Glycobiology Advance Access originally published online on October 9, 2003
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Glycobiology vol 14 no 1 pp. 65-72, 2004
© Oxford University Press 2004; all rights reserved.
Enhanced activity of serum and urinary hyaluronidases in streptozotocin-induced diabetic Wistar and GK rats
Institute of Medicinal Chemistry, Hoshi University, 2-4-41, Ebara, Shinagawaku Tokyo 142-8501, Japan
Received on August 12, 2003; revised on September 17, 2003; accepted on September 21, 2003
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Abstract |
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Using streptozotocin-induced diabetic Wistar and GK rats as models of type 1 and type 2 diabetes, respectively, we investigated the changes in serum and urinary hyaluronidase activity with the pathological progress. The serum hyaluronidase levels of streptozotocin-induced rats started to increase on the third day after injection and thereafter maintained
threefold higher levels compared with control rats; those of GK rats were already higher (twofold) from the beginning of the experiment. The increases of serum hyaluronidase activity in both diabetic rats were similar to those of blood glucose level, indicating that diabetes mellitus was accompanied by enhanced activity of circulating hyaluronidase from the early phase of its development. In zymography, every serum from diabetic and control rats gave two hyaluronidase isomers, a major 73-kDa band (Hyal-1 type) and a minor 132-kDa band, suggesting that the increases in serum hyaluronidase activity were not due to the appearance of novel isomers. The hyaluronidase activity in 24-h urine of streptozotocin-induced rats was 3-, 7-, and 11-fold higher at the 8th, 15th, and 18th week than that of control rats, respectively, and the urinary hyaluronidase activity of GK rats was not significantly different from controls. There was a good correlation between the urinary hyaluronidase activity and the albumin excretion. Thus the increase in urinary hyaluronidase activity may reflect enhanced glomerular permeability in streptozotocin-induced diabetic rats and may be a useful marker for diabetic nephropathy. Relative resistance to SDS-denaturation in zymography of rat serum and urinary hyaluronidases compared with human serum hyaluronidase are also shown. Key words: diabetes / hyaluronidase / serum / urine / zymography
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Introduction |
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Hyaluronic acid (HA), a major component of the extracellular matrix (ECM), is a high molecular weight polysaccharide composed of repeating disaccharide units of D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc). HA is implicated in basic biological processes such as cell proliferation, differentiation, and migration. In the pathological processes with enhanced ECM metabolism, such as rheumatoid arthritis and cancer, HA is recognized as one of key participants (Toole, 2001
). HA exerts different effects depending on its molecular size; for example, HA oligomers of 10–15 disaccharide units (n) are angiogenetic, whereas high molecular weight HA is antiangiogenetic (West et al., 1985; Deed et al., 1997; Slevin et al., 1998; Lokeshwar and Selzer, 2000).
Hyaluronidases (HAases), a family of enzymes involved in the degradation of HA, have also begun to attract attention. In humans, six HAase genes have been identified: HYAL1, HYAL2, and HYAL3 are clustered on chromosome 3p21.3, and PH20, HYAL4, and HYALP1 are clustered on chromosome 7q31.3. With the possible exception of HYAL4 and HYALP1, the other four are recognized as HAases that degrade HA (Csóka et al., 2001). Hyal-1, originally identified as a serum enzyme, was the first HAase to be purified to homogeneity from plasma (Frost et al., 1997). The highest levels of mRNA of HYAL1 are found in the major parenchymal tissues, such as liver, kidney, spleen, and heart. mRNA of HYAL2 is also present in most tissues, except adult brain, and the protein product Hyal-2 is considered to be a typical lysosomal enzyme originally. More recent evidence has shown that Hyal-2 can also be exposed on the cell surface bound to the plasma membrane via a glycosylphosphatidylinositol anchor (Lepperdinger et al., 1998; Rai et al., 2001). PH-20, or testicular HAase, is primarily involved in the degradation of the HA-rich cumulus layer surrounding the egg by sperm cells (Cherr et al., 2001). HYAL3 transcripts have been detected in brain and liver tissues, but the protein product Hyal3 is uncharacterized (Triggs-Raine et al., 1999). Among the three characterized HAases, Hyal-1 and Hyal-2 are acidic enzymes having an optimum pH of 3.9–4.3 and PH-20 is neutral HAase having a broad optimum pH of 5–6.
Recently, a patient with a mutation of HYAL1 termed mucopolysaccharidosis IX has been described. The clinical phenotype is surprisingly mild, indicating that other HAases such as Hyal-2 and Hyal-3 compensate for the disorder of Hyal-1 (Triggs-Raine et al., 1999). Several investigators have also demonstrated that the levels or expression of HAases are changed in tissues, sera, or urine in diseases such as cancer (Lokeshwar et al., 1996; 1999; Bertrand et al., 1997; Tamakoshi et al., 1997; Laudat et al., 2000), suggesting an important role of the enzymes.
Diabetes mellitus is a metabolic disease characterized by hyperglycemia secondary to relative or absolute insulin deficiency. Chajara et al. (1999, 2000a) have demonstrated that HA participates in the development of diabetic angiopathy. However, the involvement of HAase in diabetic situations has scarcely been reported. Therefore, we attempted to investigate the changes in serum and urinary HAase levels with the progress of diabetes, using experimental models of two types of diabetes mellitus, streptozotocin (STZ)-induced diabetic Wistar and Goto-Kakizaki (GK) rats.
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Results |
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Characterization of diabetic rats
Diabetes mellitus was induced in one group of rats by a single injection of STZ (60 mg/kg body weight). The STZ-injected rats (STZ group) were examined for blood glucose and body weight over 18 weeks after injection, compared with age-matched control rats (control group) as well as spontaneously diabetic GK rats (GK group). Figure 1 shows the changes in blood glucose and body weight in STZ, GK, and control rats. The blood glucose levels of STZ rats started to increase on the third day after injection and maintained
threefold higher levels thereafter up to the 18th week, as compared with control rats. The STZ group also showed a progressive decrease in body weight gain from the early stage and exhibited 67% of the control group body weight at the end of the experiment. These data indicate that injection of STZ resulted in a rapid development of type 1 diabetes mellitus. On the other hand, the blood glucose levels of GK rats, a genetic nonobese model of type 2 diabetes mellitus, were already elevated significantly (twofold) from the start day of the experiment, when they were the same age (8 weeks) as STZ rats. The gain in body weight of the GK group was nearly intermediate between the control and STZ groups.
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Changes of serum HAase activity in diabetic rats
Serum HAase activity was determined by the fluorimetiric Morgan-Elson method (Takahashi et al., 2003
) over the study period for both diabetic groups, in comparison with the control group (Figure 2). The serum HAase level of the STZ group again started to increase on the third day, becoming 1.6-fold higher (45.6 ± 17.4 mU/ml) than that (28.2 ± 9.0 mU/ml) of the control group, and thereafter maintained threefold higher level (p < 0.05 or better). The serum HAase level of the GK group was consistently higher (1.5- to 2-fold) from the start of the experiment, the level at age 8 weeks (0 weeks in Figure 2) being 41.7 ± 3.5 mU/ml (p < 0.001). Thus, the increases of serum HAase activity in both diabetic groups closely corresponded to those of blood glucose level, indicating that diabetes mellitus was accompanied by enhanced activity of circulating HAase from the early process of its development.
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Analysis of serum HAase activity by zymography
When the sera from STZ, GK, and control rats were subjected to zymography, every serum sample gave two HAase isomers, a major band (Hyal-1 type) of 73 kDa and a minor band of 132 kDa, irrespective of age of rats (Figure 3). These HAase isomers were found to have an acidic pH optimum around 3.5. When the loaded volume of serum to zymography was increased from 0.15 to 1.0 µl, no novel distinct HAase bands could be detected with each serum (data not shown). These results suggested that the increases in serum HAase activity in both diabetic rat models were not caused by the appearance of novel HAase isomers but that the two preexisting isomers were increased in similar proportions. As for Hyal-2 type HAase, however, this zymographic technique could not detect the enzyme, because it can cleave HA only to intermediates of
20 kDa, whereas both Hyal-1 and PH-20 type HAases can degrade HA down to the minimum tetrasaccharide.
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Analysis of HA digestion pattern with serum HAase by PAGE method
To estimate whether Hyal-2 type HAase was increased in the sera of diabetic rats, HA digestion patterns were examined by the mini-gel polyacrylamide gel electrophoresis (PAGE) method (Ikegami-Kawai and Takahashi, 2002
). As shown in Figure 4, the HA digestion patterns for the STZ and control groups appeared to differ only quantitatively, but not qualitatively. When HAase activity was determined based on the total band intensity for the oligosaccharides from n18 to n24 as described (Ikegami-Kawai and Takahashi, 2002), the serum HAase activity of the STZ group at the 13 week was 2.7 times higher than that of the control group. The result was in good agreement with the results obtained from the fluorimetric Morgan-Elson assay that the corresponding activity of the STZ group was 2.9 times higher than that of the control group. Furthermore, there were no differences between the sera from the STZ and control groups in the intensities of the saccharide bands around n50 (HA fragments of 20 kDa to be produced by Hyal-2 type enzyme) relative to those for the smaller-sized bands. Thus, the mini-gel PAGE analysis did not show any distinct increase of Hyal-2 type HAase in the process of diabetes development.
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Analysis of urinary HAase activity by zymography
Urinary HAase activity, unlike serum HAase activity, could not be precisely determined by such usual assays as the Morgan-Elson reaction-based fluorimetric (Takahashi et al., 2003
) and colorimetric (Reissig et al., 1955) methods and the turbidimetric method (Di Ferrante, 1956), because of coexisting urea for the formers and insufficient activity for the latter. Therefore, we attempted to perform zymography for urinary HAase not only qualitatively but also quantitatively. When the 24-h urine collections from STZ, GK, and control rats were examined by zymography, all the urine samples gave a broad HAase band (probably corresponding to Hyal-1 type) centered at 71 kDa. In addition, only the urine from the STZ group had a narrow HAase band of 120 kDa similar to the upper band present in all the sera from STZ, GK, and control rats (Figure 5a).
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For the determination of urinary HAase activity, various volumes (2.5–10 µl) of each urine sample were subjected to zymography and the relative intensity (RI) of the broad HAase band was determined. Figure 5b shows a representative result obtained with the urine samples from STZ, GK, and control rats, indicating an unexpectedly good linear correlation between the RI and the loaded urine volume up to at least 10 µl of even the GK rat urine with the highest HAase activity. The HAase activity in RI per 7.5 µl of urine was estimated to be 1.24 ± 0.51, 2.78 ± 0.73, and 1.83 ± 0.88 for STZ, GK, and control rats at the 18th week (26 weeks old), respectively. The volume of 24-h urine and the total urinary HAase activity thus calculated for each group are listed in Table I, in which the amount of albumin excretion in 24-h urine is also included. Because the urinary volume of STZ group was remarkably large (
20-fold) compared with those of GK and control groups, the total urinary HAase activity of STZ group was 10 times higher than that of GK or control group. It was noteworthy that there was a good correlation (R = 0.953) between the total urinary HAase activity and the amount of albumin excretion for individual rats. Figure 5c compares the total urinary HAase activity of STZ and GK groups with that of control group at three different times. At the 8th, 15th, and 18th week (age 16, 23, and 26 weeks), the total urinary HAase activity of STZ group was 3-, 7-, and 11-fold higher than that of control group, respectively, whereas the total urinary HAase activity of GK group was not significantly different. Based on the observed good correlation between urinary HAase activity and albumin excretion, it was strongly suggested that the increase in urinary HAase activity may reflect an enhanced glomerular permeability in STZ-induced diabetic rats, but not GK rats.
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Effect of incubation with sample buffer on the banding pattern of rat HAases in zymography
In zymography, the banding patterns of rat serum and urinary HAases largely depended on the incubation conditions of samples with Laemmli's sample buffer, although the banding pattern of human serum HAase did not depend at all (Figure 6). When each serum from normal and diabetic rats was subjected to electrophoresis immediately after mixing with sample buffer, it yielded four HAase bands of 132, 100, 73, and 47 kDa. A faint band between the 73- and 47-kDa bands was of residual rat serum albumin undigested by Actinase treatment, as was seen in the case with human serum as a very faint albumin band moving faster than the Hyal-1 band of 59 kDa. When the sample mixture was incubated at 37°C for up to 60 min, however, the two bands of 100 and 47 kDa disappeared completely within 20 min, and in turn the other two bands of 132 and 73 kDa were increased in intensity. The relative intensity of the 132-kDa band after incubation was increased to nearly the sum of those for the 132- and 100-kDa bands at 0 time. The intensity of the 73-kDa band after incubation was a little less than the sum of those for the 73- and 47-kDa bands at 0 time, suggesting that the intensity of the 73-kDa band was already saturated either before or after incubation. Furthermore, smearing of 100- and 47-kDa bands up to 132- and 73-kDa bands, respectively, was sometimes observed, indicating a conversion of the former bands to the respective latter bands. Such mutual conversion of the HAase bands occurred more gradually at room temperature than at 37°C.
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A similar phenomenon was also observed with rat urinary HAase. Normal rat urine gave two HAase bands of 71 and 46 kDa at 0 time incubation. Upon incubation at 37°C, the smaller band disappeared within 20 min, and in turn the intensity of the larger band was increased to nearly the sum of the intensities of both bands at 0 time, indicating a conversion of the smaller band to the larger band. Smearing of the 46-kDa band up to the 71-kDa band was observed more distinctly compared with rat serum. Because rat serum and urinary HAases were proven to change their banding patterns upon incubation, the zymographic studies described were carried out with 60-min incubation where reproducible results were obtained.
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Discussion |
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It is shown that the composition of glycosamimoglycans, including HA, alters in serum, skin, kidney, aorta, and urine in human and experimental diabetes (Ceriello et al., 1983
; Saraswathi and Vasan, 1983; Heickendorff et al., 1994; Cechowska-Pasko et al., 1996; Kozma et al., 1996; Cadaval et al., 2000; Juretic et al., 2002). Furthermore, it has been reported that a high concentration of glucose enhanced production of glycosamimoglycans including HA in kidney-derived cultured cells (Jones et al., 2001; Takeda et al., 2001). To our knowledge, however, no detailed investigation of HAase activity in diabetes has been performed; there are only two preliminary reports as follows. In sera from diabetic patients (non-insulin-dependent), activities of several lysosomal enzymes including HAase were significantly higher than in normal sera (Koscielniak-Kocurek et al., 1995). In another report, Chajara et al. (2000b) showed that serum HA and HAase activity increased in STZ-induced diabetic rats, but only in single measurements at the 13th week, and that the increases in HA and HAase were abolished by insulin treatment. In the present study, we in more detail investigated time-course changes in the serum HAase level in a type 1 diabetes model, STZ-induced diabetic rats, and a type 2 diabetes model, GK rats. Serum HAase activity increased (Figure 2) almost concomitantly with increase in the blood glucose level (Figure 1a) in the two rat models, clarifying that HAase activity increases from the very early stage of diabetes. These findings suggested that the increase in HAase activity was involved in enhanced ECM metabolism, a progression mechanism of diabetic complication.
Urinary total HAase activity also was significantly increased in the STZ group compared to that in the control group (Table I, Figure 5c). Leakage of blood HAase due to renal basement membrane impairment and deviation of HAase due to destruction of ureter or bladder cells were considered to be the causes. Of these, the most likely cause of increased urinary HAase activity in the STZ group may be leakage of blood HAase. It is known that a minute amount of urinary protein, that is, microalbuminuria, appears in the early stage of diabetic nephropathy. Phillips et al. (1999) reported that in STZ-induced daibetic rats, prominent mesangial cell proliferation and glomerular macrophage infiltration were detected from 3 days after induction. Cadaval et al. (2000) showed that the increase of 24-h urine volume as well as albuminuria remained significant from the second week. Therefore, it is quite possible that these findings support leakage of blood HAase from the early stage, resulting in an increase in urinary HAase activity.
This assumption was strongly supported by the present finding that there was a good correlation between total urinary HAase activity and albumin excretion in the urine samples from STZ, GK, and control rats (Table I), although only in single measurements at the 18th week (age 26 weeks). Namely, only the STZ group had significant increases in both urinary HAase activity and albumin excretion compared with those of the control group. Furthermore, a 120-kDa HAase band that may have been derived from blood was detected only in the STZ group urine. The measurement of total urinary HAase activity was performed only in the 8th, 15th, and 18th week in this study, but it is necessary to extensively investigate when urinary HAase starts to increase during the progression of diabetes, including the relationship with the development of nephropathy. In contrast, there were no significant differences not only in urinary HAase activity but also in albumin excretion between the GK and control groups, implying that blood HAase as well as albumin did not leak in the GK group. This is compatible with the report that although slight abnormality was noted in the glomeruli in 26-week-old GK rats, it had not reached renal dysfunction (Phillips et al., 1999). The findings of the present study suggest that total urinary HAase activity, as well as the appearance of 120-kDa HAase band, may be used as a good marker reflecting progression of nephropathy in diabetes.
Rat HAase is considered to consist of six genes, by analogy to human and mouse HAases that have been most extensively characterized among mammalian enzymes, but only rat PH20 (Hou et al., 1996) and HYAL2 (Liu et al., 2003) genes have been identified. On the other hand, the presence of HAase activity in the rat has long been known in various tissues and body fluids including serum. Recently Fiszer-Szafarz et al. (1990), using native PAGE zymography, demonstrated the polymorphism of HAase in sera from several mammalian species, including humans and rats. Fiszer-Szafarz et al. (2000) further demonstrated that human HAases from somatic tissues and body fluids were present in multiple forms and that some of the multiple forms were changed by sialidase treatment to desialylated forms, including their dimer forms. In the present work, sodium doceyl sulfate (SDS)–PAGE zymography was used, not the native PAGE zymography used by Fiszer-Szafarz et al. (1990), revealing that two of the four rat serum HAase bands at 0 time incubation were changed to the two other bands on incubation in the presence of SDS (Figure 6). These conversions of rat serum isomers, however, seemed not to be due to desialylation because sialidase activity, if present in rat serum, could scarcely act in the presence of 2% SDS. Furthermore, the possibility of desialylation was unlikely because desialylation would result, rather in an increase in electrophoretic mobility owing to the decreased molecular weight. The most probable explanation is that rat serum HAase isomers are not as easily SDS-denatured as human serum HAase (Hyal-1, 59 kDa) and they take longer to be completely SDS-denatured to finally give 132- and 73-kDa bands. This explanation is not contradictory to the smearing of the smaller bands up to the larger bands, because a completely denatured, extended form of protein moves more slowly than a native compact form of protein on SDS–PAGE gels. Thus, rat serum seemed to contain only two, but not four, forms of HAase, consisting of the major one of 73 kDa (Hyal-1 type) and the minor one of 132 kDa. Similarly, rat urine seemed to contain only one, but not two, form of HAase. Rat urinary HAase is considered to be Hyal-1 type HAase, analogous to human urine that contains Hyal-l (57 kDa) as a major HAase and its proteolytically processed smaller isoform (45 kDa) (Csóka et al., 1997). Both the 71- and 46-kDa bands of rat urine, when examined carefully, appeared not to be a simple broad band but to be a doublet-shaped broad band. This strongly suggested that these doublet-shaped broad bands consisted of two very similar but not identical sized HAases, the larger of which being Hyal-1 type HAase (73 kDa) and the smaller being a processed form (a little less than 73 kDa) of Hyal-1 type enzyme, possibly with kidney proteases. It is noteworthy that only STZ rats, but not GK rats or control rats, displayed a urinary HAase band of 120 kDa in addition to the doublet-shaped broad band centered at 71 kDa, which was commonly exhibited by every group of rats. Although the nature of the urinary HAase band of 120 kDa is unknown, it possibly results from circulating HAase isomer of 132 kDa, due to enhanced glomerular permeability in STZ rats, with concomitant processing by kidney proteases.
In the present work, we demonstrated for the first time the enhancement of circulating HAase from the early process of diabetes development. Furthermore, the increase in total urinary HAase activity, together with the appearance of urinary 120-kDa HAase, may be a useful marker for diabetic nephropathy.
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Materials and methods |
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Materials
Sodium hyaruronate from human umbilical cord, STZ, and saccharic acid 1,4-lactone were obtained from Sigma (St. Loius, MO). Alcian blue 8GX was from Fluka Chemical (Buchs, Switzerland). GlcNAc, potassium tetraborate, p-dimethylaminobenzaldehyde (DMAB), and all reagents for the polymerization of electrophoretic gels were from Wako Pure Chemical Industries (Tokyo). Molecular weight markers for electrophoresis and silver staining kit were from Bio-Rad Laboratories (Hercules, CA). Actinase E was from Kaken Pharmaceutical (Tokyo). Normal human sera were obtained from laboratory volunteers. All other chemicals were of reagent-grade.
Experimental animals
Male Wistar and GK rats (Japan Laboratory Animals, Tokyo) of 8 weeks' age were used. Wistar rats were separated into two groups, control and STZ groups. To induce type 1 diabetes mellitus, a single injection of STZ (60 mg/kg body weight) dissolved in normal saline buffered with 0.1 M sodium citrate (pH 4.5) was carried out via the tail vein of STZ group rats. Control group rats received an equivalent volume of the dissolving buffer alone. Spontaneously diabetic GK rats were used as a model of type 2 diabetes mellitus. The three groups of rats were housed three or four per cage and fed ad libitum with regular chow and tap water from 8 to 26 weeks old. All experiments were performed in accordance with the Guidelines for Animal Experiments in Hoshi University.
Blood was sampled from the jugular vein and, after standing for 30 min at 4°C, centrifuged at 500 x g for 15 min to obtain serum. Serum glucose level was determined by a glucose oxidase method with a kit (Glucose CII-Test Wako) supplied by Wako.
For 24-h urine collection, each rat was placed in a metabolic cage for 24 h. The collected urine was centrifuged to remove debris and frozen until use. Urinary albumin was measured by using a rat albumin enzyme-linked immunosorbent assay kit (Shibayagi, Gunma, Japan) with rat albumin as a standard.
Determination of HAase activity
Serum HAase activity was determined by the fluorimetric Morgan-Elson assay method as described recently (Takahashi et al., 2003). Briefly, 125 µl of the substrate solution (1.5 mg/ml HA in 0.1 M formate buffer, pH 3.9, containing 0.1 M NaCl and 1.5 mM saccharic acid 1,4-lactone) was mixed with 5 µl serum and digested at 37°C for 15 min. After heating in a boiling water bath for 5 min, the Morgan-Elson reaction was started by the addition of 25 µl 0.8 M potassium tetraborate reagent (pH 10.4) and subsequent heating for 3 min in a boiling water bath. After cooling to room temperature, 0.75 ml DMAB reagent was added and incubated at 37°C for 20 min. After centrifugation at 18,000 x g at 4°C for 10 min, the fluorescence (excitation, 545 nm; emission, 604 nm) of the clear supernatant was measured against that of a blank test, which was carried out in the same way except that the enzyme reaction mixture was incubated for 0 min. One unit of HAase activity was defined as the amount of enzyme required to produce 1 µmol of reducing terminal GlcNAc per min under the specified conditions.
Zymography of HAase
HAase was detected by HA zymography (Miura et al., 1995; Podyma et al., 1997) with slight modifications. Rat serum was diluted with nine volumes of 0.15 M NaCl and mixed with an equivalent volume of Laemmli's sample buffer (Laemmli, 1970) containing 4% SDS and no reducing reagent. After incubation for 1 h at 37°C without heating, 3 µl of the mixture (serum: 0.15 µl) were applied to 7% SDS–polyacrylamide gels containing 0.17 mg/ml HA. Rat urine was directly mixed with an equivalent volume of sample buffer and, after incubation for 1 h at 37°C, 5–20 µl of the mixture (urine: 2.5–10 µl) were applied. After electrophoretic run at 25 mA for approximately 70 min at 4°C, gels was rinsed with 2.5% Triton X-100 for 80 min at room temperature and incubated with 0.1 M formate buffer (pH 3.5) containing 0.03 M NaCl for 18 h at 37°C on an orbital shaker. Gels were then treated with 0.1 mg/ml Actinase E in 20 mM Tris–HCl buffer (pH 8.0) for 2 h at 37°C. To visualize digestion of the HA, gels were stained with 0.5% Alcian blue in 25% ethanol–10% acetic acid. After destaining, gels were counterstained with Coomassie brilliant blue R-250. For the determination of HAase activity, the stained gel was scanned on an Epi-Light 2000 image analyzer with Luminous Imager version 2.0 (Aisin Cosmos, Japan). The relative band intensities of rat serum and urinary HAases were calculated from the ratios to the band intensity of HAase from 0.5 µl of a control human serum, as a standard on the same gel.
PAGE analysis of HA digest mixtures
The procedures employed for electrophoresis using 15% polyacrylamide gel were essentially as described by Ikegami-Kawai and Takahashi (2002). HA digest samples to be examined were prepared as follows. One hundred twenty five microliters of a 1.5 mg/ml HA solution was digested with 5 µl of serum as described except that an incubation time of 30 min was used. After centrifugation at 18,000 x g for 10 min, 10 µl of the clear supernatant was mixed with 2 µl 2 M sucrose in Tris/borate/ethylenediamine tetra-acetic acid buffer and 2 µl of the mixture was applied directly to the gels. The electrophoretic run was carried out at 4°C first at 250 V for 20 min, then at 580 V for 10 min, and additionally at 450 V for approximately 15 min. After electrophoresis, oligosaccharides were fixed in the gel matrix by soaking the gel in 0.05% Alcian blue dissolved in water for 0.5 h in the dark. After destaining in water, the gel was subjected to silver staining, beginning from the oxidation step. The stained gel was scanned on an Epi-Light 2000 image analyzer with Luminous Imager version 2.0. The HAase activity was measured as a total of the relative band intensities for oligosaccharides of n18 to n24.
Statistical analysis
Results are expressed as means ± SD. For comparison of multiple means, analysis of variance was used, followed by Fisher's protected least significant difference test. The significance level was indicated in each experiment.
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Acknowledgements |
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This work was supported by the Ministry of Education, Science, Sports, and Culture of Japan.
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Footnotes |
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1 To whom correspondence should be addressed; e-mail: t-tomoko{at}hoshi.ac.jp
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Abbreviations |
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DMAB, p-dimethylaminobenzaldehyde; ECM, extracellular matrix; GK, Goto-Kakizaki; HA, hyaluronic acid; HAase, hyaluronidase; n, disaccharide unit; PAGE, polyacrylamide gel electrophoresis; RI, relative intensity; SDS, sodium dodecyl sulfate; STZ, streptozotocin
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References |
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