Intermediate Filaments from Bovine, Rat, and Human CNS: Mapping Analysis of the Major Proteins

Abstract: Intermediate filaments were isolated by an axon-flotation method from bovine, rat, and human CNS. Gel electrophoresis showed four major proteins, having molecular weights of about 50,000, 70,000, 160,000, and 210,000, to be present in filaments of all three species. Small differences in molecular weights and major differences in relative distribution of the filament proteins were observed among species. In bovine and rat brain the predominant protein was the 50,000 band, but in human brain the 70,000 band was present in greatest amount. Each filament protein of the three species was studied by peptide mapping using limited proteolysis and cyanogen bromide cleavage. Within the same molecular weight group, filament proteins from different species gave similar maps with both techniques. Some degree of heterogeneity was also observed. However, filament proteins of different molecular weights of the same species gave distinctly different maps. These studies rule out the possibility that filament proteins from different molecular weight groups are related to each other by oligomerization; nor is it likely that the lower molecular weight proteins are derived from the subunit of molecular weight 210,000.     

Plant use in Kaigani Haida culture: Correction of an ethnohistorical oversight

Until recently the use of plants for the precontact cultures of the Northwest Coast has been weakly and sporadically documented. This has led to some misconceptions about the importance of food plants in the precontact diet. This paper documents the use of plants associated with food for the Kaigani Haida of southeast Alaska who were first contacted in 1774, giving the native terms for the plants, and methods of preparation and storage both in precontact and contemporary life. Evidence is offered which indicates that plants were of great importance to the societies of the Northwest Coast and that food plants were more important than has been recognized. A map shows the territory of the Kaigani Haida prior to Euro-American contact, old village sites and the modern village of Hydaburg. Women’s knowledge of plants, their native names, economic use, and method of preparation and storage are offered as evidence that food plants were significantly important to the precontact economy. Nutritional analyses of some of the berries and sprouts indicates that such foods were necessary in the diet in order to maintain healthy, viable populations.     

Scaling threshold characters

A simple method of scaling ordered categorical responses having a joint distribution with an underlying normal variable is presented. Scores are developed that maximize heritability of the observed variate and that in the class of scores based on polychotomies: (1) maximize the correlation between score and the underlying genetic value to be predicted, and (2) minimize mean-square prediction error. Several examples suggest little is lost, in terms of heritability, by using equally spaced scores. The proposed scaling method discriminates among candidates for selection that would be tied if equally spaced scores are used and sometimes yields different rankings of candidates.     

Tightly bound calcium of adenosine triphosphatase in sarcoplasmic reticulum from rabbit skeletal muscle

Sarcoplasmic reticulum vesicles were shown to possess a class of tightly bound calcium ions, inaccessible to the chelator, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid at 0 degrees C or 25 degrees C, amounting to 4.5 nmol/mg of protein (approximately 0.5 mol/mol (Ca2+,Mg2+)-ATPase). The calcium ionophores, A23187 and X537A, induced rapid exchange of tightly bound calcium in the presence of chelator. Chelator alone at 37 degrees C, caused irreversible loss of bound calcium, which correlated with uncoupling of transport from (Ca2+,Mg2+)-ATPase activity. Uncoupling was not accompanied by increased permeability to [14C]inulin. Slow exchange of tightly bound calcium with medium calcium was unaffected by turnover of the ATPase or by tryptic cleavage into 55,000- and 45,000-dalton fragments. Binding studies with labeled calcium suggested that tight binding involves a two-step process: Ca2+ + E in equilibrium K E . Ca2+ leads to E < Ca2+ where E and < Ca2+ represent the ATPase and tightly bound calcium, and K = 1.6 X 10(3) M-1. It is suggested that tightly bound calcium is located in a hydrophobic pocket in, or in close proximity to the ATPase, and, together with tightly bound adenine nucleotides (Aderem, A., McIntosh, D. B., and Berman, M. C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3622-03632), is related to the ability of the ATPase to couple hydrolysis of ATP to vectorial transfer of calcium across the membrane.     

Proton-magnetic-resonance spectroscopic study of the histidine residues of bovine alpha-lactalbumin.

A study of the three histidine residues of bovine alpha-lactalbumin has been made using proton magnetic resonance (PMR) spectroscopy in order to obtain information on their environments in the protein and thereby to test in part the previously proposed structure. PMR titration curves are obtained for the H-4 resonances using difference spectroscopy and for the H-2 resonances and the 1-H-2-H exchange rates of the H-2 protons have been measured. The assignment of resonances to particular histidine residues is achieved by utilising their selective reaction with iodoacetate in conjunction with a PMR study of the carboxymethylation of alpha-N-acetyl-L-histidine. The H-2 and H-4 resonances labelled 1, 2 and 3 starting from the downfield end of the spectrum are assigned to histidine residues 107, 68 and 32 respectively. Their apparent pK values at low ionic strength and 20 degrees C are 5.78, 6.49 and 6.51 respectively. The experimental results on two histidine residues are consistent with the predictions of the proposed structure, which indicate that histidine-68 is an external residue and histidine-32 is partially buried and in the vicinity of aromatic residues. The experimental data on histidine 107 can also be rationalised with less certainty in terms of the proposed structure, which indicates a partially buried residue that may be involved in hydrogen bonding.     

Evidence for essential lysyl residues in ribulosebisphosphate carboxylase by use of the affinity label 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate.

A previous study from our laboratory suggested that 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate is an affinity label for spinach ribulosebisphosphate carboxylase. To identify the essential residues that react with the reagent we have isolated and characterized the labeled peptides that are present in tryptic digests of inactivated enzyme but lacking in digests of the substrate-protected enzyme. Peptides representing two sites of modification have been obtained from the inactivated carboxylase. Both sites of reaction have been identified as lysyl residues based on the conversion of the derivatives to free lysine by oxidation with sodium metaperiodate. Sodium dodecyl sulfate-gel electrophoretic experiments show that both essential lysyl residues are contained within the large subunit of ribulosebisphosphate carboxylase. In addition to lysyl residues, sulfhydryl groups of the carboxylase are also modified, but their modification seems to play little role in the inactivation process. The carboxylase modified in the presence of substrate contains sulfhydryl derivatives but is essentially lacking in lysyl derivatives. By comparing the profiles from ion exchange chromatography of labeled peptides in digests of inactivated and substrate-protected enzyme, we conclude that the same sulfhydryl groups are modified in the absence and presence of substrate.     

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Strategies for assignments.

Natural abundance 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes) of aqueous native proteins yield numerous narrow single carbon resonances of nonprotonated aromatic carbons. Techniques for the assignment of these resonances are presented. Each technique is applied to one or more of the following proteins: ferricytochrome c from horse heart and Candida krusei, ferrocytochrome c and cyanoferricytochrome c from horse heart, lysozyme from hen egg white, cyanoferrimyoglobins from horse and sperm whale skeletal muscle, and carbon monoxide myoglobin from horse. In all of the protein spectra we have examined, methine aromatic carbons give rise to broad bands. Studies of the narrow resonances of nonprotonated aromatic carbons of proteins are facilitated by removal of these broad bands by means of the convolution-difference method, preferably from spectra recorded under conditions of noise-modulated off-resonance proton decoupling. We present a summary of the chemical shift ranges for the various types of nonprotonated aromatic carbons of amino acid residues and hemes of diamagnetic proteins, based on our results for hen egg white lysozyme, horse heart ferrocytochrome c, horse carbon monoxide myoglobin, and carbon monoxide hemoglobins from various species...