A myofibrillar form of smooth muscle myosin light chain phosphatase (MLCPase) was purified from turkey gizzard myofibrils, and it was found to be closely associated with the myosin light chain kinase (MLCKase). For this reason we have named this phosphatase the kinase-and myosin-associated protein phosphatase (KAMPPase). Subunits of the KAMPPase could be identified during the first ion exchange chromatography step. After further purification on calmodulin (CaM) and on thiophosphorylated regulatory myosin light chain affinity columns we obtained either a homogenous preparation of a 37-kDa catalytic (PC) subunit or a mixture of the PC subunit and variable amounts of a 67-kDa targeting (PT) subunit. The PT subunit bound the PC subunit to CaM affinity columns in a Ca 2؉ -independent manner; thus, elution of the subunits required only high salt concentration. Specificity of interaction between these subunits was shown by the following observations: 1) activity of isolated PC subunit, but not of the PTC holoenzyme, was stimulated 10 -20-fold after preincubation with 5-50 M of CoCl 2 ; 2) the pH activity profile of the PC subunit was modified by the PT subunit (the specific activity of the PTC holoenzyme was higher at neutral pH and lower at alkaline pH); and 3) affinity of the holoenzyme for unphosphorylated myosin was 3-fold higher, and for phosphorylated myosin it was 2-fold lower, in comparison with that of the purified PC subunit. KAMPPase was inhibited by okadaic acid (K i ‫؍‬ 250 nM), microcystin-LR (50 nM) and calyculin A (1.5 M) but not by arachidonic acid or the heat-stable inhibitor (I-2), which suggested that this is a type PP1 or PP2A protein phosphatase.

We show that a myofibrillar form of smooth muscle myosin light chain phosphatase (MLCPase) forms a multienzyme complex with myosin light chain kinase (MLCKase). The stability of the complex was indicated by the copurification of MLCKase and MLCPase activities through multiple steps that included myofibril preparation, gel filtration chromatography, cation (SPSepharose BB) and anion (Q-Sepharose FF) exchange chromatography, and affinity purification on calmodulin and on thiophosphorylated regulatory light chain columns. In addition, the purified complex eluted as a single peak from a final gel filtration column in the presence of calmodulin (CaM). Because a similar MLCPase is present in varying amounts in standard preparations of both MLCKase and myosin filaments, we have named it a kinase-and myosin-associated protein phosphatase (KAMPPase).The KAMPPase multienzyme complex was composed of a 37-kDa catalytic ( Phosphorylation of myosin by myosin light chain kinase (MLCKase) 1 represents the key activation step leading to contraction of smooth muscle (for reviews, see Refs. 1-4). Relaxation or inactivation of myosin is accomplished by a myosin light chain phosphatase (MLCPase) that has a controversial identification and subunit composition (see Ref. 5). In numerous previous studies (for references, see Ref. 6), many types of cytosolic MLCPases have been purified exhibiting different specific activities toward phosphorylated myosin or isolated myosin regulatory light chain (ReLC). A common feature of all of these phosphatases seems to be the presence of not only a catalytic (PC) subunit of about 35-38 kDa but also another subunit in the range of 55-72 kDa. The function of the latter subunit has not been established. Initial attempts to classify these serine/threonine phosphatases were not very successful (7), and it appears that smooth muscle MLCPases could be either the PP1 or the PP2A type.Several years after our initial report on the first myofibrillar MLCPase (8), we and others again turned our attention to MLCPases from smooth muscle. In our new approach, the phosphatase was purified by CaM affinity chromatography and was shown to be composed of 37-kDa catalytic and 67-kDa targeting subunits (9). The only other myofibrillar phosphatase known so far was purified and partly characterized by Alessi et al. (Ref. 10; see also Refs. 11 and 12). It is composed of three subunits: a 37-kDa catalytic subunit and two regulatory subunits of 130 and 20 kDa. Although the sequence of all three subunits has been determined, the role of the regulatory subunits is not understood (see Ref. 5). In this report, we describe further results on our myofibrillar smooth muscle protein phosphatase, which is closely associated with MLCKase and myosin filaments and is called, therefore, a kinase-and myosin-associated protein phosphatase (KAMPPase). We show for the first time that this association results in a functional multienzyme complex between these two key regulatory enzymes of smooth muscle.

The study investigated whether changes in body surface temperature in a sprint interval testing protocol (SITP) correlated with aerobic capacity in cyclists. The study involved 21 welltrained cyclists. Maximal aerobic power and maximal oxygen uptake relative to lean body mass (LBM-Pmax and LBM-VO2max, respectively) were determined by incremental exercise testing on a cycle ergometer. SITP was administered 48 hours later and involved four 30-s maximal sprints interspersed with 90-s active recovery. Body surface temperature was recorded at the temple and arm and the delta difference between baseline temperature and temperature measured immediately after the first sprint (ΔTt1 and ΔTa1, respectively) and 80 seconds after the fourth sprint (ΔTt4 and ΔTa4, respectively) was calculated. Significant correlations were found between ΔTt4 and LBM-Pmax and respectively) with no significant change in ΔTa1 or ΔTa4. Body surface temperature, measured at the temple region, can be used to indirectly assess aerobic capacity during maximal sprint exercise.

The aim of this study was to determine differences in glycolytic metabolite concentrations and work output in response to an all-out interval training session in 23 cyclists with at least 2 years of interval training experience (E) and those inexperienced (IE) in this form of training. The intervention involved subsequent sets of maximal intensity exercise on a cycle ergometer. Each set comprised four 30 s repetitions interspersed with 90 s recovery periods; sets were repeated when blood pH returned to 7.3. Measurements of post-exercise hydrogen (H+) and lactate ion (LA-) concentrations and work output were taken. The experienced cyclists performed significantly more sets of maximal efforts than the inexperienced athletes (5.8 ± 1.2 vs. 4.3 ± 0.9 sets, respectively). Work output decreased in each subsequent set in the IE group and only in the last set in the E group. Distribution of power output changed only in the E group; power decreased in the initial repetitions of set only to increase in the final repetitions. H+ concentration decreased in the third, penultimate, and last sets in the E group and in each subsequent set in the IE group. LA- decreased in the last set in both groups. In conclusion, the experienced cyclists were able to repeatedly induce elevated levels of lactic acidosis. Power output distribution changed with decreased acid–base imbalance. In this way, this group could compensate for a decreased anaerobic metabolism. The above factors allowed cyclists experienced in interval training to perform more sets of maximal exercise without a decrease in power output compared with inexperienced cyclists.

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