Effects of estradiol, progestogens, and of tibolone on breast proliferation and apoptosis.

AIM: To study the effects of estrogen therapy, alone or combined with progestogens, and of tibolone on the expression of proliferation and apoptosis markers in normal breast tissue. METHODS: Thirty 250-day-old Wistar rats were castrated and 3 weeks later received one of the following treatments by gavage for 5 weeks: (1) estradiol benzoate; (2) estradiol benzoate + medroxyprogesterone acetate; (3) estradiol benzoate + norethisterone acetate; (4) estradiol benzoate + dydrogesterone; (5) tibolone; (6) placebo. Following treatment, the expression of proliferating cell nuclear antigen (PCNA) and caspase-3 was analyzed by quantitative immunohistochemistry in the breast tissue, and proliferation and apoptosis were analyzed semiquantitatively by microscopic imaging. RESULTS: There was a statistically significant difference among the groups for PCNA, caspase-3 and the caspase-3 : PCNA ratio. Tibolone was associated with the lowest proliferative activity, followed by estradiol benzoate + dydrogesterone; however, estradiol benzoate + dydrogesterone showed the greatest rate of apoptosis. CONCLUSIONS: The various progestogens can have more or less proliferative and pro-apoptotic effects than estradiol alone. Among the treatment schemes analyzed, the estradiol + dydrogesterone combination resulted in a higher apoptosis rate in relation to the proliferation rate and tibolone was associated with the lowest proliferation.

Is expression of rat breast matrix components influenced by estrogen, progestins and tibolone?

AIM: To study the effects of estrogen therapy, alone or combined with progestogens, and of tibolone on the expression of heparanase (HSPE), extracellular matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), perlecan and proliferating cell nuclear antigen (PCNA) in normal breast tissue. METHODS: Thirty 250-day-old Wistar rats were castrated and 3 weeks later received one of the following treatments by gavage for 5 weeks: (1) estradiol benzoate; (2) estradiol benzoate + medroxyprogesterone acetate; (3) estradiol benzoate + norethisterone acetate; (4) estradiol benzoate + dydrogesterone; (5) tibolone; (6) placebo. Following treatment, the expressions of mRNA for HSPE, MMP-2 and MMP-9 were analyzed by real-time PCR and the protein expressions of HSPE, MMP-2, MMP-9, perlecan and PCNA were quantified by immunohistochemistry. RESULTS: There was a statistically significant difference among the groups for the expression of HSPE mRNA due to high levels in the tibolone group. The groups differed in terms of PCNA, with lower levels found in the tibolone group followed by the estradiol benzoate + dydrogesterone group. A statistically significant positive correlation was observed for PCNA versus perlecan and MMP-9. CONCLUSIONS: There was no difference in the effects of combinations of estradiol and different progestogens on extracellular matrix components, and breast cell proliferation was associated with increases in perlecan and MMP-9.

Effect of estrogen therapy on vascular perlecan and metalloproteinases 2 and 9 in castrated rats.

AIM: To study the effects of estrogen therapy on the expression of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) and perlecan in the vascular wall. METHODS: Twenty 180-day-old Wistar rats were castrated and treated 1 week later for a period of 4 weeks with one of the following: (1) placebo; (2) 0.5 µg/day estradiol benzoate (E(2)B); (3) 5 µg/day E(2)B; (4) 50 µg/day E(2)B. A fifth group consisted of rats that had not been castrated. Following treatment, expression of MMP-2 and MMP-9 mRNA (MMP-2([RNA]) and MMP-9([RNA]), respectively) was analyzed by real-time PCR, and expression of MMP-2 (MMP-2([IH])), MMP-9 (MMP-9([IH])) and perlecan was quantified by immunohistochemistry, in carotid walls. RESULTS: There were no differences among castrated groups for MMP-2([RNA]) (p = 0.1969) and for MMP-9([RNA]) (p = 0.1828); however, a correlation was observed between E(2)B dose and MMP-9([RNA]) levels (r = 0.471, p = 0.018). Differences among groups were observed for MMP-2([IH]), MMP-9([IH]) and perlecan (p < 0.0001), wherein higher levels were observed in animals treated with estrogen therapy, correlating with E(2)B doses in the case of MMP-9 (r = 0.441, p = 0.026) and perlecan (r = 0.574, p = 0.005). CONCLUSIONS: Estrogen therapy correlates with higher levels of MMP-2, MMP-9 and perlecan in the extracellular matrix of carotid walls in castrated rats, in a dose-dependent manner. There was a dose-response effect of E(2)B on the expression of MMP-9 mRNA and, possibly, MMP-2 mRNA.

Methylene blue photodynamic therapy in malignant melanoma decreases expression of proliferating cell nuclear antigen and heparanases.

BACKGROUND: Malignant melanoma (MM) is a very aggressive tumour. Although surgical excision of MM in the early stages has a very good prognosis, it often fails to completely inhibit tumour progression. Methylene blue photodynamic therapy (MB-PDT) is a technique that induces tissue damage by reactive oxygen species (ROS). AIM: To investigate the efficacy of and potential use of MB-PDT in restraining the aggressiveness of MM by analysing levels of proliferating cell nuclear antigen (PCNA) and heparanase (HPSE, a molecular marker of cell invasion) in a mouse model. METHODS: Expression of PCNA and two HPSE isoforms were analysed using immunohistochemistry (IHC) after MB-PDT in mice. Tumour volume and weight were also measured. RESULTS: Two treatments with MB-PDT promoted a decrease of 99% decrease in tumour volume and 75% in tumour weight compared with untreated mice (P < 0.05). Using IHC, a decrease in expression of 75% for PCNA and 95% for both HPSE isoforms (P < 0.05) was found. CONCLUSION: MB-PDT is a cheap and efficient method of decreasing MM volume and thus disease progression. This reduction is mediated by downregulation of PCNA and heparanases.

EJ-ras oncogene transfection of endothelial cells upregulates the expression of syndecan-4 and downregulates heparan sulfate sulfotransferases and epimerase.

The EC rabbit endothelial cell line was transfected with the EJ-ras oncogene (EJ-ras EC). EJ-ras EC cells display over expression of the Ras oncogene, morphological changes and deregulation of the cell cycle, becoming more densely populated and serum-independent. In addition, EJ-ras-transfectant cells show higher levels of the syndecan-4 mRNA. In addition to the increase in the core protein, a parallel increase in the glycosylation of the syndecan-4 protein, a proteoglycan that bears heparan sulfate chains, also occurs. This increase is observed both for the heparan sulfate proteoglycan synthesized by the cells and for that secreted to the culture medium. This enhancement in heparan sulfate synthesis was observed through metabolic labeling of the cells, immunoprecipitation of syndecan-4 and heparitinases treatment. Furthermore, the EJ-ras-transfectant cells do not exhibit decreased synthesis of heparan sulfate during the G(1)-S phase transition, as observed for the parental cell line. Also, heparan sulfate synthesis is not stimulated by PMA as displayed by parental endothelial cells. Significant structural changes of heparan sulfate, such as decreased O-sulfation, were observed in the EJ-ras-transfected cells. Decreases in the mRNA levels of some enzymes (glucuronosyl C-5 epimerase, iduronosyl-2-O-sulfotransferase, glucosaminyl-6-O-sulfotransferase-1 and N-deacetylase/N-sulfotransferase-1), involved in the biosynthetic pathway of heparan sulfate, were also observed. The results suggest that overexpression of the EJ-ras oncogene alters the cell cycle, through signal transduction cascades, upregulates the expression of syndecan-4, and downregulates enzymes involved in the heparan sulfate biosynthesis related to chain modification, leading to the structural changes of the heparan sulfate syndecan-4 proteoglycan in endothelial cells.

Immunohistochemical expression of heparanase isoforms and syndecan-1 proteins in colorectal adenomas.

The proteoglycan syndecan-1 and the endoglucuronidases heparanase-1 and heparanase-2 are involved in molecular pathways that deregulate cell adhesion during carcinogenesis. Few studies have examined the expression of syndecan-1, heparanase-1 and mainly heparanase-2 proteins in non-neoplastic and neoplastic human colorectal adenoma tissues. The aim of this study was to analyze the correlation among the heparanase isoforms and the syndecan-1 proteins through immunohistochemical expression in the tissue of colorectal adenomas. Primary anti-human polyclonal anti-HPSE and anti-HPSE2 antibodies and primary anti-human monoclonal anti-SDC1 antibody were used in the immunohistochemical study. The expressions of heparanase-1 and heparanase-2 proteins were determined in tissue samples from 65 colorectal adenomas; the expression of syndecan-1 protein was obtained from 39 (60%) patients. The histological type of adenoma was tubular in 44 (67.7%) patients and tubular-villous in 21 (32.3%); there were no villous adenomas. The polyps were <1.0 cm in size in 54 (83.1%) patients and ≥1.0 cm in 11 (16.9%). The images were quantified by digital counter with a computer program for this purpose. The expression index represented the relationship between the intensity expression and the percentage of positively stained cells. The results showed that the average of heparanase-1, heparanase-2 and syndecan-1 expression index was 73.29 o.u./µm², 93.34 o.u./µm², and 55.29 o.u./µm², respectively. The correlation between the heparanase-1 and syndecan-1 expression index was positive (R=0.034) and significant (P=0.035). There was a negative (R= -0.384) and significant (P=0.016) correlation between the expression index of heparanase-1 and heparanase-2. A negative (R= -0.421) and significant (P=0.008) correlation between the expression index of heparanase-2 and syndecan-1 was found. We concluded that in colorectal adenomas, the heparanase-1 does not participate in syndecan-1 degradation; the heparanase-2 does not stimulate syndecan-1 degradation by the action of heparanase-1, and the heparanase-2 may be involved in the modulation of the heparanase-1 activity.

Profile of hMSH2 expression in breast tumors and lymph nodes: a preliminary study.

OBJECTIVE: The mismatch repair (MMR) genes play a central role for the onset of cancer. One of these genes is hMSH2. A differential hMSH2 protein expression has been detected in the mononuclear fraction of peripheral blood of patients with breast cancer when compared to healthy women. This work aims to evaluate the expression of hMSH2 in patients diagnosed with breast cancer undergoing treatment at various stages of the disease to verify its potential use as a prognostic marker. PATIENTS AND METHODS: Immunohistochemical expression of hMSH2 at different stages of breast cancer in 40 patients biopsy samples were analyzed. RESULTS: hMSH2 has a considerable increased expression in all groups of patients with tumors, when compared to patients without tumors. CONCLUSIONS: immunohistochemistry indeed can be a great tool for the diagnosis of breast cancer, as it is an easy and versatile technique.

Minimum fragments of the heparin molecule able to produce the accumulation and change of the sulfation pattern of an antithrombotic heparan sulfate from endothelial cells.

Heparin and low molecular weight heparins stimulate two to three fold the accumulation of an antithrombotic heparan sulfate secreted by endothelial cells in culture. This led us to search for the minimum structural requirements of the heparin molecule able to elicit the enhancement of the heparan sulfate. Fragments were prepared from heparin by degradation with bacterial heparinase and heparitinases. A heparin pentasulfated tetrasaccharide was shown to be the minimum structural sequence able to enhance two to three fold the secretion of heparan sulfate by endothelial cells. The stimulation is specific for the endothelial cell, is concentration dependent and the effect is already noticed after one hour of exposure of the cells to heparin and the tetrasaccharide. Degradation of the [35S]-heparan sulfate synthesized in the presence of heparin or the tetrasaccharide has shown a higher degree of sulfation of its iduronic acid residues.

Heparin and a cyclic octaphenol-octasulfonic acid (GL-522-Y-1) bind with high affinity to a 47-kda protein from vascular endothelial cell surface and stimulate the synthesis and structural changes of heparan sulfate proteoglycan.

The effect of a cyclic octaphenol-octasulfonic acid (GL-522-Y-1), upon the synthesis of a heparan sulfate proteoglycan synthesized by endothelial cells (rabbit aorta and human umbilical vein) were studied. The cells were exposed to the compounds at various concentrations for different periods of time and the synthesized heparan sulfates analyzed by a combination of agarose gel electrophoresis and enzymatic degradation. The GL-522-Y-1, like heparin, change the sulfation pattern and stimulate two- to three-fold the synthesis of heparan sulfate proteoglycan secreted by rabbit and human endothelial cells in culture. GL-522-Y-1, besides being 100 times more active than heparin, also produces a significant enhancement of cell surface heparan sulfate in human vein endothelial cells. The effect of GL-522-Y-1 is completely abolished by methylation or acetylation of its free hydroxyl groups. Both heparin and GL-522-Y-1 have high affinity for a 47-kDa protein present at the surface of endothelial cells. These and other results lead us to speculate that the antithrombotic activity of heparin and GL522 "in vivo" could be related, at least in part, to the increased production of the heparan sulfate proteoglycan by endothelial cells.

Antithrombotic agents stimulate the synthesis and modify the sulfation pattern of a heparan sulfate proteoglycan from endothelial cells.

Low molecular weight heparins, namely CY 216 and CY 222 (Sanofi/Choay); OP 622 and OP 386 (Opocrin); PK 10169 (Pharmuka); an oligosaccharide prepared from heparin by heparitinase II digestion; chemically sulfated glycosaminoglycans and polysaccharide namely Suleparoid (Syntex), Aprosulate (Luitpold-Werk); chemically modified glycosaminoglycans GAGPS and MPS (Luitpold-Werk) as well as unmodified heparin stimulate two to three fold the synthesis of a heparan sulfate with antithrombotic activity secreted by endothelial cells in culture. The stimulation is concentration dependent and specific for the endothelial cell. The [35S]-heparan sulfate synthesized in the presence of heparin and/or the tested antithrombotic agents has shown a high degree of sulfation of the iduronic acid residues as revealed by the analyses of the disaccharide products formed from the heparan sulfate by the action of bacterial heparitinases. The features of the above compounds in common with heparin are their polymeric nature and a high change density, as well as their pharmacological activities as potent antithrombotic agents "in vivo". These combined observations reinforce the proposition that the antithrombotic activity of heparin, low molecular weight heparins and the chemically modified polysaccharides could be related to the increased production of this peculiar heparan sulfate by endothelial cells.

Binding of heparin and compound Y to endothelial cells stimulates the synthesis of an antithrombotic heparan sulfate proteoglycan.

The mechanism by which heparin and antithrombotic agents, including a cyclic octaphenolsulfonic acid (compound Y), stimulate the synthesis of an antithrombotic heparan sulfate by endothelial cells in culture was investigated. Compound Y increases the amount of heparan sulfate from the cell surface and secreted to the medium by endothelial cells by three-fold. Binding experiments have shown saturation of the endothelial cell receptors at a concentration of 0.16 microM for heparin and 2.7 microM for compound Y. The kinetic binding constants (Ks) for compound Y and heparin were 1,333 nM and 42 nM, respectively. It was also shown that both compounds bind to the same receptors. The Scatchard plots indicated that 1,319 nmoles compound Y and 35 nmoles heparin bound per microgram cell protein, indicating that 40-fold more molecules of compound Y bound to the receptors when compared to heparin. No significant internalization of the compounds was observed.

Development of new heparin-like compounds and other antithrombotic drugs and their interaction with vascular endothelial cells.

The anticlotting and antithrombotic activities of heparin, heparan sulfate, low molecular weight heparins, heparin and heparin-like compounds from various sources used in clinical practice or under development are briefly reviewed. Heparin isolated from shrimp mimics the pharmacological activities of low molecular weight heparins. A heparan sulfate from Artemia franciscana and a dermatan sulfate from tuna fish show a potent heparin cofactor II activity. A heparan sulfate derived from bovine pancreas has a potent antithrombotic activity in an arterial and venous thrombosis model with a negligible activity upon the serine proteases of the coagulation cascade. It is suggested that the antithrombotic activity of heparin and other antithrombotic agents is due at least in part to their action on endothelial cells stimulating the synthesis of an antithrombotic heparan sulfate.

PP123. Evaluation of glycosaminoglycans in placental of pregnant women with and without preeclampsia.

INTRODUCTION: Preeclampsia (PE) is a major cause of maternal and perinatal mobility and mortality and its etiology is not yet completely understood. Recently studies have shown the association between increased expression of glycosaminoglycans (GAGs) in placental of women with PE and its physiopathology. OBJECTIVES: Identify and quantify GAGs in placental of pregnant women in healthy pregnancy and PE. METHODS: Case-control study with 44 patients, control group (CG) n=29 and n=15 PE group. All patients were submitted to placental tissue resection (sample size of 5×5cm with the umbilical cord insertion in the center). The tissue was conserved in acetone. The GAGs' analysis consisted of centrifugation, proteolysis, precipitation, and electrophoresis RESULTS: Average age and gestational age in CG and PE were 27.33years and 39.02weeks and 24.17 and 36.90, respectively. In CG 68.96% (20/29) were Caucasian and 80.00% (12/15) in PE. We found in CG 34.48% (10/29) of primiparous and 40.00% (6/15) in PE. The average 24-hour proteinuria in PE was 554.28g/24hs. The average birth weight was 3333.31g in CG and 2972.66 in PE. The mean ± standard deviation of dermatan sulfate (DS), heparan sulfate (HS) and hyaluronic acid (HA) in CG and PE were: 0,100µg/mg of tissue ±0,005 and 0,144 ±0,071; 0,077±0,041 and 0,113±0,061; 1,281±1,857 and 3,076±4,930. CONCLUSION: GAGs are increased in PE when compared to normal pregnancy. The expression of HA and HS was twice higher in PE. More studies are needed to determine the correlation between GAGs and physiopathology of PE.

[Prostate cancer: promising biomarkers related to aggressive disease]. / Cáncer de próstata: biomarcadores prometedores relacionados con la enfermedad agresiva.

BACKGROUND: Although a rapidly growing number of candidate biological markers of prognosis and/or response to specific treatments in prostate cancer, none have to date showed ability to completely prognosticate prostate cancer on evidence based urology. OBJECTIVE: To review the pertinent literature on the issue. ACQUISITION OF EVIDENCE: A comprehensive review of the current literature was done focusing on promising biomarkers related to aggressive prostate cancer. SUMMARY OF EVIDENCE: Combined with the heterogeneous nature of the disease, mixed case series are the most common study design, impeding robust results and the development of an effective therapeutic strategy. Improvement in prostate cancer patient survival requires not only the identification of new therapeutic target based on detailed understanding of the biological mechanisms involved in metastatic dissemination and tumor growth but strong clinical studies as well. CONCLUSION: Better study design involving potential markers and including well-classified and staged patients with robust methodology and adequate outcomes (mainly survival) are necessary to the field evolution.

Enzyme interactions in heparan sulfate biosynthesis: uronosyl 5-epimerase and 2-O-sulfotransferase interact in vivo.

The formation of heparan sulfate occurs within the lumen of the endoplasmic reticulum-Golgi complex-trans-Golgi network by the concerted action of several glycosyltransferases, an epimerase, and multiple sulfotransferases. In this report, we have examined the location and interaction of tagged forms of five of the biosynthetic enzymes: galactosyltransferase I and glucuronosyltransferase I, required for the formation of the linkage region, and GlcNAc N-deacetylase/N-sulfotransferase 1, uronosyl 5-epimerase, and uronosyl 2-O-sulfotransferase, the first three enzymes involved in the modification of the chains. All of the enzymes colocalized with the medial-Golgi marker alpha-mannosidase II. To study whether any of these enzymes interacted with each other, they were relocated to the endoplasmic reticulum (ER) by replacing their cytoplasmic N-terminal tails with an ER retention signal derived from the cytoplasmic domain of human invariant chain (p33). Relocating either galactosyltransferase I or glucuronosyltransferase I had no effect on the other's location or activity. However, relocating the epimerase to the ER caused a parallel redistribution of the 2-O-sulfotransferase. Transfected epimerase was also located in the ER in a cell mutant lacking the 2-O-sulfotransferase, but moved to the Golgi when the cells were transfected with 2-O-sulfotransferase cDNA. Epimerase activity was depressed in the mutant, but increased upon restoration of 2-O-sulfotransferase, suggesting that their physical association was required for both epimerase stability and translocation to the Golgi. These findings provide in vivo evidence for the formation of complexes among enzymes involved in heparan sulfate biosynthesis. The functional significance of these complexes may relate to the rapidity of heparan sulfate formation.

Cloning, Golgi localization, and enzyme activity of the full-length heparin/heparan sulfate-glucuronic acid C5-epimerase.

While studying the cellular localization and activity of enzymes involved in heparan sulfate biosynthesis, we discovered that the published sequence for the glucuronic acid C5-epimerase responsible for the interconversion of d-glucuronic acid and l-iduronic acid residues encodes a truncated protein. Genome analysis and 5'-rapid amplification of cDNA ends was used to clone the full-length cDNA from a mouse mastocytoma cell line. The extended cDNA encodes for an additional 174 amino acids at the amino terminus of the protein. The murine sequence is 95% identical to the human epimerase identified from genomic sequences and fits with the general size and structure of the gene from Drosophila melanogaster and Caenorhabditis elegans. Full-length epimerase is predicted to have a type II transmembrane topology with a 17-amino acid transmembrane domain and an 11-amino acid cytoplasmic tail. An assay with increased sensitivity was devised that detects enzyme activity in extracts prepared from cultured cells and in recombinant proteins. Unlike other enzymes involved in glycosaminoglycan biosynthesis, the addition of a c-myc tag or green fluorescent protein to the highly conserved COOH-terminal portion of the protein inhibits its activity. The amino-terminally truncated epimerase does not localize to any cellular compartment, whereas the full-length enzyme is in the Golgi, where heparan sulfate synthesis is thought to occur.

Transport of UDP-galactose into the Golgi lumen regulates the biosynthesis of proteoglycans.

The lumen of the Golgi apparatus is the subcellular site where galactose is transferred, from UDP-galactose, to the oligosaccharide chains of glycoproteins, glycolipids, and proteoglycans. The nucleotide sugar, which is synthesized in the cytosol, must first be transported into the Golgi lumen by a specific UDP-galactose transporter. Previously, a mutant polarized epithelial cell (MDCKII-RCAr) with a 2% residual rate of transport of UDP-galactose into the lumen of Golgi vesicles was described (Brandli, A. W., Hansson, G. C., Rodriguez-Boulan, E., and Simons, K. (1988) J. Biol. Chem. 263, 16283-16290). The mutant has an enrichment in glucosyl ceramide and cell surface glycoconjugates bearing terminal N-acetylglucosamine, as well as a 75% reduction in sialylation of cell surface glycoproteins and glycosphingolipids. We have now studied the biosynthesis of galactose containing proteoglycans in this mutant and the corresponding parental cell line. Wild-type Madin-Darby canine kidney cells synthesize significant amounts of chondroitin sulfate, heparan sulfate, and keratan sulfate, while the above mutant synthesizes chondroitin sulfate and heparan sulfate but not keratan sulfate, the only proteoglycan containing galactose in its glycosaminoglycan polymer. The mutant also synthesizes chondroitin 6-sulfate rather than only chondroitin 4-sulfate as wild-type cells. Together, the above results demonstrate that the Golgi membrane UDP-galactose transporter is rate-limiting in the supply of UDP-galactose into the Golgi lumen; this in turn results in selective galactosylation of macromolecules. Apparently, the Km for galactosyltransferases involved in the synthesis of linkage regions of heparan sulfate and chondroitin sulfate are significantly lower than those participating in the synthesis of keratan sulfate polymer, glycoproteins, and glycolipids. The results also suggest that the 6-O-sulfotransferases, in the absence of their natural substrates (keratan sulfate) may catalyze the sulfation of chondroitin 4-sulfate as alternative substrate.

Biosynthesis of glycosaminoglycans in the endometrium during the initial stages of pregnancy of the mouse.

Significant changes in the synthesis of glycosaminoglycans occur during the transformation of stromal cells of the endometrium into decidual cells which takes place during the initial stages of pregnancy in mice. Hyaluronic acid, which is practically absent in the endometrium of virgin mice, increases dramatically on the fifth day of pregnancy, reaching its maximal concentration on day 6 followed by a 50% decrease on day 7. Changes in hyaluronic acid concentration also occur in pseudopregnant mice indicating that they are not related to the presence of the embryo in the uterus. The absolute concentration of the sulfated glycosaminoglycans, e.g., heparan sulfate, dermatan sulfate and chondroitin sulfate in the decidua did not change significantly. There was, however, a striking decrease of their biosynthesis in pregnant and pseudopregnant mice when compared to virgin mice, as shown by the use of radioactive inorganic sulfate as a precursor for the study of in vivo synthesis. A radioautographical analysis confirmed that the highest incorporation of radioactive sulfate was observed in virgin endometria when compared to pregnant ones. These studies also have shown a characteristic pattern of labeling in different regions of the endometrium that repeats itself during the different days of pregnancy.

Purification and substrate specificity of heparitinase I and heparitinase II from Flavobacterium heparinum. Analyses of the heparin and heparan sulfate degradation products by 13C NMR spectroscopy.

The purification of two heparitinases and a heparinase, in high yields from Flavobacterium heparinum was achieved by a combination of molecular sieving and cation-exchange chromatography. Heparinase acts upon N-sulfated glucosaminido-L-iduronic acid linkages of heparin. Substitution of N-sulfate by N-acetyl groups renders the heparin molecule resistant to degradation by the enzyme. Heparitinase I acts on N-acetylated or N-sulfated glucosaminido-glucuronic acid linkages of the heparan sulfate. Sulfate groups at the 6-position of the glucosamine moiety of the heparan sulfate chains seem to be impeditive for heparitinase I action. Heparitinase II acts upon heparan sulfate producing disulfated, N-sulfated and N-acetylated-6-sulfated disaccharides, and small amounts of N-acetylated disaccharide. These and other results suggest that heparitinase II acts preferentially upon N,6-sulfated glucosaminido-glucuronic acid linkages. The total degradation of heparan sulfate is only achieved by the combined action of both heparitinases. The 13C NMR spectra of the disaccharides formed from heparan sulfate and a heparin oligosaccharide formed by the action of the heparitinases are in accordance to the proposed mode of action of the enzymes. Comparative studies of the enzymes with the commercially available heparinase and heparitinase are described.