Tripolyphosphate hydrolysis by bovine myosin

Marie Yamazaki, Purdue University

Abstract

Polyphosphates are often used in the meat industry to increase the water holding capacity and tenderness of meat products, and tripolyphosphate (TPP) is a commonly used polyphosphate. However, the mechanism of how polyphosphates improve these qualities is poorly understood. It has been shown that TPP is metabolized into monophosphate in meat. Pyrophosphate (PPi) is an intermediate in this metabolism and it dissociates the acto-myosin complex and favors myosin extraction. The enzyme responsible for TPP hydrolysis has not been clearly determined. However, it can be hypothesized that myosin, or more specifically the motor domain or the myosin ATPase, is responsible and that the kinetic scheme for TPP is similar to that of ATP. The overall purpose of this research was to characterize the biochemical mechanism of TPP hydrolysis by fast and slow isoforms of bovine myosin in comparison with ATP hydrolysis. Protein purification protocols were modified to increase the yield and quality of bovine masseter, or slow subfragment-1 (S1). Fast and slow bovine myosin S1 hydrolyzed TPP, showing that myosin is most likely the major TPPase in meat. Fast bovine S1 (Cutaneous trunci) hydrolyzed both substrates faster compared to slow bovine S1 (Masseter), and ATP was hydrolyzed at a higher rate than TPP by both isoforms (1.66 and 5.85 nmole Pi/mg S1 protein/min for TPPase activity of slow and fast S1; 10.1 and 18.5 nmole Pi/mg S1 protein/min for ATPase activity of slow and fast S1). Analysis of enzyme activity as a function of TPP concentration showed that the Vmax was 0.937 and 4.99 nmole Pi/mg S1 protein/min while the Km was 0.380 and 0.898 mM TPP for slow and fast S1, respectively. PPi was found to be a strong inhibitor of TPPase activity with a Ki of 88.4 and 18.2 μM PPi for fast and slow S1 isoforms, respectively. Data from these kinetic studies support the proposed kinetic scheme for TPP hydrolysis. Both ATPase and TPPase activities were only modestly influenced by pH, with activity being higher at low pH for both fast and slow S1 isoforms. These results were contrary to our hypothesis that the activities of both ATP and TPPase would increase at high pH and that the activities would be increased by at least 3 fold. The activity at pH 5.40 was approximately 145% of that at pH 7.60. With isolated myofibrils from fast and slow muscles, calcium had a strong influence on ATP hydrolysis at pCa 4.0 compared to pCa 9.0 (41.2 and 56.7 nmole Pi/mg myofibril protein/min for slow and fast myofibrils at pCa 4.0; 6.08 and 8.26 nmole Pi/mg myofibril protein/min for slow and fast myofibrils at pCa 9.0). However, calcium only modestly influenced TPP hydrolysis for both isoforms (0.638 and 0.481 nmole Pi/mg myofibrils protein/min for slow and fast myofibrils at pCa 4.0; 0.437 and 0.380 nmole Pi/mg myofibril protein/min for slow and fast myofibrils at pCa 9.0). Lastly, although both S1 and myofibrils hydrolyze TPP, studies with myofibers and myofibrils at high calcium showed that TPP did not result in sarcomere shortening. Overall, these data showed that myosin S1 is the catalytic site for TPP hydrolysis and likely the main TPPase in meat; TPPase activity of fast and slow myosin is lower compared to the ATPase activity of the same proteins, fast myosin has a higher TPPase rate compared to slow myosin; PPi strongly inhibits TPP hydrolysis; the influence of calcium and pH on TPPase activity was modest; and although TPP is hydrolyzed much like ATP, the lack of nucleotide most likely prevents the necessary conformational changes from occurring, thereby preventing contraction. Results from these studies may benefit the meat industry by characterizing the major protein that hydrolyzes TPP and giving insight into explaining how TPP may increase the water holding capacity and tenderness of meat products.

Degree

M.S.

Advisors

Swartz, Purdue University.

Subject Area

Physiology

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