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W H Massover

Publications and source records attributed to W H Massover.

At least 19 recordsLinked to original sources
PubMed2001

Negative staining permits 4.0 A resolution with low-dose electron diffraction of catalase crystals.

Low-dose electron diffraction of thin single crystals of catalase that are negatively stained with the light-atom compound, dipotassium glucose-1,6-diphosphate, reveals Bragg reflections extending to 4.0A (= 0.40 nm). Under the same conditions, negative staining with the traditional heavy-metal salt, ammonium molybdate, also gives diffraction spots extending to 4.0 A. These results establish that negative staining of protein crystals preserves periodic structural information into the high-resolution range, unlike the widely accepted current belief that this methodology can give a resolution limited to only 20-25 A.

Catalase↗
PubMed2000

Light atom derivatives of structure-preserving sugars are unconventional negative stains.

Although glucose and certain other sugars are known to greatly reduce distortion and denaturation of proteins during drying, use of this monosaccharide as an experimental negative stain does not permit imaging of lattice periodicities in test specimens of thin catalase crystals. However, the potassium and sodium salts of several forms of monophosphorylated glucose (200 mM), diphosphorylated glucose, monosulfated glucose, maltose-1-phosphate, and trehalose-6-phosphate, all dry into a glassy layer and scatter transmitted electrons sufficiently to show the 86 A major periods in catalase crystals. Glucose-6-phosphate provides sufficient image contrast at concentrations from 2 mM (=0.067%) to 500 mM (= 16.8%). Underfocusing increases visualization of the periodic lattice, indicating a large contribution of phase contrast to these images. Upon exposure to the electron beam, thicker regions of derivatized saccharides or pure glucose develop bubbling; this redistribution of dried stain largely can be precluded by imaging with low-dose exposures. Power spectra of images of catalase crystals contained within 200 mM disodium glucose-6-phosphate show that periodic information can be recorded to 21 A; some individual features of dipotassium glucose-6-phosphate distribution within the protein lattice have a measured width of around 5 A. The experimental results demonstrate that structure-preserving mono- and di-saccharides also serve successfully as negative stains after they are coupled to light atom scatterers.

Animals↗
PubMed1996

Novel properties of L-type polypeptide subunits in mouse ferritin molecules.

Properties of the L- and H-type polypeptide subunits forming ferritin 24-mer molecules in mice were investigated, using the products of in vitro transcription and translation from the two cloned genes, and recombinant ferritin molecules (H24L0 or H0L24) produced by transformation in Escherichia coli. Several different conditions for analytical electrophoresis reproducibly show that the relative migration position of the two mouse ferritin subunits is reversed from that reported for ferritin H- and L-subunits in all other mammals; since mouse and human H-polypeptides almost co-migrate, this unusual relative mobility is due largely to novel properties of the murine L-subunit. This unusual electrophoretic property of the mouse L-subunit has led to conflicting reports about the subunit composition of natural mouse ferritin. Here, we show that the single major electrophoretic band given by liver ferritin purified from mice having a short-term iron overload matches that produced by the genetically defined L-polypeptide and that some bona fide H-subunits are also detected. In conclusion, it is reasonable to assume that, when mouse ferritin samples will be analyzed under the same conditions as those described here, the slower species will correspond to the L-type subunit. However, when dealing with ferritin from species other than human or mouse, it should be kept in mind that upon electrophoretic analysis of ferritin polypeptide, the designation of an electrophoretic band as being H- or L-type subunits will be very uncertain without corroboration from genetic, immunological, or amino acid sequencing data.

Animals↗
PubMed1989

Rabbit serum alpha-2-macroglobulin binds to liver ferritin: association causes a heterogeneity of ferritin molecules.

Rabbit liver ferritin is unusual since it forms two discrete electrophoretic bands at the beta position of molecular dimers (Santambrogio & Massover, 1987). The present studies have sought to identify the nature of a 170 kDa non-ferritin polypeptide that is uniquely present in the larger beta band. Ultrastructural, immunological and biochemical results all indicate that this polypeptide is a subunit of the plasma protein. alpha-2-macroglobulin. Experimental results show that rabbit serum alpha-2-macroglobulin will bind liver ferritin, and this association induces the de novo formation of the larger beta band. These results thus demonstrate that molecular heterogeneity of ferritin can be caused by its association with a non-ferritin protein. We conclude that alpha-2-macroglobulin is a binder of rabbit tissue ferritin in the circulation; this binding could provide additional means for the receptor-mediated uptake of circulating ferritin.

Animals↗
PubMed1988

Iron binding proteins and their roles in the tobacco hornworm, Manduca sexta (L.).

Manduca sexta larvae accumulate large amounts of iron during their larval feeding period. When 59Fe was fed to 5th instar larvae, it was evenly distributed among the hemolymph, gut and carcass until the cessation of feeding. By pupation 95% of the labelled iron was found in the fat body. In the adult a significant portion of this iron was found in flight muscle. Studies of the hemolymph disclosed two iron-containing proteins. The first was composed of a single polypeptide chain of 80 kD, containing one atom of iron. This protein bound ionic iron in vitro and was able to transfer this iron to ferritin when incubated with fat body in vitro. Therefore, it appeared to serve a transport function. The second protein had a molecular weight of 490 kD with subunits of 24 and 26 kD and contained 220 micrograms of iron/mg protein. Its chemical and ultrastructural characteristics were those of ferritin. These studies demonstrate the presence of both a transport protein and a unique circulating ferritin in Manduca sexta, the latter serving a storage function during development and possibly also a transport function.

Animals↗
PubMed1987

Protein heterogeneity in rabbit liver ferritin: two types of molecular dimers.

Native pore-gradient polyacrylamide gel electrophoresis of rabbit liver ferritin reveals the usual single band of molecular monomers, but shows two bands at the position of molecular dimers. The proteins in these three bands were purified by excision from preparative slab gels. All three bands (1) contain considerable amounts of iron-rich ferritin when examined by electron microscopy, (2) show complete identity when reacted with anti-rabbit-ferritin antibodies, and (3) have similar amounts of H-type and L-type ferritin subunits with denaturing polyacrylamide gel electrophoresis. These results establish that there are two classes of ferritin molecular dimers. The larger dimer band uniquely also contains a polypeptide with Mr = 170,000. This unusual type of ferritin heterogeneity seems to be due to the presence of a non-ferritin protein associated only with one class of dimers.

Animals↗
PubMed1985

Molecular size heterogeneity of ferritin in mouse liver.

As much as 4% of the total protein in pure liver ferritin from mice with short-term parenteral iron overload produces a minor band migrating anodally to the major (alpha) band of holoferritin with non-denaturing polyacrylamide gel electrophoresis. The components in this minor band and the alpha band have been isolated to purity by preparative electrophoretic fractionation. The protein in the minor band is ferritin, since it contains ferric iron and fulfills defining criteria at the level of biochemistry, immunology and ultrastructure. Native polyacrylamide electrophoresis with pore-size-gradient gels shows that the ferritin molecules in the minor band have a slightly smaller diameter than the holoferritin in the alpha band. Isoelectric focusing reveals that the smaller ferritin has an identical number and range of charge isomers (pI 4.9-5.3) as the larger ferritin, but the relative amount of each size class within some isoferritin bands differs. The smaller ferritin molecules are structurally intact and are made from polypeptide subunits with Mr 18 000; the larger ferritin molecules have subunits with Mr 22 000. The minor species of hepatic ferritin thus has a smaller molecular size because it is made mainly from smaller subunits. No minor electrophoretic band can be detected in liver ferritin obtained from mice with normal iron levels. These results demonstrate that siderosis induces the formation of molecular size polymorphism (macroheterogeneity) in mouse liver ferritin. The new smaller hepatic ferritin could serve to redistribute excess iron into the main storage organs during the early response to iron overload, since it appears to be identical to one of the two types of serum ferritin molecules present in these siderotic mice.

Animals↗
PubMed1979

Mouse hepatoma and liver ferritins. Comparative structural studies.

Pure ferritin from male mouse liver produces a single band of monomers (RF = 0.199) with electrophoresis in polyacrylamide gels at pH 9.0. The five sub-bands within this monomeric band appear to represent charge isomers having the same molecular size. Ferritin from BH3 transplantable mouse hepatoma shows two overlapping bands of monomers (RFA = 0.208 and RFB = 0.240); further electrophoretic studies show that these bands represent two subpopulations of molecules differing both in charge and size. Sub-bands are not found in this hepatoma ferritin. The larger tumor ferritin reaches the same end migration position as all liver isoferritins on gradient gels, signifying a very similar or identical molecular size; however, the absence of sub-bands indicates that this hepatoma ferritin differs in charge from the homologous liver proteins. Liver and hepatoma ferritins both produce a single prominent subunit band corresponding to nominal molecular weights of 22 250 and 21 700, with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. With electrophoresis on polyacrylamide gradient slabs containing sodium dodecyl sulfate and dithiothreitol, both liver and hepatoma ferritins now reveal two subunits bands situated at identical positions. The polypeptides of these two closely spaced bands have a nominal molecular weight difference of less than 1000. Neither the hepatoma nor the liver seems to produce the ferritins found in the other tissue. Nevertheless, all these ferritins are composed of the same two types of subunits, albeit in different relative amounts. Observed distinctions in the ferritins from these normal or neoplastic cells must reflect differences in assembly and processing, as well as in the regulated expression of the same ferritin genes.

Animals↗
PubMed1978

Multiple isoferritins in mouse liver: demonstration by polyacrylamide gel electrophoresis.

Unfractionated pure ferritin isolated from the livers of female or male mice forms five narrow protein-positive bands on standard 5% polyacrylamide gel disc electrophoresis (pH 9.0). Since all of these sub-bands also contain iron, they are interpreted as being isoferritins. The multiple sub-bands are very unlikely to be artifactually generated by the analytical procedure used since they are not found in horse spleen ferritin when this is coelectrophoresed with female mouse liver ferritin. The present results provide an independnet indication that many isoferritins indeed can be found within a single organ.

Animals↗
PubMed1973

The ultrastructure of ferritin macromolecules. The lattice structure of the core crystallites.

The ultrastructure of the crystalline ferric mineral that forms the central core of ferritin macro-molecules has been examined by means of ultrahigh resolution electron microscopy at 100 kV. Very high magnification dark-field images reveal the presence of either a single large crystal or several smaller crystallites within many of the cores. When the highly crystalline core contents are suitably oriented to transmit their Bragg reflections through the objective aperture, regular fringes separated by 2-9.5 A have been visualized. The geometrical relations of lattice fringes and of periodically organized point details in these individual crystallites largely confirm the structural model proposed by Towe and Bradley (1967). The highly variable occupancy of ferric ions in certain planes of the lattice suggests that 20-33% of the iron content of fully saturated ferritin should undergo more rapid physiological release than does the remainder, and that iron uptake will have kinetics that depend upon more than only the maximal rate of crystallization.

Crystallography↗