscope. The distribution of the angles between the Fab arms of the monoclonal antibodies forming the circular dimers has been measured for the different subclasses. Most were open at wide angles (> 100 degrees) but some formed very shallow angles, with the Fab arms being nearly parallel to each other. The free energy for this transition was calculated from the ratio of open/closed angles, and it was found to be proportional to the length of the upper hinge of the monoclonal antibody, in agreement with previous nanosecond depolarization results (Dangl et al., Eur. molec. Biol. Org. J. 7, 1989-1994, 1988).

[11] Isolation and characterization of apolipoprotein B-100

Publisher Summary Two physically separable macromolecules carry the designation apolipoproteinB. The long recognized form of apoB, large apoB or B-100, is the predominant protein in LDL and the major apoB protein found in human VLDL. This chapter describes the isolation and characterization of apolipoprotein B-100. ApoB-100 is generally an abundant apolipoprotein in plasma, and constitutes over 95% of the protein mass of low-density lipoprotein (LDL) and approximately one-third of the protein mass of very low-density lipoprotein. It is readily isolated from LDL. ApoB may be modified chemically with lysines and arginines, which changes the biological properties of the apolipoprotein. The chapter suggests that the amino acid analysis of apoB is not particularly useful in characterizing a preparation, and it has been shown that up to 30% of the protein may be removed enzymatically with little change in the amino acid composition of the remaining apoB. A minimal criterion for characterizing an apoB preparation is demonstration of a single band on a 4 or 5% SDS-polyacrylamide gel, and a second criterion is the measurement of the sedimentation coefficient.

The free energy of angular position of the fab arms of IgG antibody

Abstract From the observed distribution of ring-shaped complexes formed with bivalent antibody and bivalent hapten, we have calculated the free energies of angular position of the Fab arms at different angles about the hinge region. These positional energies are found to be small, less than RT, except for the case of complete closure of the hinge angle. When complete closure occurs with the formation of a dimeric complex, we calculate that the strain energy is approximately 700 cal/dimer.