| Abstrakt: |
Studies were designed to examine the effects of isotonic sucrose and hypertonic KCl on the extraction and properties of phosphatases (acid optima) from long bones of suckling rats. Low-speed supernatants (S) of homogenates in sucrose were dialyzed against sucrose or deionized HO. Free enzyme was recovered from both retentates by ultracentrifugation before (S) or after sequential treatment with KCl and Triton X-100 (S), protamine (S), and a final dialysis (S). Activity was measured with p-nitrophenylphosphate ( p-NPP), β-glycerophosphate (β-GP), ATP, and casein. S was evaluated for latent enzyme with hypertonic KCl and classic lysing methods. Fractionation of activity in final extracts was accomplished with CM-52 cellulose or electrophoresis. Results showed that: (a) sucrose did not alter the types or properties of enzymes extracted, but did decrease yield of activities; (b) activity in S was increased approximately 20% by lysing methods, 80% by hypertonic KCl, and was unstable unless salt and detergent were present; and (c) chromatography or electrophoresis of S resolved only two enzymes: a tartrate-sensitive phosphomonoesterase (E) responsible for 15% of the total activity in S, and a tartrate-insensitive enzyme (E) which accounted for 85% of the activity, was unstable in isotonic medium, had high affinity for ATP and p-NPP, and had low affinity for casein and β-GP. It is concluded that sucrose is not necessary for the isolation of total bone acid phosphatase activity, that hypertonic KCl does not negatively affect the properties of the enzymes isolated, and that E and E show different latencies. [ABSTRACT FROM AUTHOR] |
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