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Nuclear glycoproteins recognized by Concanavalin A have been isolated from pig, rabbit and chicken tissues. Mono and bidimensional electrophoresis patterns of proteins loosely and tightly bound to DNA have been examined. The tissue specificity rather than species-specificity appears to be a quite general property of these proteins, suggesting for them a role in the mechanism of regulation of chromatin functions.

We have investigated the distribution of tissue-specific tightly bound non-histone proteins in the first and third levels of chromatin organization. The proteins of this class have been extracted from whole chromatin and chromatin fractions prepared from pig liver or kidney. The tissue-specific proteins have high molecular mass (ranging from 135 KDa to 70 KDa in liver, over 135 KDa in kidney) and in kidney a more basic isoelectric point. These proteins are mainly located outside the core particles, and are instead present only in the chromatin matrix, become more intense after extensive digestion of the matrix with DNAase I.

Microcalorimetric titrations of whole chromatin and histones with sodium dodecylsulfate were performed at pH 7 and 25 degrees C. Enthalpy variations at low detergent concentration (less than 0.02%) are much more negative for histones than for chromatin. At 0.065% sodium dodecylsulfate the difference between the two curves becomes constant and, after correction for monomerization effects, amounts to +130 kcal/mol of nucleosomal unit. Core particles show heat effects similar to those of histones. These findings suggest that the chromatin structure is not stabilized exclusively by electrostatic interactions and that hydrogen bonds responsible for the additional stability may be contributed by non histone chromatin proteins.

Non-histone proteins, tightly bound to DNA, have been extracted from whole chromatin and core particles prepared from pig liver or kidney. We have investigated by bidimensional slab gel electrophoresis the distribution of this protein class in the first level of repeating structure of chromatin. Our results reveal that non-histone proteins tightly bound to DNA are a heterogeneous protein class. Some of them, particularly in the core particles, appear to be essentially the same in both tissues, though having differences in their isoelectric point, which may be attributed to postsynthetic modifications. We have calculated that this protein class is associated to only 10% of nucleosomes, these nucleosomes having, on the average, one protein molecule for each core DNA. The tissue-specific proteins have high molecular mass (ranging from 135 kDa to 70 kDa in liver, over 135 kDa in kidney) and, in kidney, a more basic isoelectric point. These proteins are mainly located outside the core particles; they could be situated in the spacer regions and/or be involved in determining higher levels of chromatin organization.

The tissue specificity of a particular class of non-histone chromatin proteins characterized by strong bonds to DNA, tightly bound non-histone chromatin proteins (t.b. NHCp), was investigated. In connection with its possible role in the cell differentiation, its distribution in chromatin fractions obtained by digestion with DNAase II and precipitation with MgCl2 was studied. It was shown by bidimensional gel electrophoresis that some proteins of this class are tissue specific for pig liver compared to kidney chromatin. For both tissues, most tissue specific proteins were found to be peculiar to the chromatin fraction that remains insoluble after prolonged digestion with DNAase II. The proteins found in this chromatin fraction are common to the nuclear matrix proteins and, considering the chromatin organization, may be involved in the attachment point for loops in the axial matrix structure. It is possible that proteins of this class, directly or indirectly, play an important role in the regulation of transcription and differentiation in eukaryotes, by modulating the supercoiled state of DNA.

Digestion of pig liver chromatin with DNAse II afforded three different fractions which were characterized in terms of their DNA, RNA and tightly bound non-histone protein content, their DNA fragment size and their template activity. Two of these fractions are soluble after digestion with DNAase II and have been separated on the basis of their different solubility in MgCl2. A third fraction is not solubilized even after extensive digestion, although the size of its DNA is comparable to that of the enzyme solubilized fractions. The three fractions show qualitatively and quantitatively different distribution of tightly bound non-histone proteins, with specific protein components in each fraction; furthermore the non-solubilized fraction is greatly enriched in proteins tightly bound to DNA. From all the data obtained it can be suggested that the tightly bound proteins of the insoluble fraction may play, directly or indirectly, a role in maintaining an organized chromatin structure.

Total half-cystine residues in proteins of pig liver chromatin have been measured. About half of them are present in the reduced state. Thiol groups of non-histone chromatin proteins, which amount to about 40 nmol/mg of protein, are preferentially located in chromatin fragments which are more easily solubilised either by DNAse I or by DNAse II. The data obtained are compatible with an involvement of SH and SS groups in chromatin structure and function.

Chromatin glycoproteins recognized by Concanavalin A have been isolated from pig liver, kidney and heart by the use of immobilized lectin. Two groups of proteins differing in affinity for DNA have been analysed. Glycoproteins are mainly present in the group of proteins which are tightly bound to DNA. Mono and bidimensional electrophoretic patterns of total tightly bound proteins reveal a similarity among the three organs examined, while the corresponding patterns of the glycoproteins are typical for each organ. The tissue specificity of chromatin glycoproteins, together with their capability to interact not only with DNA but possibly also with other nuclear components, suggest a role for these proteins in the mechanism of genome expression.