**2.7. NA radiolabeling**

NA were 5'-endlabeled *in vitro* with -[32P]ATP and T4 polynucleotide kinase (Maxam A., et al. 1977). Dephosphorylation was carried out in 50 mM Tris-HCL (pH 9.0), 10 mM MgCl2, 10 mM spermidine, 1 mM ZnCl2, 0.1 units of alkaline phosphatase at 370С. The enzyme was removed by treating the samples twice with phenol. NA was precipitated with ethanol and collected by centrifugation in a minicentrifuge at 12,000 rpm for 5 min. The pellet was washed with 70% ethanol to remove phenol, dried, and dissolved in a necessary volume of bidistilled water. The resulting solution was combined with a kinase buffer (50 mM Tris-HCl, pH 7.6), 10 mM MgCl2, 50 mM dithiothreitol, 1 mM spermidine), 1-2 MBq of -[32P]ATP, and 2 units of T4 polynucleotide kinase. The mixture was incubated at 37С for 30 min. The reaction was terminated by adding EDTA to 20 mM, and the enzyme was removed by phenol treatment. NA was precipitated with ethanol, and the nonbound label was removed by repeated precipitation with ethanol or by gel filtration on Sephadex G-25.

## **2.8. Dot hybridization**

BA83 and BA85 filters (Schleicher & Shuell, Germany) with immobilized DNA and polysaccharides (20 g per dot) were hybridized with radiolabeled NA probes at 37C for 18-24 h. The hybridization buffer contained 5 х Denhardt's solution (0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, 0.02% Ficoll), 0.1% SDS (Denhardt D.T. 1966). Before being applied onto a filter, DNA was denatured in boiling water for 5 min. Filters were dried and exposed with a РМ-В X-ray film for 1-7 days.

### **2.9. Neutral sugar assays**

Neutral sugars (monosaccharides contained in protein-polysaccharide complexes) were assayed by the anthrone method according to (Jermyn M.A. 1975, Chaplin M.F., et al. 1986). A sample (0.5 ml) was combined with 4 ml of an anthrone reagent (20 mg of anthrone, 100 ml of 80% sulfuric acid). The mixture was heated in boiling water for 5 min, chilled in water to room temperature, and tested for A630. As a control, we used an aqueous solution water treated with the anthrone reagent as above. Neutral sugars were quantitated using a calibration plot, which was constructed with galactose solutions.

To study the GAG composition, a homogenate and the nuclear fraction of the liver were obtained from intact adult rats after fasting over 24 h. To estimate the contents of glycans (HA, CS, and HS), alkaline lysis, deoxyribonuclease treatment, gel filtration on Sephadex G-25, and ion-exchange chromatography on DEAE cellulose were performed as above. Polysaccharide elution profiles were obtained with a gradient of NaCl concentration (0-1.5 M). DNA was assayed in nuclei and liver homogenates as in (Slutskii L.I. 1969).

In the next series of experiments, non-inbred white rats weighing 190-210 g were injected intraperitoneally with [14С]glucose, labeled at C1, at 1 g per kg body weight (4 х 108 Bq per rat). Rats were sacrificed and the liver frozen in liquid nitrogen 15, 30, 60, 120, and 360 min after the injection. Differential centrifugation was used to obtain the nuclear and microsomal fractions (Orekhovich V.N. 1968), which were tested for radioactivity. Tissue homogenates were obtained similarly; their GAG fractions were isolated and tested for radioactivity.

The results were statistically processed using the software package STATISTICA 6.0 for WINDOWS. The processing included computation of the mean and standard deviation, tests for significance of differences; and correlation, regression, and factor analyses as well as analysis of variance.
