The properties and the regulation of adenosine 5'-phosphosulfate sulfotransferase extracted from cell suspension cultures ofNicotiana sylvestris was investigated. Optimal adenosine 5'-phosphosulfate sulfotransferase activity was obtained from the cells by extraction with 0.1 M tris-HCl, pH8.0, containing 2 M MgSO4 and 10 mM dithioerythritol. The K m for adenosine 5'-phosphosulfate in the sulfotransferase reaction was about 11 μM. Adenosine 5'-phosphosulfate in concentrations above 50 μM were inhibitory. The extratable adenosine 5'-phosphosulfate sulfotransferase activity decreased during cultivation with sulfate as the sole sulfur source, but after about 3 days it reached a constant level (50 to 100 nmol activated sulfate transferred h(-1) mg(-1) protein) which was maintained for at least 24 h. Addition of 0.5 mM cysteine to the culture medium decreased the extractable adenosine 5'-phosphosulfate sulfotransferase activity and blocked growth completely. With 0.1 mM cysteine an enzyme level of about 10% of the initial value was reached within 6 to 12 h without significant inhibition of growth. The added cysteine was absorbed rapidly and after 24 h cysteine could no longer be detected in the medium. Before the cysteine was completely depleted, the activity of adenosine 5'-phosphosulfate sulfotransferase started to increase, reaching ultimately a level which was comparable to the initial value.

Adequate prestimulation is considered a requirement for a fast, gentle, and complete udder emptying at machine milking. Reduced vacuum or reduced liner-open phase of pulsation (or both) may replace prestimulation and consequently reduce work load of the milker and increase parlor efficiency. In the present study we compared 2 milking routines (MR) with manual prestimulation (LPrep = long preparation: 15 s of forestripping, teat cleaning, and stimulation; SPrep = short preparation: 5 s of teat cleaning) followed by 1-min latency period and milking at standard vacuum and pulsation settings (claw vacuum 44 kPa, pulsation rate 60 cycles/min, pulsation ratio 65/35) with 2 MR consisting of 5 s of teat cleaning, immediate cluster attachment and milking at reduced vacuum with or without shortened liner-open phase of pulsation until milk flow exceeded 400 g/min (RP = reduced pulsation: pulsation ratio 30/70, pulsation rate 50 cycles/ min, claw vacuum 44 kPa; RPV = reduced pulsation and vacuum: pulsation ratio 30/70, pulsation rate 50 cycles/min, claw vacuum 34 kPa). Cluster detachment was performed at 400 g/min in all MR. Ten Holstein dairy cows were milked twice daily at 14-h and 10-h milking intervals. Milk flow and electrical conductivity (EC) were recorded throughout milking. During the first 2 min of each milking ultrasound cross-section images of the gland cistern of one front quarter were recorded, and 5 min after the end of milking teat tissue thickness of both front teats was measured by using a cutimeter. Most milking characteristics such as total milk yield, average milk flow, and machine-on time reached higher values at 14-h than 10-h milking intervals, but did not differ among MR. However, the occupancy time (time from the first touch of the udder until cluster detachment) was considerably shorter in RP and RPV compared with LPrep and SPrep. Ultrasound cross section areas of the gland cistern were larger in LPrep than in RP and RPV indicating that milk ejection already occurred at cluster attachment in LPrep. This assumption is also supported by the lower EC at cluster attachment in LPrep than in RP and RPV, which was caused by the presence of alveolar milk in the gland cistern after milk ejection. The MR RP and RPV increase parlor efficiency and are worksaving alternatives to MR, which include an adequate prestimulation (LPrep). However, shortening prestimulation to a 5-s teat cleaning followed by a latency period and milking at regular vacuum and pulsation is not adequate to save occupancy time. Because milking was performed at a relatively low vacuum (44 kPa) and at a detachment level of 400 g/min, teat tissue thickness did not differ among MR, and the vacuum reduction in RPV did not cause an additional advantage for teat condition compared with RP.