Estimating the initial reaction velocity of a soluble dehydrogenase in situ

✍ Scribed by Nakae, Yoshiko ;Stoward, Peter J.

• Publisher: Springer
• Year: 1993
• Tongue: English
• Weight: 611 KB
• Volume: 25
• Category: Article
• ISSN: 0018-2214
• DOI: 10.1007/bf00163815

✦ Synopsis

The initial reaction velocities (v~) of lactate dehydrogenase in single hepatocytes were determined, by microdensitometry or computer-assisted image analysis, in sections of unfixed mouse liver incubated at 37~ on substrate-containing agarose gel films. They were found to fit the equations v i = 2.82~ and v i = v + 2~ where v and ~ are, respectively, the gradients (or steady-state linear velocities) and the intercepts on the absorbance axis of the linear regression lines of the absorbance (A) on incubation time plots for incubation times between I and 3 min. Both equations were independent of section thickness between 4 and 14 gin. The observed and calculated values of v i agreed within 11.5% (n = 7I). The validity of the equations for vi was confirmed by showing that the calculated v i was proportional to the thickness of the section and hence the amount of enzyme present. Thus, v i can be determined from measurements of either ~ alone or v and ~