A cyclic AMP-dependent phosphorylation of spectrin dimer

In contrast to the properties of spectrin obtained from [³²P]phosphate-labeled red cells, purified spectrin dimer could be phosphorylated by a cAMP-dependent protein kinase from bovine heart. Both spectrin bands were phosphorylated. Spectrin band 2 contained in addition to autophosphorylated peptides several phosphopeptides that were distinct from autophosphorylated ones. The cAMP-dependent phosphorylation of spectrin band 1 was modulated by reducing agent and the concentration of spectrin. At high concentrations spectrin band 2 was predominantly labeled. The cAMP-dependent phosphoform of spectrin band 2 had a pI slightly higher than that of autophosphorylated spectrin band 2, but lower than that of ankyrin.     

Photosensitized cross-linking of erythrocyte membrane proteins. Evidence against participation of amino groups in the reaction

Exposure of human erythrocyte ghosts (pH 8, 10°C) to visible light in the presence of the photosensitizer, methylene blue, results in a relatively rapid loss of spectrin (bands 1 and 2 on sodium dodecyl sulfate gel electropherograms) and the appearance of high molecular weight cross-linked derivatives. Isolated spectrin also undergoes photosensitized cross-linking, indicating that the reaction is not lipid-dependent.Extensive cross-linking was neither reversed by dithiothreitol nor prevented by prior blocking of SH groups with $\text{N-}\text{ethylmaleimide}$, suggesting that cysteine residues are not crucial bridging sites. The possible requirement for NH₂ groups, as suggested by previous model studies (Dubbelman, T.M.A.R., de Goeij, A.F.P.M. and van Steveninck, J. (1978) Biochim. Biophys. Acta 511, 141–151), was tested. Succinylation of spectrin protected against cross-linking, but this effect is attributed to the disruption of quaternary structure, as deduced from sedimentation measurements. However, virtually complete blocking of NH₂ groups by amidination perturbed overall structure relatively little, and had no effect on cross-linking. Moreover, exogenous amines such as ethylamine, added in large excess to spectrin prior to irradiation, did not interfere with cross-link formation. These results suggest that NH₂ groups are not involved in the reaction.     

Organisation of the proteins of the chromaffin granule membrane

The organisation of the protein components of bovine chromaffin granules has been investigated by labelling or digesting intact granules or broken membranes with the following reagents: lactoperoxidase/Na¹²⁵I as a reagent for tyrosine residues, $\text{N-}\text{(iodoacetylaminoethyl)-5-naphthylamine-1-sulphonic}$ acid as a reagent for cysteine residues, pronase, and galactose oxidase/KB³H₄. Following treatment, membranes were purified and washed and proteins were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Rather more than 60 bands were resolved, of which about 40 were relatively intense and reproducible. The bands were classified according to their molecular weights and sensitivity to reagents. Penetration of the membranes by the reagents was assessed by examination of intragranular proteins.The majority of chromaffin granule membrane polypeptides became labelled when intact granules were treated with impermeant reagents. Eleven were probably protected in the intact granules, reactive sites becoming exposed only on membrane lysis. By contrast, carbohydrate moieties of glycoproteins appear to be exposed only on the matrix side of the membrane. Two proteins were shown to span the membrane, although this is probably an underestimate.     

An F-actin- and calmodulin-binding protein from isolated intestinal brush borders has a morphology related to spectrin

A high molecular weight protein from the brush border of chicken intestinal epithelial cells has been purified. This protein (TW $\text{260}\text{240}$), a complex of two polypeptides with apparent molecular weights of 260,000 and 240,000, accounts for a significant amount of the terminal web organization. TW $\text{260}\text{240}$ is an F-actin-binding protein that also interacts with calmodulin. Rotary shadowing reveals long flexible rods of double-stranded morphology tightly connected at each end. TW $\text{260}\text{240}$ is quite distinct from smooth muscle filamin and macrophage actin-binding protein (APB), but, in spite of its higher contour length (265 nm), seems to be related to erythrocyte spectrin (194 nm for the tetramer). Immunofluorescence microscopy with antibodies against TW $\text{260}\text{240}$ indicates the existence of a submembranous organization distinctly different from that of stress fibers. We have compared TW $\text{260}\text{240}$ with fodrin, a brain protein known to occur in submembranous organization but not previously characterized in molecular terms. TW $\text{260}\text{240}$ and fodrin are clearly distinct molecules but are similar in many aspects. Ultrastructural, biochemical and immunological results indicate three distinct classes of rod-like high molecular weight actin-binding proteins, possibly reflected by the prototypes filamin (ABP), spectrin and TW $\text{260}\text{240}$ (fodrin). The latter group may be responsible for calmodulin control of submembranous microfilament structures in various nonmuscle cells.     

Investigation of the quaternary structure of beef liver glutamate dehydrogenase with bifunctional reagents

The six identical polypeptide chains of the smallest enzymatically active unit of beef liver glutamate dehydrogenase are shown to be arranged in two trimers. Cross-linking with bifunctional reagents of varying chain length and subsequent SDS polyacrylamide gel electrophoresis of the protein shows main bands corresponding to molecular weights of 168,000 and 336,000 daltons which is three times and six times, respectively, the molecular weight of the polypeptide chain (56,000 daltons). This finding supports a model for the quaternary structure of the glutamate dehydrogenase proposed by Reisler and Eisenberg (Biopolymers $\text{9}$, 877 (1970)).     

The determination of the helical screw angle of a helical particle from its diffraction pattern

A Fourier analysis of the distributions of different types of amino acids in the sequence of tropomyosin shows strong 14th-order peaks in the profiles of both negatively charged and non-polar amino acids, with a period of $\text{19}\text{2}\text{3}$ residues and an overall repeat length of 275 ± 2 amino acids, which is shorter than the sequence length of 284 amino acids. Both peaks are statistically significant and confirm Parry's work (1974, 1975b). The regularities are analysed in terms of an assumed supercoil structure in which two α-helices lie parallel and in register to form a supercoil with a pitch of 137 Å. These molecules are then assumed to overlap end-to-end by eight to nine amino acids so that the periodicity is continuous along an extended filament of linked tropomyosin molecules. The periodic features are stronger in the outer surface of the molecule away from the core of the supercoil. The sequence divides into 14 bands which each have a narrow zone of net positive charge and a broader negatively charged zone. Overlapping every positive zone is a hydrophobic zone which always has at least one non-polar group on the outer surface. Anomalies in the charge distribution are found near the molecular ends and close to Cys190. These are attributed to the end-to-end overlap site and the troponin binding site.In the thin filament the 137 Å pitch supercoil would make seven half-twists relative to the twisted actin helix along a 385 Å length, so that a pair of adjacent bands would be oriented equivalently with respect to a pair of actins 28 Å apart. We therefore suggest that the bands (each containing one zone of each type) should be divided alternately into two series, α and β. Every pair of bands is $\text{39}\text{1}\text{3}$ residues long and each of the seven pairs corresponds with one segment of the 42-residue gene duplication repeat observed previously in the sequence. The disparity between the periods of 42 and $\text{39}\text{1}\text{3}$ is overcome by deletions and insertions. The $\text{39}\text{1}\text{3}$-residue periodicity is not simply a consequence of the supercoil structure or gene duplication but is probably a result of adaptation to the spatial periodicity of the actin helix in muscle. Although the α and β bands are alike in general, they differ systematically in detail and the α bands are more regular than the β.We propose that the seven α and seven β bands are alternative sets of sites which bind equivalently to complementary groups of sites on seven actins in the “relaxed” and “active” states of muscle, respectively. In each band the negative zone probably attaches to actin by magnesium bridges and the hydrophobic zone by direct contacts with the narrow outer edge of the supercoil. Since the supercoil twists 90 ° relative to actin on passing between adjacent α and β bands, a quarter rotation of the whole tropomyosin molecule would detach one set of seven sites and attach the other, allowing a highly co-operative switch mechanism.     

The solubilization of cytoskeletons of human erythrocyte membranes by p-mercuribenzene sulphonate

The disruption of erythyrocyte membrane cytoskeletons brought about by treatment with $\text{p-}\text{mercuribenzene sulphonate}$ (PMBS) has been followed by measurements of turbidity and the binding of ²⁰³Hg-labelled PMBS. After pretreatment with $\text{N-}\text{ethylmaleimide}$ to block readily reactive sulphydryl groups, incubation with [²⁰³Hg]PMBS showed incorporation of approximately 4 moles radiolabel per mole of spectrin and one per mole of actin. The incorporation of radiolabel paralleled the decrease in turbidity, and the labelling of spectrin paralleled that of actin. The kinetics were pseudo first order, and the pH dependence of the observed rate constant indicated a normal $\text{p}\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ value for the sulphydryl group involved. The calculated second-order rate constant for the reaction of the sulphydryl anion with PMBS, however, was several orders of magnitude less than expected from model compound studies. The results suggest that association between spectrin and actin may result in the steric hindrance of reactivity of a limited number of sulphydryl groups in each protein. Disruption of the spectrin-actin association may then be linked to the modification of the sulphydryl groups.     

The two components of spectrin, filamin, and the heavy chain of smooth muscle myosin show no detectable homologies to one another by two-dimensional mapping of iodinated tryptic peptides.

The possible structural relationships among four high molecular weight mechanochemical proteins has been examined using two-dimensional mapping of the tryptic peptide fragments prepared from ¹²⁵I-labeled proteins (Elder et al., J. Biol. Chem. $\text{252}$:6510–6515 (1977)). Erythrocyte spectrin bands 1 and 2 protein, the heavy chain of smooth muscle (uterine) myosin, a filamin from human and rabbit were studied. The maps of the four proteins within each species differed considerably from each other, with no apparent homologies evident among them, whereas maps of the same individual protein between the two species showed a high degree of homology.     

Purification and characterization of two forms of a low-affinity Ca2+-ATPase from erythrocyte membranes

A low-affinity Ca²⁺-ATPase from erythrocyte membranes has been purified by agarose suspension electrophoresis and polyacrylamide gel electrophoresis in the absence of detergents. For maximal activity a calcium concentration above 10 mM is required. The activity is independent of magnesium. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP is about 60 μM. The enzyme appears in two forms (A and B) with similar amino acid composition. The specific activity of A is higher than that of B. Gel electrophoresis in SDS of A gives a pattern consisting of two bands. B gives the same pattern; the only difference between the patterns is the ratio of the amounts of protein in the bands. The apparent molecular weight of the proteins in the two SDS bands has been estimated at 23 000 and 21 000, respectively. The results obtained can be explained by assuming that the two proteins corresponding to the two bands obtained in SDS electrophoresis have a similar structure and can associate to complexes A and B. We have also shown that electrophoretic and chromatographic supporting media can induce aggregation of (membrane) proteins. Artificial complexes can thus be formed and cause misinterpretation of the data obtained. This may be the reason why some authors have speculated that Ca²⁺-ATPase is active only in complex with other proteins such as spectrin and actin.     

Prostaglandin F2α receptors in bovine corpus luteum cell membranes. Effect of enzymes and protein reagents

Various enzymes and proteins reagents inhibited [³H]prostaglandin F₂α binding to bovine corpus luteum cell membranes. Studies were undertaken (a) to explore further on the dose response relationships with the above agents, (b) to investigate the mechanism of inhibition of binding with respect to receptor affinities and number and (c) to assess whether decreased binding reflected changes in receptors and/or other membrane components.Preincubation of membranes with phoshpolipase A, trypsin, pronase, lipase, tetranitromethane, dinitrofluorobenzene, acetic anhydride and $\text{N-}\text{ethylmaleimide}$ resulted in moderate to drastic inhibitions of [³H]prostaglandin F₂α binding. The dose-dependent inhibition of binding by enzymes, but not by protein reagents (except for $\text{N-}\text{ethylmaleimide}$), exhibited a biphasic pattern: at lower concentrations, the loss of binding was low and relatively plateaued, but at higher concentrations, the losses were dramatic. The drastic reduction in binding by trypsin was due to destruction rather than solubilization of receptors from membranes. Phospholipase A was intrinsically more effective than phospholipases C and Ca²⁺ was not required for its inhibition of [³H]prostaglandin F₂α binding. Protein reagents inhibition of binding was differently influenced by added Ca²⁺ i.e., loss of binding increased with some ($\text{N-}\text{ethylmaleimide}$), decreased with others (tetranitromethane, dinitrofluorobenzene and azobenzene sulfenylbromide). These results are interpreted to indicate that Ca²⁺ induced conformational changes in membranes which may result in exposure of new groups and burying of already exposed modifiable groups.Treatment of membranes wiht trypsin and $\text{N-}\text{ethylmaleimide}$ selectively abolished high affinity prostaglandin F₂α receptors. The low affinity receptors were present but their numbers as well as their affinity were decreased. Lipase, phospholipase A, acetic anhydride, dinitrofluorobenzen and tetranitromethane appear to decrease binding by totally abolishing all prostaglandin F₂α receptors or by severely reducing their affinities.The occupancy of receptors by prostaglandin F₂α afforded considerable protection against trypsin, phospholipase A, lipase and dinitrofluorobenzene. These data indicated that the inhibition of binding by the above agents, at least in part, can be attributable to changes in receptor sites alone.     

The interaction of spectrin · actin and synthetic phospholipids

Using differential scanning calorimetry and freeze fracture electron microscopy interactions were studied between lipids and a spectrin · action complex isolated from human erythrocyte membranes. With dispersions of $\text{1,2-}\text{dimyristoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$, $\text{1,2-}\text{dimyristoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphoglycerol}$ and mixtures of these two compounds, which for experimental reasons were chosen as the lipid counterpart, such an interaction could clearly be deduced from changes in the temperature and the enthalpy of the phase transition. Furthermore it was demonstrated that the interaction with this membrane protein protects the bilayer against the action of Ca²⁺ and Mg²⁺ and prevents fusion of lipid vesicles which easily occurs in some of the systems when divalent ions were added to the pure lipid vesicles.     

Motility of the N-terminal tail of phosphorylase b as revealed by crosslinking.

There are lysyl-ε-NH₂ groups within about 3.5 Å distance across the intersubunit contact area of rabbit muscle phosphorylase $\text{b}$, as shown by cross-linking with malonic diimidate. These include the lysines of N-terminal region as revealed by limited tryptic digestion, but the contribution of the tail lysines to overall formation of covalent dimers is small. The fine structure of dimer band on dodecylsulfate-gelelectrophoretograms of crosslinked phosphorylases suggests that the tail retains its freedom in the phosphorylase $\text{b}$-AMP complex. Amidination induces the dissociation of phosphorylase $\text{b}$ dimer, which is slow relative to crosslinking.     

Studies on the fragmentation of erythrocyte ghost membrane with p-chloromercuribenzoate in the micromolar range

The effects of nonsaturating amounts (5–60 nmol/mg membrane protein) of $\text{p-}\text{chloromercuribenzoate}$ on the stability of unsealed erythrocyte ghosts were studied by turbidimetric measurements and direct observation by phase contrast microscopy. The organic mercurial provokes drastic disorganization of the membrane involving vesicle formation by inter- and externalization of the bilayer. These effects are not associated with a release in solution of membrane proteins which was shown in previous studies to occur at higher $\text{p-}\text{chloromercuribenzoate}$ concentration. Attempts have been made to identify the proteins involved in this phenomenon by the use of nonsaturating amounts of radioactively-labelled $\text{p-}\text{chloromercuribenzoate}$. Actin and band 3 protein which are the first to be labelled, represent plausible candidates as sensitive targets for the disrupting organic mercurial. Stroma obtained from spherocytes did not show significant differences with normocytes in their stability with regard to $\text{p-}\text{chloromercuribenzoate}$. Other reagents including $\text{N-}\text{ethylmaleimide}$, diamide and DNAase I were also studied. The results suggest strongly that the integrity of the sulfhydryl groups of actin, as well as those of band 3 protein, is essential for the stability of the erythrocyte membrane.     

Binding of steroids to P.testosteroniΔ5-3-ketosteroid isomerase: Measurement of the number of binding sites by equilibrium dialysis

The binding of progesterone, 17β-estradiol and 19-nortestosterone acetate to the Δ⁵-3-ketosteroid isomerase from $\text{Pseudomonas}$$\text{testosteroni}$ has been investigated by the technique of equilibrium dialysis. Under the conditions used, all three steroids formed 2:1 complexes with each molecule of enzyme dimer (M.W. = 26,788). No evidence of any cooperative binding phenomena was obtained. The dissociation constants of the enzyme steroid complexes at 25°C were: progesterone, 2.2 μ$\text{M}$; estradiol, 2.5 μ$\text{M}$; 19-nortestosterone acetate, 9.2 μ$\text{M}$.     

The argECBH-bearing EcoRI segment of the Escherichia coli chromosome

The transducing phage λd$\text{arg}\text{14}$, carrying a portion of the $\text{E. coli}$ chromosome including $\text{argECBH}$, is derived from the heat-inducible, lysis-defective strain λy199, which has the $\text{b}\text{519}$ and $\text{b}\text{515}$ deletions. Cleavage of λy199 DNA by $\text{Eco}$RI endonuclease, followed by agarose slab gel electrophoresis, results in bands corresponding to the known C, D, E, and F segments of λ, and a segment A′ (A plus B minus $\text{b}\text{519}$ minus $\text{b}\text{515}$, the cleavage site between A and B being eliminated). Cleavage of λd$\text{arg}\text{14}$ DNA by $\text{Eco}$RI yields the expected D, E, and F segments of λ and four other segments, termed 14-1 through 14-4, whose length is 17.5, 6.2, 3.0, and 2.0 kilobases, respectively, as determined by electron microscopy and corroborated by electrophoretic mobility. Heteroduplex analysis shows that the $\text{E. coli argECBH}$ cluster is on the 14-1 segment.     

Crystallographic studies of immunoglobulins: crystallization of the Fc fragment of rabbit IgG with and without cleavage of the inter-chain disulphide bridge

The Fc fragment was prepared from rabbit immunoglobulin G by digestion with papain, both with the inter-chain disulphide bond intact, and after reduction and alkylation. These two types of Fc crystallized in different, yet related forms, each with one dimer in the asymmetric unit. The covalently linked dimer crystallized in space group $\text{P2}{}_1\text{; a = 68.85 ± 0.05}\text{A}\text{?}\text{, b = 72.50 ± 0.05}\text{A}\text{?}\text{, c = 60.40 ± 0.05}\text{A}\text{?}$ and β = 105.1 ± 0.2 °. The reduced, non-covalently linked dimer also crystallized in space group $\text{P2}{}_1\text{; a = 81.55 ± 0.05}\text{A}\text{?}\text{, b = 55.65 ± 0.05}\text{A}\text{?}\text{, c = 68.85 ± 0.05}\text{A}\text{?}$ and β = 1051 ± 0.2 °. A non-crystallographic 2-fold axis relating the two identical polypeptide chains is clearly visible in the h0l projection of the second crystal form.     

Influence of coupling conditions on activity and operational stability of glucoamylase immobilized on titanium (IV)-activated controlled pore glass

Glucoamylase (EC 3.2.1.3) was coupled to controlled pore glass by using titanium(IV) chloride. The drying conditions used during the activation step were studied, and the highest activity (237 units/g of matrix) of immobilized enzyme was obtained when the support and the titanium(IV) chloride solution were dried at 45°C in vacuo for 16 h. After several washing cycles, the specific activity of the immobilized enzyme was ～13 units/mg of protein irrespective of the washing cycle used. However, this immobilized enzyme preparation was also the least stable ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1}\text{h}$). Investigation of the possibility of the stabilization of the linkage of the enzyme to the support by crosslinking with bifunctional reagents showed that the stabilization of the enzyme ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=100}\text{h}$) was achievable by treatment with a 5% glutaraldehyde solution at pH 7.0 for 2 h (product activity 67 units/g of matrix, specific activity 4 units/mg of protein); this product also showed no release of protein during use. A higher activity (296 units/g of matrix was achieved by stabilization by treatment with a 5% tannic acid solution at pH 7.0 for 2 h. The combined use of glutaraldehyde and tannic acid was effective in stabilizing the bound enzyme ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=80 to 120 h}$) with an initial activity of 116 units/g of matrix. When use was made of the same support in presilanized (3-aminopropyltriethoxy silane) form followed by glutaraldehyde coupling a similar initial activity (112 units/g of matrix) was obtained, but the operational stability was much better ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 640}\text{h}$.     

Biosynthesis of 2-methylalkanes in the crickets Nemobius fasciatus and Gryllus pennsylvanicus.

The biosynthesis of 2-methylalkanes was studied in the crickets $\text{Nemobius}\text{fasciatus}$ and $\text{Gryllus}\text{pennsylvanicus}$. Labelled acetate, valine, and isobutyric acid were incorporated into the cuticular hydrocarbon of $\text{N.}\text{fasciatus}$ at levels of 6.0 ± 1, 6.5 ± 2, and 1.5 ± 0.7 percent respectively. The hydrocarbons of this insect are 20 percent 2-methylalkanes, primarily of even numbered carbon chain lengths, and 80% n-alkanes. Of the label incorporated into the hydrocarbon fraction, 28 ± 2 percent of sodium [1-¹⁴C] acetate, 98 ± 1 percent of L-[G-³H] valine, and 75 ± 10 percent of [1-¹⁴C] isobutyric acid were incorporated into the 2-methylalkanes. This suggests that valine is converted to isobutyric acid and is incorporated into the even numbered carbon chain length 2-methylalkanes during the initial stages of chain elongation. Similar data obtained in $\text{G.}\text{pennsylvanicus}$ suggests that leucine is converted to isovaleric acid which is then incorporated into the odd numbered carbon chain length 2-methylalkanes.     

Novel heme: O2 bonding of possible relevance to oxygen utilizing heme and other proteins

Solid dipyridine hemes which are unreactive toward oxygen lose both pyridine ligands upon heating under vacuum to give a solid which takes up O₂, reversibly, one O₂ per heme. Replacement of ¹⁶O₂ by ¹⁸O₂ reduces only infrared bands near 1660 and 1590 cm^?1, frequencies near the vibrational band for gaseous O₂. No FeO bands are detected. EPR spectra reveal a free radical and ferric iron; Mössbauer, NMR and infrared spectra support an iron(III) oxidation state. Limited molecular weight data indicate a dimer. Possibly two dioxygen molecules are held sandwich fashion between two porphyrins via donor-acceptor interactions, which are facilitated by electron transfer from iron(II) into the porphyrin forming a π-anion. Such O₂ bonding is not found in oxy Hb and Mb or in oxyhemerythrin but may occur with cytochrome $\text{c}$ oxidase and other oxygen utilizing (or producing) heme and other proteins.