Sphingomyelinase in Pig and Human Epidermis

Abstract

The enzyme sphingomyelinase (sphingomyelin phosphorylcholine phosphohydrolase E.C.3.1.4.12) which hydrolyzes sphingomyelin to ceramide (N-acylsphingosine) and phosphorylcholine was identified in the subcellular fractions of pig and human epidermis. The enzyme has an optimum pH of 4.5 to 5 and is activated by Triton X-100 (0.1% w/v). Approximately two-thirds of the enzyme activity in both the pig and human epidermal homogenates was in the soluble subcellular fraction and more than half of the enzyme activity in the subcellular particulate fraction was solubilized by freeze-thawing. The pH optimum suggests that epidermal sphingomyelinase is probably a lysozomal enzyme. The enzymes in both pig and human epidermis exhibited Michaelis-Menten kinetics. The soluble sphingomyelinase in pig epidermis had an apparent Km, 4.5 X 10(-5) M and that in human epidermis an apparent Km 7.7 X 10(-5) M. The pig epidermal sphingomyelinase had no special requirement for either divalent or heavy metal ions and was not inhibited by sulfydryl group-blocking agents but it was moderately inhibited by dithiothreitol. No evidence was found in either pig or human epidermis for the presence of a phospholipase C (E.C.3.1.4.3) which hydrolyzes phosphatidylcholine to diglyceride and phosphorylcholine but there was suggestive evidence of another catabolic pathway for phosphatidylcholine.

Secretory sphingomyelinase in health and disease

“…Both S-ASM activities in saliva and tear fluid peak at pH 4-5 and are markedly decreased at pH 6, with negligible residual activities at pH 7 (Takahashi et al, 2000). This behavior corresponds well to the pH profiles described for tissue L-ASM (Schneider and Kennedy, 1967;Bowser and Gray, 1978), ASM released from human fibroblasts (optimal pH 4.4) and mouse L-cells (pH 4.8) (Weitz et al, 1983) and our data for S-ASM in human CSF , serum, plasma, saliva and urine, which indicate < 5% of maximal Zn 2+ -dependent activity at pH 6.5 and nearly background levels at pH 7.0 (C. Mühle, unpublished data). Purified recombinant human ASM from CHO cells binds tightly to SM at pH 4.0, while binding is not detectable at pH 8.0 (He et al, 1999).…”

“…MichaelisMenten kinetic analysis revealed roughly similar values of the Michaelis constant K m for SM affinity despite the use of differently labeled substrates: 77 μm for soluble ASM in the human epidermis (Bowser and Gray, 1978), 5-25 μm for ASM purified from placenta (Jones et al, 1981Sakuragawa, 1982), 65 μm for ASM from Bacillus cereus (Fujii et al, 2004), 47 μm for ASM from human fibroblasts (Sato et al, 1988), 25 μm for recombinant ASM from the conditioned medium of infected insect cells (Bartelsen et al, 1998), 20 μm for ASM from human CSF , 0.2-0.6 μm for ASM released from human fibroblasts and mouse L-cells, respectively (Weitz et al, 1983), 11 μm with a V MAX of 21 nmol/h/mg protein for HEK 293 whole cell lysates and 2 μm and a V MAX of 4.3 μmol/h/mg protein for ASM immunoprecipitated from Jurkat cells (Gulbins and Kolesnick, 2000). Specific activities ranged from 600 to 2500 μmol/h/mg (Sakuragawa, 1982;Yamanaka and Suzuki, 1982;Jones et al, 1983;Weitz et al, 1985;Lansmann et al, 1996).…”

Roles for tumor necrosis factor receptor p55 and sphingomyelinase in repairing the cutaneous permeability barrier

“…This assumption is based on the high activity of the enzyme present, the colocalization of ASMase and sphingomyelin in the lamellar bodies (10,11,51,52), and the increase in predominantly C 24 ceramides that are abundant in the SC permeability barrier (37)(38)(39). In detail, we found that the activity of A-SMase in the epidermis was 60-fold higher than that…”

The vitamin D3 analogue, calcipotriol, induces sphingomyelin hydrolysis in human keratinocytes

“…In keratinocytes, the existence of the SM cycle has not been [25,26] and the importance of ceramides in the skin barrier system has been discussed [27]. Pulse medium was removed and the cells were incubated for 5 h with KGM containing 5 mM unlabelled serine.…”