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  • br Chondroitin sulfate proteoglycans CSPGs are major


    Chondroitin sulfate proteoglycans (CSPGs) are major components of the extracellular matrix in cartilage. Each CSPG comprises a single core protein and attached chondroitin sulfate (CS), belonging to the glycosaminoglycan family of sugar chains. CSPGs and CS are involved in numerous biological and pathological processes [,]. Some CS-degrading enzymes such as hyaluronidase (HAase) and chondroitinase (CHase) have been described in animals and bacteria [,]. However, the metabolism of CS remains poorly understood in mammals because the involved enzymes are expressed in restricted tissues or are localized in lysosomes. The screening of CS-degrading enzymes may have therapeutic applications as they could be applied in the treatment of spinal cord injuries [, , ]. Gel zymography is a highly sensitive method that is used to detect catabolic enzymes such as proteases and HAases [,]. However, to detect CS-degrading enzymes, CS requires modification for its immobilization in the gel because its molecular size is too small for it be used as a substrate []. Here, we present a simple procedure using salmon proteoglycans (PG) instead of modified CS. The salmon PG fraction was one of the commercially available CSPGs isolated from its nasal cartilage. In our previous analysis, the salmon PG fraction was shown to comprise various sizes of CSPGs, although most are approximately 540 kDa []. The disaccharide composition of CS in salmon PG was approximately 14.4% of ΔDi-0S [glucuronic sesamin (GlcUA)- -acetylgalactosamine (GalNAc)], 27.0% of ΔDi-4S [GlcUA-GalNAc(4S)], 57.8% of ΔDi-6S [GlcUA-GalNAc(6S)], 0.8% of ΔDi-SD [GlcUA(2S)-GalNAc(6S)], and 0.0% of ΔDi-SE [GlcUA- GalNAc(4S, 6S)] () []. The fractions contained less HA and other glycosaminoglycans such as heparan sulfate and keratan sulfate []. These characteristics of the salmon PG fraction should make it a suitable substrate for zymography to detect CS-degrading enzymes. The zymography procedure using salmon PG has been described in the Supplemental Information. CHase ABC and BTH, which are sodium dodecyl sulfate (SDS)-resistant enzymes, were clearly detected in the PG-substrate SDS polyacrylamide gel at each predicted size by staining with Alcian Blue (). The minimum detectable amounts for CHase ABC and BTH under our experimental conditions were 0.08 mU and 7.8 ng, respectively (). We then tested different pH conditions to compare enzyme activities (). The activity of each enzyme from pH 5.0 to neutral was found to be similar to that described in a previous report []. We recommend the use of 0.1–0.5 mg/ml PG as the initial substrate concentration. Thus, salmon PG is a useful alternative material to detect CS-degrading enzymes by gel zymography. However, the salmon PG-substrate gel has some limitations. Owing to the disaccharide composition of CS in salmon PG mentioned above, the gel is not suitable to detect the lyases for CS-B (dermatan sulfate), CS-D, and/or CS-E. Similar to conventional zymography, PG-substrate gel zymography cannot detect SDS-sensitive enzymes. However, the PG-substrate gel may be suitable for an electro-transfer method to detect SDS-sensitive enzymes in a native gel [,]. In addition, PG-substrate gel may be used to detect inhibitors of CS-degrading enzymes through reverse zymography like HA-substrate gel []. Indeed, BTH treatment (0.5 mg/ml, 37 °C, 16 h) of the gel (0.1 mg/ml PG) was sufficient to induce complete failure of Alcian Blue staining.
    Introduction A common complication of the kinetics of the NAD(P)+-dependent aldehyde dehydrogenase (ALDH) enzymes is substrate inhibition by the aldehyde, but, in spite of its high occurrence, it has been neglected in most of the reported kinetic studies. Most likely, inhibition by the aldehyde substrate is a general feature of these enzymes as a consequence of both their ordered steady-state kinetic mechanism and structural features. A search in the literature showed that inhibition of ALDHs by high concentrations of the aldehyde substrate has been reported for decades [[1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29]], but a complete kinetic characterization of this behavior was seldom performed. A few studies addressed the mechanism involved in substrate inhibition, which was proposed to be caused by (i) formation of the non-productive ternary complex E-NAD(P)H-aldehyde from which NAD(P)H can be released [3,4,7,12]; (ii) binding of the aldehyde to the free enzyme in competition with the coenzyme [10,14,26]; (iii) non-productive binding [24] or binding of two aldehyde molecules [19] in the aldehyde-binding site; and (iv) binding of the inhibitor substrate molecule to an inhibitory allosteric site [6]. Regardless of the mechanism and possible physiological relevance, if any, of substrate inhibition, and of its value as a tool to establish a kinetic mechanism—three aspects which are out of the scope of this work—we reasoned that if this phenomenon is not taken into account in kinetic studies of ALDHs, and of enzymes in general, important errors in the determination of the kinetic parameters and even in the determination of the kinetic mechanism will ensue. Here, we explored the kind and extent of these errors using theoretical simulations of the substrate saturation kinetics with different possible mechanisms of substrate inhibition in monosubstrate and Bi Bi ordered steady-state reactions. In addition, we exemplify these errors with experimentally determined initial velocity data obtained studying the betaine aldehyde dehydrogenase from Spinacia oleracea (SoBADH), an enzyme belonging to the family 10 of the ALDH superfamily.