Interactions of actin and tubulin with human deoxyhemoglobin. Their possible occurrence within erythrocytes

Short actin filaments are an essential component of the red-cell membrane skeleton, and microtubules are also present in nucleated erythrocytes as a marginal band. Actin and tubulin share the property of possessing a very anionic terminal peptide. Since deoxyhemoglobin (Hb) is known to be a strong polyanion-binding protein, we have considered how it may interact with actin and tubulin within the intact cell. Here we demonstrate that actin and tubulin form in vitro a high-affinity complex with Hb. This is shown by measuring, by stopped-flow experiments, the decrease of the binding rate constant of CO to Hb in the presence of increasing amounts of actin and tubulin. One tetramer of Hb is bound by an actin monomer, and about two tetramers by an alpha, beta-tubulin heterodimer. Binding assays in batch experiments with immobilized tubulin give the same stoichiometry. Formation of the complexes involves the 2,3-bisphosphoglycerate-binding site of Hb and a negatively charged domain, most likely the highly acidic N and C-terminal peptides of actin and tubulin. In addition to providing new opportunities to study the structural and functional properties of actin and tubulin, these results support the idea that in the case of partial metabolic depletion of bisphosphoglycerate and ATP in erythrocytes, Hb may interact with oligomeric actin and tubulin present in the cytoskeleton.     

Role of nucleoside components and internucleotide phosphate groups of oligodeoxyribonucleotide template in its binding to human DNA polymerase alpha

Affinity labelling of human placenta DNA polymerase alpha (EC 2.7.7.7) with the reactive oligodeoxyribonucleotide d(pT)2pC[Pt2+(NH3)2OH](pT)7 was used for quantitative analysis of enzyme interaction with oligodeoxyribonucleotides as templates. Dissociation constants and Gibb's energy values for different oligothymidylates d(pT)nT where n = 1-14 have been evaluated by competitive experiments of these ligands with Pt2+ reagent. The data obtained prove the formation of one Me2+-dependent electrostatic contact and a hydrogen bond between the enzyme and one phosphate of these templates. One may suppose that the hydrophobic interaction of any other monomeric link of oligodeoxyribonucleotides with the enzyme template site takes place.     

Characterization of the nucleotide tight-binding sites of the isolated mitochondrial F1-ATPase

The properties of the nucleotides tightly bound with mitochondrial F1-ATPase were examined. One of three bound nucleotide molecules is localized at the site with Kd approximately 10(-7) M and released with koff approximately 0.1 s-1. The second nucleotide molecule is bound with the enzyme with Kd approximately 10(-8) M and koff for its dissociation is 3 X 10(-4) s-1. The third is never released even in the presence of 1 mM ATP or ADP. The last two nucleotides are believed to be bound at the noncatalytic sites of F1-ATPase. Pyrophosphate promotes liberation of two releasable nucleotide molecules, decreasing the affinity of the enzyme to AD(T)P. From the results obtained it follows that the only suitable criterion for localization of the nucleotide at the F1-ATPase catalytic site is the high rate (koff greater than or equal to 0.1 s-1) of its spontaneous release.     

On secondary hyperparathyroidism in orang-utans, Pongo satyrus (Linnaeus, 1758)

Orang-utan crania with alterations in bone structure which could be determined morphologically were studied and evaluated. The alterations in bone structure were referred to in the literature up to about 1939 as "rickets"; when vitamin D was given to the animals, the alterations diminished, until they were almost unnoticeable. From about 1941/52, the alterations were diagnosed as "Morbus Paget". Research on orang-utan crania has become possible through comparison of a larger number of single symptoms, occurring in a number of individuals. Out of a larger sample, the study was carried out on 5 individuals, showing these alterations in varying degrees. The individuals also covered various age groups, both sexes and both subspecies of orang-utans. The findings permit a diagnosis of secondary (or tertiary) hyperparathyroidism (= Morbus Engel-von Recklinghausen = fibrous cystic osteitis). The study also showed that orang-utans fall prey to Morbus Engel-von Recklinghausen in a shorter period and suffer more severely than humans. The frequency of orang-utans suffering from this disease, which are kept captive in zoos, is statistically far higher than the occurrence in humans. Orang-utans living under natural conditions do not suffer from the disease at all; according to the study of 500 animals. The authors also believe that there is a psychogenic basis for the occurrence of Morbus Engel-von Recklinghausen in zoo animals; psychological conditions such as apathy, disinterest, etc. are part of the illness, and these symptoms are also shown by animals having no outward signs of fibrous cystic osteitis. The authors believe that improved prophylaxis of orang-utans in zoos would lead to discovery of Morbus Engel-von Recklinghausen in early stages, and allow early therapy to arrest the disease. This is necessary for preservation of the species.     

Characterization of a heterogeneous camel milk whey non-casein protein

A milk protein, occurring in the whey fraction, has been characterized from camel milk. Determination of the primary structure reveals the existence of two related types of chain with residue differences in at least the N-terminal region. A fragment representing an N-terminal part of the protein was also recovered (heterogeneous at the same positions). The absence of cysteine residues in the protein shows that no disulphide bridges are present. The pattern of fragments and a parent protein resembles that for casein and its fragments, showing that fragments and a multiplicity of forms may be typical for different milk proteins.     

Cytological studies on six cyprinid fishes

The chromosome complements of six cyprinid fishes were studied, using the routine air-drying Giemsa staining technique. The diploid chromosome number recorded is 2n = 50 (8m+18sm+14st+10t) with NF = 90 in Aspidoparia morar, 2n = 50 (8m+12sm+12st+18t) with NF = 82 in Crossocheilus latius latius, 2n = 50 (6m+12sm–16st+16t) with NF = 90 in Labeo pangusia, 2n = 70 (16m+6sm+16st–32t) with NF = 108 in Perilampus atpar, 2n = 48 (4m+6st+38t) with NF = 58 in Puntius chrysopterus and 2n = 50 (2m+2sm+4st+42t) with NF = 58 in P. tetrarupagus. Sex chromosomes are not identifiable in any of these species. A pair of marker chromosomes has been observed in all species excepting A. morar.     

The impact of different fixation procedures on staining of macromolecules in a microtiter system

Summary The effects of formaldehyde, glutaraldehyde, methanol, Clarke's fixative and microwave irradiation on the quantitative staining of proteins (Naphthol Yellow S) and nucleic acids (Ethyl Green—Pyronin) in a cell culture system have been investigated. Overall, glutaraldehyde rapidly yielded the highest and most consistent levels of staining when compared to all other chemical fixatives. Although microwave irradiation was found to be uneven, 4 min exposure to 700W was found to give higher levels of protein staining than those achieveable with glutaraldehyde. Time-dependent processes were observed with all procedures. In addition, dissociations in the trends of protein and nucleic acid staining were observed. It is suggested that these results domonstrate fixation events that have not previously been resolved from the effects of reagent penetration into tissue blocks.     

Genetics and evolution of the Lpm-system in American mink. VI. Identification, genetic control and phylogenesis of the new allotype Lpm13

Data on immuno- and biochemical identification, genetic control and phylogenesis of new allotype Lpm13 of the Lpm system in domestic mink are presented. This allotype is encountered in mink populations with the frequency 0.9 and higher. The availability of Lpm13 genetic marker permitted another haplotype to be revealed, in addition to the eight known Lpm haplotypes by means of genetic analysis. It was established that, alongside with the earlier described haplotype Lpm3,4,6,8,9,10,11 (abbreviation H3), there exists a similar haplotype, Lpm3,4,6,8,9,10,11,13 (abbreviation H3.13), containing the Lpm13 gene. Of the rest seven haplotypes, five have the Lpm13 gene and two do not. Taking into account this gene and corresponding antigenic marker, the differentiation of 28, instead of 25, phenotypes and 45, instead of 36, genotypes for the Lpm system became possible. Lpm13 antigenic specificity was found with no exception in all individual serum samples taken from ten species and interspecific hybrids of Mustelidae which are closely related to domestic mink. The data obtained give grounds to refer the newly identified Lpm13 gene to the first evolutionary conservative category of genes of the multigenic Lpm system which is also represented by the Lpm6, Lpm9, Lpm10 and Lpm11 genes. The hypotheses of instantaneous formation of polymorphism of the Lpm system in domestic mink are briefly regarded.