Development and application of a method for the detection, elution, and characterization of rat submandibular proteinases separated on isoelectric focusing gels reveals male/female differences.
Submandibular gland homogenates from age-matched male and female rats were focused on pH 4-6.5 isoelectric focusing gels. At least 14 bands (A to N) showing amidolytic activity against three different oligopeptide-7-amino-4-trifluoromethylcoumarin (AFC) derivatives, immobilized on cellulose diacetate overlay membranes, were observed under ultraviolet light on focused gels. All the proteinases were optimally eluted, from excised gel pieces, into 20 mM ammonium bicarbonate buffer, pH 9.8, containing 0.1% Triton X-100; the protein content of the band eluants was measured after selective precipitation of protein from interfering substances. Differences were observed in the substrate specificities of the proteinases such that enzymes in bands A and K showed increased reactivity to ZVKKR-AFC, and those in bands H and J to ZR-AFC and DVLR-AFC, respectively. While the ability of aprotinin to inhibit the enzymes showed an inverse relationship to their isoelectric points, soybean trypsin inhibitor was most potent against bands B to I and least effective against bands K to N. Extracts from the glands of female rats contained the same mixture of proteinases as their male counterparts but the concentrations of all the proteinases apart from band K were reduced by approximately 25-50%. Where suitable substrates are available these methods have general applications for the rapid identification and characterization of other enzymes fractionated on IEF gels.
Shori
D K
DK
Secretory and Soft Tissue Research Unit, King's College School of Medicine and Dentistry, London, United Kingdom.
Proctor
G B
GB
Garrett
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D005260:Female; D007525:Isoelectric Focusing; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D017208:Rats, Wistar; D012697:Serine Endopeptidases / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D012727:Sex Characteristics; D013363:Submandibular Gland / Q000201:enzymology*
D012697:Serine Endopeptidases