Partial purification and characterization of cysteine proteinases in eccrine sweat

1987 ◽  
Vol 252 (6) ◽  
pp. R1119-R1129
Author(s):  
H. Yokozeki ◽  
T. Hibino ◽  
K. Sato
Keyword(s):  
Concanavalin A ◽  
Cathepsin B ◽  
Ethylenediaminetetraacetic Acid ◽  
Sweat Glands ◽  
Partial Purification ◽  
Aminopeptidase Activity ◽  
Specific Substrate ◽  
Cysteine Proteinases ◽  
Eccrine Sweat

Attempts were made to purify and characterize cysteine proteinases in human eccrine sweat and further clarify their origin. Benzoyl-DL-arginine-beta-naphthylamide (BANA) and L-leucine beta-naphthylamide (LeuNA) hydrolases in thermally induced sweat were sequentially purified by Sephacryl S-200 chromatography and chromatofocusing, which yielded two major peaks of BANA hydrolase activity, BANA-I and BANA-II. Both enzymes are cysteine proteinases as evidenced by stimulation of enzymic activity by dithiothreitol and ethylenediaminetetraacetic acid and its inhibition by iodoacetic acid, (PCMB), and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane (E-64). Unlike BANA-II, BANA-I showed an additional aminopeptidase activity, an affinity to concanavalin A-Sepharose but no affinity to organomercurial sepharose and failed to hydrolyze benzyloxycarbonyl-phenylalanyl-arginine 4-methyl 7-coumarylamide (Z-Phe-Arg-NMec), a specific substrate for cathepsin B, which is poorly sensitive to leupeptin [inhibitor constant (Ki) = 1 X 10(-5) M] and relatively heat resistant. These and other characteristics such as its isoelectric points (PI) (= 5.8) and the Km for Arg-NMec (0.1 mM) and BANA (0.71 mM) all support the possibility that BANA-I is closely related to cathepsin H. In contrast, BANA-II is sensitive to Zn2+, leupeptin (Ki = 5.5 X 10(-9) M), is not adsorbed by concanavalin A- (Con-A)Sepharose, but is bound to organomercurial sepharose. It has a specificity to Z-Phe-Arg-NMec but not to Arg-NMec, has the molecular weight of 27, PI of 5.2, the pH optima for BANA (6.0), and the Km for BANA of 3.3 mM and the Km for Z-Phe-Arg-NMec of 0.1 mM. These features resemble those of liver cathepsin B. Leupeptin-sensitive BANA hydrolase was observed in the glandular extract of isolated sweat glands, which was increased after stimulation with methacholine and isoproterenol in vitro. The data are consistent with the notion that cathepsins B- and H-like enzymes are present in eccrine sweat and the former may be derived from the sweat gland.

Pharmacologic responsiveness of isolated single eccrine sweat glands

1981 ◽  
Vol 240 (1) ◽  
pp. R44-R51 ◽  
Author(s):  
K. Sato ◽  
F. Sato
Keyword(s):  
Dose Response ◽  
Dissociation Constants ◽  
Sweat Rate ◽  
Sweat Glands ◽  
Adrenergic Agonist ◽  
Beta Adrenergic ◽  
Adrenergic Agents ◽  
Eccrine Sweat Glands ◽  
Eccrine Sweat

Pharmacologic responsiveness of the eccrine sweat gland has never been studied under well-defined in vitro experimental conditions. Using isolated cannulated single monkey palm eccrine sweat glands, the dose response to both cholinergic and alpha- and beta-adrenergic agents and the effects of various antagonists on agonists were studied. The maximal sweat rate was highest after stimulation with cholinergic agonists, was lower with the beta-adrenergic agonist, and was least with the alpha-adrenergic agonist. Each secretory response was inhibited by its specific antagonist. Attempts to demonstrate the spare receptor, if any, by means of preincubation of the glands with N-(2-chlorethyl)dibenzylamine (Dibenamine) were unsuccessful. From the hyperbolic dose-response curves the values for KA and KB, dissociation constants for agonists and antagonists, respectively, were thus tentatively estimated according to Clark's classical receptor theory. Schild plots for each agonist-antagonist interaction produced straight lines with slopes of near unity, indicating the adequacy of the methodology. It was concluded that the isolated eccrine sweat glands retain their pharmacologic viability in vitro and show responsiveness to cholinergic as well as both alpha- and beta-adrenergic stimulations.

Cathepsin B and aminopeptidase in the posterior midgut of Phymata wolffii Stål (Hemiptera: Phymatidae)

1985 ◽  
Vol 63 (6) ◽  
pp. 1288-1291 ◽  
Author(s):  
Jon G. Houseman ◽  
P. E. Morrison ◽  
A. E. R. Downe
Keyword(s):  
Molecular Weight ◽  
Trypsin Inhibitor ◽  
Cathepsin B ◽  
Ethylenediaminetetraacetic Acid ◽  
Soybean Trypsin Inhibitor ◽  
Aminopeptidase Activity ◽  
Chloromethyl Ketone ◽  
Posterior Midgut ◽  
Hydrolysis Of

The posterior midgut of the phymatid Phymata wolffii Stål contains cathepsin B and aminopeptidase activity. Identification of cathepsin B was based on maximal hydrolysis of benzoyl-DL-arginine-2-naphthylamide and benzoyl-DL-arginine-p-nitroanilide at pH 5.8 and 5.5, respectively. Cathepsin B hydrolysis of the tested substrates was activated by thiol chemicals and ethylenediaminetetraacetic acid (EDTA) and inhibited by tosyl-L-lysine chloromethyl ketone, iodoacetamide, and soybean trypsin inhibitor. Aminopeptidase hydrolyzed leucine-p-nitroanilide maximally at pH 7.8 and hydrolysis of the substrate was activated by magnesium and inhibited by EDTA, dithiothreitol, glutathione, and cysteine. The molecular weight of cathepsin B was 40 000 and was greater than 150 000 for aminopeptidase.

Cyclic GMP accumulation during cholinergic stimulation of eccrine sweat glands

1984 ◽  
Vol 247 (3) ◽  
pp. C234-C239 ◽  
Author(s):  
K. Sato ◽  
F. Sato
Keyword(s):  
Time Course ◽  
Sweat Glands ◽  
Cgmp Level ◽  
Ionophore A23187 ◽  
Cholinergic Stimulation ◽  
Sweat Secretion ◽  
Transient Elevation ◽  
Eccrine Sweat Glands ◽  
Eccrine Sweat

The possibility that guanosine 3'5'-cyclic monophosphate (cGMP) may be an intracellular mediator of cholinergic stimulation [methacholine chloride (MCh)] was explored by comparing the relationship between the time course of cGMP accumulation and sweat secretion by use of isolated monkey palm eccrine sweat glands. Isolated sweat glands were incubated with MCh or other agents, and tissue levels of cGMP were determined by radioimmunoassay. In parallel experiments, sweat secretion was induced from cannulated single sweat glands in vitro. Stimulation with MCh produced a Ca-dependent transient elevation of cGMP level from 10 to 80 fmol/gland, peaking at 1-2 min but returning to the basal level by 5 min. The MCh-induced cGMP level was dose dependent and was inhibited by atropine. Ionophore A23187 (2 X 10(-4) M), however, caused persistent elevation of cGMP level for at least 20 min. Neither 10(-4) M MNNG, which elevated the cGMP level comparably with MCh stimulation, nor 8-bromo-cGMP (2 mM) induced sweat secretion. Thus although a parallelism between the cGMP level and sweating rate appears to hold for the initial stage of MCh-induced sweating, it does not hold for the steady state of sweat secretion. Data could not be interpreted to favor the notion that cGMP may be the intracellular mediator of cholinergic sweat secretion.

Individual variations in structure and function of human eccrine sweat gland

1983 ◽  
Vol 245 (2) ◽  
pp. R203-R208 ◽  
Author(s):  
K. Sato ◽  
F. Sato
Keyword(s):  
Sweat Gland ◽  
Sweat Rate ◽  
Unit Length ◽  
Secretory Activity ◽  
Eccrine Sweat Gland ◽  
Sweat Glands ◽  
And Function ◽  
Eccrine Sweat

The mechanisms underlying variations in perspiration rate at the glandular level are still poorly understood. Human eccrine sweat glands were dissected from the back of 12 adults, cannulated, and stimulated in vitro with methacholine (Mch). The maximal sweat rate and pKA for Mch determined from the dose-response curve for each individual were compared with the anatomic dimensions of the isolated secretory tubules. There was significant correlation between Mch sensitivity (pKA) and the size of the sweat gland, sweat rate per gland, sweat rate per unit length of the secretory tubule, and sweat rate per unit glandular volume. The sweat glands from individuals judged to be poor sweaters exhibited smaller size, lower secretory activity both in vivo and in vitro, and decreased Mch sensitivity compared with glands from physically fit individuals. We conclude that the increased Mch sensitivity and glandular hypertrophy are the two important features of functionally active sweat glands and infer that these parameters could improve as a result of acclimatization to physical exercise and/or heat.

scholarly journals Surface morphology and agglutinability with concanavalin A in normal and transformed murine fibroblasts.

1976 ◽  
Vol 68 (1) ◽  
pp. 101-112 ◽  
Author(s):  
J G Collard ◽  
J H Temmink
Keyword(s):  
Surface Morphology ◽  
Concanavalin A ◽  
Ethylenediaminetetraacetic Acid ◽  
Simian Virus 40 ◽  
Cell Surfaces ◽  
3T3 Cells ◽  
Con A ◽  
Transformed Fibroblasts ◽  
Murine Fibroblasts

The surface morphology of attached and suspended normal and transformed fibroblasts has been studied with the scanning electron microscope. Normal murine fibroblasts (3T3) grow in vitro with widely extended leading lamellae. During most parts of the cell cycle the surfaces of these cells are practically free of microvilli. When the cells round up for mitosis, their cell surfaces become adorned with many microvilli. In contrast, simian virus 40-transformed fibroblasts (SV3T3) grow more compact, and their cell surfaces remain smooth throughout the life cycle. When confluent 3T3 and SV3T3 cells are suspended with ethylenediaminetetraacetic acid (EDTA) for agglutination assays, similar differences in surface morphology are found: 3T3 cells always bear many microvilli, whereas most SV3T3 cells are essentially free of microvilli. The addition of concanavalin A (Con A) does not influence the surface morphology of the suspended cells. The morphological differences described here may be important for the agglutination process of the normal and transformed 3T3 cells, because they affect the real cell surface area and thus the density of Con A-binding sites.

Cysteine proteinase inhibitor in eccrine sweat is derived from sweat gland

1991 ◽  
Vol 260 (2) ◽  
pp. R314-R320 ◽  
Author(s):  
H. Yokozeki ◽  
T. Hibino ◽  
T. Takemura ◽  
K. Sato
Keyword(s):  
Liquid Chromatography ◽  
Molecular Mass ◽  
Sweat Gland ◽  
Proteinase Inhibitors ◽  
Sweat Rate ◽  
Cysteine Proteinase ◽  
Sweat Glands ◽  
Cysteine Proteinase Inhibitor ◽  
Eccrine Sweat

Although cysteine proteinases have been reported to be present in human eccrine sweat, their endogenous inhibitors, cysteine proteinase inhibitors (CPIs), have remained unstudied. We now present evidence that CPIs are indeed a true ingredient of human eccrine sweat. Sweat induced in sauna was collected over a Vaseline barrier placed on the skin to minimize epidermal contamination. The absence of major epidermal contamination of the sweat was further ensured by monitoring an epidermal marker, high-molecular-mass aminopeptidase. Sweat CPI was purified sequentially by chromatography with Sephacryl S-200, carboxymethylated papain-Sepharose, and anion-exchange Mono Q fast-protein liquid chromatography columns. Sweat CPI has a molecular mass of approximately 15 kDa, is stable for temperature (up to 80 degrees C) and pH (from 3 to 10), and inhibits papain, ficin, and sweat cathepsin B- and H-like enzymes. Sweat CPI may be of sweat gland origin because 1) the rate of CPI output in sweat (CPI concentration x sweat rate) is constant over 45 min; 2) antibody against epidermal CPI, which cross-reacts with sweat CPI, localized immunoreactivity in the sweat duct; 3) CPI activity was present in the glandular extracts of control and methacholine-stimulated (for 1 h in vitro) human sweat glands; and 4) the peaks of CPI activity in the glandular extract and sweat CPI were both eluted (by high-pressure liquid chromatography) at around 15 kDa. Sweat CPI may be very similar to epidermal CPI (which belongs to the stefin family of CPIs) because of many shared characteristics. The identity and function of sweat CPI remain to be studied.

Role of calcium in cholinergic and adrenergic mechanisms of eccrine sweat secretion

1981 ◽  
Vol 241 (3) ◽  
pp. C113-C120 ◽  
Author(s):  
K. Sato ◽  
F. Sato
Keyword(s):  
Sweat Rate ◽  
Sweat Glands ◽  
Hyperbolic Function ◽  
Ionophore A23187 ◽  
Secretory Rate ◽  
Sweat Secretion ◽  
Eccrine Sweat Glands ◽  
Eccrine Sweat

The role of Ca2+ in eccrine sweat secretion was studied using isolated cannulated monkey palm eccrine sweat glands in vitro. Removal of Ca2+ from the incubation medium promptly abolished sweat secretion induced by methacholine or phenylephrine. In contrast, isoproterenol-induced sweat secretion lasted from 40 to 220 min in a Ca2+-free medium. The methacholine-induced maximal sweat rate was a hyperbolic function of the Ca2+ concentration in the bath and reached a plateau at 1 mM Ca2+. Higher Ca2+ concentrations rather suppressed the secretory rate. The Ca2+ ionophore A23187, but not X537A, at 3 X 10(-6) M induced copious prolonged sweat secretion after a latent period of 10 min. A23187-induced sweat secretion was not inhibited by either atropine or propranolol. D 600 (methoxyverapamil) at 10(-3) M inhibited sweat secretion induced by methacholine or by isoproterenol, although the latter lasted longer than methacholine sweating (20 vs. 5 min) in the presence of D 600. The data support the notion that Ca2+ influx into the cell plays a crucial role in cholinergic and alpha-adrenergic sweating, whereas a partial supply of Ca2+ for isoproterenol-induced sweating is derived from an intracellular store.

Influence of calcium and cyclic nucleotides on beta-adrenergic sweat secretion in equine sweat glands

1984 ◽  
Vol 247 (1) ◽  
pp. C10-C13 ◽  
Author(s):  
J. Bijman ◽  
P. M. Quinton
Keyword(s):  
Cyclic Nucleotides ◽  
Ethylenediaminetetraacetic Acid ◽  
Sweat Rate ◽  
Sweat Glands ◽  
Ionophore A23187 ◽  
Beta Adrenergic ◽  
Cyclic Monophosphate ◽  
Sweat Secretion ◽  
Adrenergic Antagonist

The effects of Ca2+, the cyclic nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP), and other parameters of sweat secretion from single equine sweat glands were examined in vitro. Extracellular Ca2+, the Ca2+ ionophore A23187, and the Ca2+ channel antagonist verapamil were all without effect on sweat secretion. Prolonged rinsing of the glands in Ca2+-free Ringer solution with 5 mM ethylenediaminetetraacetic acid decreased the secretion to 30% of the control sweat rate in response to the beta-adrenergic agonist isoproterenol; the sweat response was restored upon adding Ca2+ to the Ringer. cAMP but not cGMP analogues were as effective in stimulating sweat rates as isoproterenol, which elicited maximal secretory rates in vitro. cAMP stimulation was not inhibited by the beta-adrenergic antagonist propranolol. Because the equine sweat gland is predominantly stimulated via the beta-adrenergic receptor, we conclude that cAMP is a principal intracellular messenger in coupling this type of stimulus to the fluid secretion response in this tissue.

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