A relationship between trypsin and plasmin inhibitors and sialyltransferase activity

Nadkarni, Sheila (1994) A relationship between trypsin and plasmin inhibitors and sialyltransferase activity. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

The incubation of rat jejunal slices in Kreb’s-Ringer bicarbonate buffer (RRS) resulted in a time dependent release of soluble sialyltransferase (STase) into the incubation medium for up to 6 h. However, the STase released was susceptible to proteolysis and in order to measure the STase activity released there was a requirement for either heat-inactivated serum, α₁ proteinase inhibitor (A1PI) or α₂ antiplasmin in the incubation medium. Trypsin and plasmin activities higher in medium obtained from KRB incubations, compared to incubations where KRB supplemented with either heat-inactivated horse serum (HHS) heat inactivated rat serum (HRS). -- Addition of heparin to jejunal incubations supplemented with HRS or HHS resulted in decreased STase activity and increased trypsin and plasmin activities in the medium. It was determined that the heparin-binding fraction (HSF) from HHS or HRS was the serum component required in order to measure STase activity in the medium. -- HSF exhibited inhibitory activity towards trypsin and plasmin, but did not inhibit either elastase, thrombin, chymotrypsin, kallikrein or papain. A trypsin-binding protein (TBP) was isolated from HSF by trypsin agarose affinity chromatography. TBP was able to inhibit trypsin and plasmin and on 50s-Page showed a single major band and an apparent molecular weight of 67 kDa. When TBP was used to supplement jejunal incubations, it was as effective as HSF in protecting the STase activity released during jejunal incubations. -- Galactosyltransferase (GTase), which was also released in the soluble form during jejunal incubations, was not dependent on the proteolytic activity of the medium. GTase activity in the incubation medium remained similar whether incubations were carried out in KRB alone or in KRB supplemented with either HHS, HRS, HBF or TBP. Addition of heparin to incubations in either KRB or KRB supplemented with HRS did not cause a decrease in GTase activity, further suggesting that, GTase in contrast to STase was not dependent on the proteolytic activity of the medium. -- Heat-inactivated serum from turpentine treated rats had higher trypsin and plasmin inhibitory activities compared to heat-inactivated control rat serum. When heat-inactivated serum from turpentine treated rats was used to supplement KRB during jejunal incubations there was an increase in the STase activity released into the medium compared to incubations where heat-inactivated serum from control rats was used. In contrast, GTase activity remained similar whether incubations were carried out in KRB alone or in KRB supplemented with heat-inactivated serum from either control or turpentine treated rats. -- Trypsin and plasmin when added individually to a mixture of pure STase (5) and GTase preferentially inhibited STase activity. This effect was observed for both the STases used (α2-6[N] and α2-3 [O]). TBP was able to protect STase against the action of trypsin and plasmin. -- Serum from turpentine treated and control rats when incubated for 4 h at 37°C showed a progressive decline in STase activity over the time of incubation. Trypsin and plasmin inhibitory activities also decreased over the 4 h of incubation. Serum STase, as well as trypsin and plasmin inhibitory activities remained higher in the turpentine treated rats compared to control rats. However, serum GTase activity was similar in both groups. -- In incubations with hepatocytes, STase activity released into the medium was also dependent on the balance between trypsin and plasmin inhibitory / trypsin and plasmin activities of the incubation medium. STase activity was higher in the medium when heat-inactivated serum from either turpentine treated or control rats, HBF or TBP were used as supplements compared to incubations carried out in buffer alone. GTase activity remained similar when incubations were carried out in buffer alone or in buffer supplemented with antiproteases. -- The results indicate that increased STase activity was associated with increased trypsin and plasmin inhibitory activities or decreased trypsin and plasmin activities. This effect was not observed for GTase activity. Soluble STase is susceptible to proteolysis and trypsin and plasmin inhibitors therefore play a key role in determining measurable STase activity.

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