Phosphatidylglycerol Acts as a Switch for Cholesterol-Dependent Membrane Binding of ApoE Signal Peptide.

The apolipoprotein E (ApoE) signal peptide is a short stretch of N-terminal amino acids that direct the ApoE protein to the endoplasmic reticulum after synthesis. Previous studies have shown that this peptide can bind to lipid membranes in a cholesterol-dependent manner; however, the mechanism of this interaction is yet to be clarified. In this study, we aimed to investigate how the composition of neighboring lipids affects the membrane-binding of the ApoE signal peptide. We found that a negatively charged lipid, such as phosphatidylglycerol, can act as a switch that reduces the binding efficiency of the peptide to cholesterol-rich membranes. Interestingly, phosphatidylethanolamine does not activate the cholesterol-dependent binding of the ApoE signal peptide yet acts synergistically to enhance the cholesterol sensitivity in phosphatidylglycerol-containing membranes. To the best of our knowledge, this is the first report of modulation of the affinity of a peptide for a membrane by a neighboring lipid rather than by the lipid-binding domain of the peptide. Our findings revealed a novel role of lipid diversity in modulating the membrane binding of the ApoE signal peptide and its potential implications in the unidirectional trafficking of a newly synthesized protein from the ribosomes to the endoplasmic reticulum.

Lipid composition dependent binding of apolipoprotein E signal peptide: Importance of membrane cholesterol in protein trafficking.

Soluble secretory and membrane proteins contain a short stretch of signal peptide (SP) at their N-terminal end, which gets cleaved after reaching the destination organelle. However, the importance of SP in protein trafficking is not fully understood. The lipid compositions of cellular organelles are highly heterogeneous, and the preference of SP toward a particular lipid composition might play a key role in unidirectional trafficking of protein. In order to understand the preference of Apolipoprotein E (ApoE) toward endoplasmic reticulum (ER), we have studied the interaction of its SP with membranes of varying lipid compositions. The importance of cholesterol is paramount as subcellular organelles contain differential amount of cholesterol; endoplasmic reticulum (ER) contains the least amount of cholesterol. We have utilized batteries of steady-state and time-resolved fluorescence techniques to understand the affinity of ApoE signal peptide toward membranes of varying lipid compositions. We observed that the ApoE signal peptide binds tightly with membranes devoid of cholesterol, and binding affinity reduces with increasing concentration of membrane cholesterol. Our results clearly suggest the importance of membrane composition in the unidirectional movement of ApoE toward ER. This property of SP can further be utilized for the development of organelle specific cargo delivery.

Effect of variations in the conserved residues E371 and S359 on the structural dynamics of protein Z dependent protease inhibitor (ZPI): a molecular dynamic simulation study.

Protein Z (PZ) dependent protease inhibitor (ZPI) is a natural anticoagulant inhibiting blood coagulation proteases fXa and fXIa. Despite being a member of the serpin superfamily, it possesses unique structural features such as activation by PZ, regulating its inhibitory function. In order to understand the Reactive Centre Loop (RCL) dynamics of ZPI, which is absolutely critical for its activity, we performed Molecular Dynamics (MD) simulation on ZPI and its E371 and S359 variants located at important conserved functional sites. Unexpectedly, the RCL of E371 variants, (E371K, E371R, and E371Q), were shown to be very stable due to compensatory interactions at the proximal end of RCL. Interestingly, RCL flexibility was shown to be enhanced in the double mutant K318E-E371K due to the repulsive effect of increased negative charge on top of the breach region. Principal component analysis (PCA) coupled with residue wise interaction network analysis(RIN) revealed correlated motion between the RCL and the PZ binding regions in the WT. However, a loss of regulation in correlated motion between RCL and PZ binding hotspot Tyr240 in the double mutant was also observed. Additionally, the S359F and S359I mutations resulted in increased RCL flexibility owing to the disruption of stabilizing hydrogen bonding interaction at the distal end of strand S5A. Thus, the current study proposes that the overall stabilizing interactions of S5A is a major regulator of proper loop movement of ZPI for its activity. The results would be beneficial to engineer activity compromised ZPI as a prophylactic agent for the treatment of hemophilia.Communicated by Ramaswamy H. Sarma.

Role of vitamin D in treating COVID-19-associated coagulopathy: problems and perspectives.

Aggressive inflammatory response leading to hypercoagulability has been found to be associated with disease severity in COVID-19 patients and portends bad treatment outcome. A state of acute disseminated intravascular coagulation (DIC), along with pulmonary embolism and/or deep vein thrombosis, has been observed in critically ill ICU patients. Autopsy reports of COVID-19 patients demonstrated microthrombi in lungs and in other organs, as well as marked inflammatory changes, characteristic clinicopathological features that exacerbate disease severity. Vitamin D supplementation was recommended by many clinicians across the globe to improve clinical symptoms of COVID-19 patients, mainly because of its immunomodulatory roles on immune cells. Furthermore, vitamin D and its associated molecules are also known to directly or indirectly regulate various thrombotic pathways. We propose that vitamin D supplementation not only attenuates the risk of Acute Respiratory Disease Syndrome (ARDS) but it also may have a role in reducing coagulation abnormalities in critically ill COVID-19 patients. The overarching goal of this review is to discuss the effects of vitamin D on coagulation pathways and other intertwined processes leading to thrombosis. Many clinical trials are currently investigating the efficacy of vitamin D supplementation in reducing the risk of COVID-19 infection. However, randomized placebo control clinical trials are also necessary to ascertain the effect of vitamin D supplementation on reducing the risk of coagulopathy in COVID-19 patients.

Phosphatidylserine and phosphatidylethanolamine regulate the structure and function of FVIIa and its interaction with soluble tissue factor.

Cell membranes have important functions in many steps of the blood coagulation cascade, including the activation of factor X (FX) by the factor VIIa (FVIIa)-tissue factor (TF) complex (extrinsic Xase). FVIIa shares structural similarity with factor IXa (FIXa) and FXa. FIXa and FXa are regulated by binding to phosphatidylserine (PS)-containing membranes via their γ-carboxyglutamic acid-rich domain (Gla) and epidermal growth-factor (EGF) domains. Although FVIIa also has a Gla-rich region, its affinity for PS-containing membranes is much lower compared with that of FIXa and FXa. Research suggests that a more common endothelial cell lipid, phosphatidylethanolamine (PE), might augment the contribution of PS in FVIIa membrane-binding and proteolytic activity. We used soluble forms of PS and PE (1,2-dicaproyl-sn-glycero-3-phospho-l-serine (C6PS), 1,2-dicaproyl-sn-glycero-3-phospho-ethanolamine (C6PE)) to test the hypothesis that the two lipids bind to FVIIa jointly to promote FVIIa membrane binding and proteolytic activity. By equilibrium dialysis and tryptophan fluorescence, we found two sites on FVIIa that bound equally to C6PE and C6PS with Kd of ∼ 150-160 µM, however, deletion of Gla domain reduced the binding affinity. Binding of lipids occurred with greater affinity (Kd∼70-80 µM) when monitored by FVIIa proteolytic activity. Global fitting of all datasets indicated independent binding of two molecules of each lipid. The proteolytic activity of FVIIa increased by ∼50-100-fold in the presence of soluble TF (sTF) plus C6PS/C6PE. However, the proteolytic activity of Gla-deleted FVIIa in the presence of sTF was reduced drastically, suggesting the importance of Gla domain to maintain full proteolytic activity.

COVID-19: a probable role of the anticoagulant Protein S in managing COVID-19-associated coagulopathy.

The COVID-19 pandemic has caused monumental mortality, and there are still no adequate therapies. Most severely ill COVID-19 patients manifest a hyperactivated immune response, instigated by interleukin 6 (IL6) that triggers a so called "cytokine storm" and coagulopathy. Hypoxia is also associated with COVID-19. So far overlooked is the fact that both IL6 and hypoxia depress the abundance of a key anticoagulant, Protein S. We speculate that the IL6-driven cytokine explosion plus hypoxemia causes a severe drop in Protein S level that exacerbates the thrombotic risk in COVID-19 patients. Here we highlight a mechanism by which the IL6-hypoxia curse causes a deadly hypercoagulable state in COVID-19 patients, and we suggest a path to therapy.

Phosphatidylserine and Phosphatidylethanolamine Bind to Protein Z Cooperatively and with Equal Affinity.

Protein Z (PZ) is an anticoagulant that binds with high affinity to Protein Z-dependent protease inhibitor (ZPI) and accelerates the rate of ZPI-mediated inhibition of factor Xa (fXa) by more than 1000-fold in the presence of Ca2+ and phospholipids. PZ promotion of the ZPI-fXa interaction results from the anchoring of the Gla domain of PZ onto phospholipid surfaces and positioning the bound ZPI in close proximity to the Gla-anchored fXa, forming a ternary complex of PZ/ZPI/fXa. Although interaction of PZ with phospholipid membrane appears to be absolutely crucial for its cofactor activity, little is known about the binding of different phospholipids to PZ. The present study was conceived to understand the interaction of different phospholipids with PZ. Experiments with both soluble lipids and model membranes revealed that PZ binds to phosphatidylserine (PS) and phosphatidylethanolamine (PE) with equal affinity (Kd~48 µM); further, PS and PE bound to PZ synergistically. Equilibrium dialysis experiments revealed two lipid-binding sites for both PS and PE. PZ binds with weaker affinity to other phospholipids, e.g., phosphatidic acid, phosphatidylglycerol, phosphatidylcholine and binding of these lipids is not synergistic with respect to PS. Both PS and PE -containing membranes supported the formation of a fXa-PZ complex. PZ protection of fXa from antithrombin inhibition were also shown to be comparable in presence of both PS: PC and PE: PC membranes. These findings are particularly important and intriguing since they suggest a special affinity of PZ, in vivo, towards activated platelets, the primary membrane involved in blood coagulation process.

The transmembrane domain peptide of vesicular stomatitis virus promotes both intermediate and pore formation during PEG-mediated vesicle fusion.

We propose mechanisms by which the transmembrane domain of vesicular stomatitis virus (VSV-TMD) promotes both initiation of fusion and formation of a fusion pore. Time courses of polyethyleneglycol (PEG)-mediated fusion of 25 nm small unilamellar vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), bovine brain sphingomyelin, and cholesterol (35:30:15:20 molar ratio) were recorded at pH 7.4 at five different temperatures (from 17°C to 37°C) and compared with time courses obtained with the same vesicles containing the fusion-active TMD of the G protein of VSV. Multiple time courses were fitted globally to a one-intermediate ensemble kinetic model to estimate the rate constants for conversion of the aggregated state to an intermediate hemifused state (k1, stalk, or I1) that rapidly transits to an unstable intermediate (I2 state) that converts to a final fusion pore state with a combined rate k3. The probabilities of lipid mixing, contents mixing, and contents leakage in the three states were also obtained from this analysis. The activation thermodynamics for each step were consistent with previously published models of lipid rearrangements during intermediate and pore formation. The influences of VSV-TMD, hexadecane, and VSV-TMD + hexadecane on the kinetics, activation thermodynamics, and membrane structure support the hypothesis that these two agents do not catalyze fusion by a common mechanism, except possibly at the lowest temperatures examined. VSV-TMD primarily catalyzed initial intermediate formation, although it substantially increased the probability of contents mixing in the intermediate state. Our results support the hypothesis that the catalytic influence of VSV-TMD on the initial-intermediate- and pore-forming steps of PEG-mediated fusion derives from its ability to impose a positive intrinsic curvature and thereby stress small unilamellar vesicle outer leaflets as well as the periphery of intermediate microstructures.

pH Alters PEG-mediated fusion of phosphatidylethanolamine-containing vesicles.

Here, we examine the different mechanisms of poly(ethylene glycol)-mediated fusion of small unilamellar vesicles composed of dioleoylphosphatidylcholine/dioleoylphosphatidylethanolamine (DOPE)/sphingomyelin/cholesterol in a molar ratio of 35:30:15:20 at pH 7.4 versus pH 5. In doing so, we test the hypothesis that fusion of this lipid mixture should be influenced by differences in hydration of DOPE at these two pH values. An examination of the literature reveals that DOPE should be less hydrated at pH 5 (where influenza virus particles fuse with endosome membranes) than at pH 7.4 (where synaptic vesicles or HIV virus particles fuse with plasma membrane). Ensemble kinetic experiments revealed substantial differences in fusion of this plasma membrane mimetic system at these two pH values. The most dramatic difference was the observation of two intermediates at pH 5 but loss of one of these fusion intermediates at pH 7.4. Analysis of data collected at several temperatures also revealed that formation of the initial fusion intermediate (stalk) was favored at pH 7.4 due to increased activation entropy. Our observations support the hypothesis that the different negative intrinsic curvature of DOPE can account for different fusion paths and activation thermodynamics in steps of the fusion process at these two pH values. Finally, the effects of 2 mol % hexadecane on fusion at both pH values seemed to have similar origins for step 1 (promotion of acyl chain or hydrocarbon excursion into interbilayer space) and step 3 (reduction of interstice energy leading to expansion to a critical stalk radius). Different hexadecane effects on activation thermodynamics at these two pH values can also be related to altered DOPE hydration. The results support our kinetic model for fusion and offer insight into the critical role of phosphatidylethanolamine in fusion.

Soluble phosphatidylserine binds to two sites on human factor IXa in a Ca2+ dependent fashion to specifically regulate structure and activity.

Clinical studies have demonstrated a correlation between elevated levels of FIX and the risk of coronary heart disease, while reduced plasma FIX causes hemophilia B. FIXa interacts with FVIIIa in the presence of Ca2+ and phosphatidylserine (PS)-containing membranes to form a factor X-activating complex (Xase) that is key to propagation of the initiated blood coagulation process in human. We test the hypothesis that PS in these membranes up-regulates the catalytic activity of this essential enzyme. We used a soluble form of phosphatidylserine, 1, 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), as a tool to do so. C6PS and PS in membranes are reported to regulate the homologous FXa nearly identically. FIXa binds a molecule of C6PS at each of with two sites with such different affinities (∼100-fold) that these appear to be independent. A high affinity C6PS binding site (Kd∼1.4 µM) regulates structure, whereas a low-affinity binding site (Kd∼140 µM) regulates activity. Equilibrium dialysis experiments were analyzed globally with four other data sets (proteolytic and amidolytic activities, intrinsic fluorescence, ellipticity) to unequivocally demonstrate stoichiometries of one for both sites. Michaelis-Menten parameters for FIXa proteolytic activity were the same in the presence of C6PS or PS/PC membranes. We conclude that the PS molecule and not a membrane surface is the key regulator of both factors Xa and IXa. Despite some minor differences in the details of regulation of factors Xa and IXa, the similarities we found suggest that lipid regulation of these two proteases may be similar, a hypothesis that we continue to test.

Inhibition of intrinsic Xase by protein S: a novel regulatory role of protein S independent of activated protein C.

OBJECTIVE: Protein S is a vitamin K-dependent plasma protein that functions in the feedback regulation of thrombin generation. Our goal was to determine how protein S regulates the intrinsic pathway of blood coagulation. METHODS AND RESULTS: We used plasma, including platelet-rich plasma, and in vitro methods to determine how the intrinsic pathway of blood coagulation is regulated by protein S. We obtained the following results: (1) activated partial thromboplastin time assays with protein S-supplemented plasma confirmed that protein S prolongs clotting time; (2) a modified activated partial thromboplastin time assay with factor IX (fIX)-deficient plasma confirmed that protein S affects fIX-initiated clotting; (3) a fIXa/factor VIIIa (fVIIIa)-mediated thrombin generation assay with either platelet-rich plasma or factor-deficient plasma, initiated with a limiting amount of tissue factor, was regulated by protein S; (4) in the presence of phosphatidylserine vesicles, protein S inhibited fIXa in the absence and presence of fVIIIa; and (5) protein S altered only the K(M) for factor X activation by fIXa in the absence of fVIIIa and both k(cat) and K(M) in the presence of fVIIIa. CONCLUSIONS: From our findings, it can be concluded that protein S inhibits fIXa in the presence or absence of fVIIIa in an activated protein C-independent way.

Activation thermodynamics of poly(ethylene glycol)-mediated model membrane fusion support mechanistic models of stalk and pore formation.

Membrane fusion, essential to eukaryotic life, is broadly envisioned as a three-step process proceeding from contacting bilayers through two semistable, nonlamellar lipidic intermediate states to a fusion pore. Here, we introduced a new, to our knowledge, experimental approach to gain insight into the nature of the transition states between initial, intermediate, and final states. Recorded time courses of lipid-mixing, content-mixing, and content-leakage associated with fusion of 23 nm vesicles in the presence of poly(ethylene glycol) at multiple temperatures were fitted globally to a three-step sequential model to yield rate constants and thereby activation thermodynamics for each step of the process, as well as probabilities of occurrence of lipid-mixing, content-mixing, or content-leakage in each state. Experiments with membranes containing hexadecane, known to reduce interstice energy in nonlamellar structures, provided additional insight into the nature of fusion intermediates and transition states. The results support a hypothesis for the mechanism of stalk formation (step-1) that involves acyl chain protrusions into the interbilayer contact region, a hypothesis for a step-2 mechanism involving continuous interconversion of semistable nonlamellar intermediates, and a hypothesis for step-3 (pore formation) mechanism involving correlated movement of whole lipid molecules into hydrophobic spaces created by geometry mismatch between intermediate structures.

Local and global effects of a cavity filling mutation in a metastable serpin.

The serpins are an unusual class of protease inhibitors which fold to a metastable form and subsequently undergo a massive conformational change to a stable form when they inhibit their target proteases. The driving force for this conformational change has been extensively investigated by site directed mutagenesis, and it has been found that mutations which stabilize the metastable form frequently result in activity deficiency. Here, we employ hydrogen/deuterium exchange to probe the effects of a cavity filling mutant of alpha(1)AT. The Gly117 --> Phe substitution fills a cavity between the F-helix and the face of beta-sheet A, stabilizes the metastable form of alpha(1)AT by approximately 4 kcal/mol and results in a 60% reduction in inhibitory activity against elastase. Globally, the G117F substitution alters the unfolding mechanism by eliminating the molten globule intermediate that is seen in wild type unfolding. Remarkably, this is accomplished primarily by destabilizing the molten globule rather than stabilizing the metastable native state. Locally, conformational flexibility in the native state is reduced in specific regions: the top of the F-helix, beta-strands 5A, 1C, and 4C, and helix D. Except for strand 4C, all of these regions mediate or propagate conformational changes. The F-helix and strand 5A must be displaced during protease inhibition, displacement of strand 1C is required for polymer formation, and helix D is a site (in antithrombin) of allosteric regulation. Our results indicate that these functionally important regions form a delocalized network of residues that are dynamically coupled and that both local and global stability mediate inhibitory activity.

The structural basis of serpin polymerization studied by hydrogen/deuterium exchange and mass spectrometry.

The serpinopathies are a group of inherited disorders that share as their molecular basis the misfolding and polymerization of serpins, an important class of protease inhibitors. Depending on the identity of the serpin, conditions arising from polymerization include emphysema, thrombosis, and dementia. The structure of serpin polymers is thus of considerable medical interest. Wild-type alpha(1)-antitrypsin will form polymers upon incubation at moderate temperatures and has been widely used as a model system for studying serpin polymerization. Using hydrogen/deuterium exchange and mass spectrometry, we have obtained molecular level structural information on the alpha(1)-antitrypsin polymer. We found that the flexible reactive center loop becomes strongly protected upon polymerization. We also found significant increases in protection in the center of beta-sheet A and in helix F. These results support a model in which linkage between serpins is achieved through insertion of the reactive center loop of one serpin into beta-sheet A of another. We have also examined the heat-induced conformational changes preceding polymerization. We found that polymerization is preceded by significant destabilization of beta-sheet C. On the basis of our results, we propose a mechanism for polymerization in which beta-strand 1C is displaced from the rest of beta-sheet C through a binary serpin/serpin interaction. Displacement of strand 1C triggers further conformational changes, including the opening of beta-sheet A, and allows for subsequent polymerization.

Characterization of a low-molecular-mass stimulator protein of Mg2+-independent Ca2+-ATPase: effect on phosphorylation/dephosphorylation, calcium transport and sperm-cell motility.

A 14 kDa cytosolic protein purified from bovine brain homogenate has been recently reported as a stimulator of goat spermatozoa Mg2+-independent Ca2+-ATPase. In the present study, we demonstrate the formation of the [gamma-32P]ATP-labelled phosphoenzyme as the 110 kDa phosphoprotein and its rapid decomposition in presence of the stimulator protein. Together with the cross-reactivity of this 110 kDa protein with an anti-SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 2a antibody, the ATPase can now be conclusively said to belong to the SERCA family, which is activated by the stimulator. The ability of the stimulator to enhance the Ca2+ transport has been elucidated from 45Ca2+ uptake studies and was found to be sensitive to Ca2+ channel blockers. CD revealed an alpha-helical structure of the stimulator. The amino acid analysis suggests that it is composed primarily of hydrophobic and some acidic amino acid residues. The pI of 5.1 has been re-confirmed from two-dimensional electrophoresis. Immuno-cross-reactivity studies indicate that the stimulator or similar proteins are present in cytosolic fractions of liver, kidney or testes in different species, but brain is the richest source. Proteomic analyses of its trypsinized fragments suggest its similarity with bovine THRP (thyroid hormone-responsive protein). The physiological significance of the stimulator has been suggested from its ability to activate sperm-cell motility.

Structural and functional characterization and physiological significance of a stimulator protein of Mg2+-independent Ca2-ATPase isolated from goat spermatozoa.

Recently a low-molecular-mass protein purified from goat testes cytosol has been reported from our laboratory which is found to stimulate Mg2+ -independent Ca2+-ATPase without any significant effect on Mg2+-dependent Ca2+-ATPase. In the present study, detailed structural and functional characterization, as well as the physiological significance of the protein has been described. The stimulatory effect is found to be inhibited by known inhibitors of P-type ATPases, vanadate and lanthanum chloride. Monitoring of the phosphoenzyme intermediate by autoradiography has shown that the stimulation of the ATPase is due to the enhancement in the rate of dephosphorylation of the overall reaction step. Along with the stimulation of the enzyme activity, the protein is found to enhance the calcium uptake. Amino acid analysis data show that the stimulator contains about 26% non-polar amino acid facilitating easy penetration to the hydrophobic core of the membrane bound ATPase. Circular dichroism analysis of the protein suggested the presence of all secondary structural elements. The Western-blotting experiment shows its expression level is the highest in goat testes. Peptide fragments obtained in MALDI-MS analysis when subjected to MSDB database search by MASCOT search engine reveals that the proteins of close similarity with the protein under study are actin related protein 2/3 complex subunit, peptidyl-prolyl cis-trans isomerase and gastrin releasing peptide precursor. Besides, the protein under study is also shown to decrease the forward motility of goat sperm without having any significant effect on the total motility indicating its possible role in fertility regulation.

Stimulation of Mg2+-independent form of Ca2+-ATPase by a low molecular mass protein purified from goat testes cytosol.

A low molecular mass protein purified from goat (Capra hircus) testes cytosol following gel filtration and anion exchange chromatographic separation stimulates Mg(2+)-independent Ca(2+)-ATPase activity without any significant effect on Mg(2+)-dependent Ca(2+)-ATPase. Stimulation of the ATPase is due to an increase in the rate of dephosphorylation of the overall reaction step of the enzyme. Binding of the stimulator increases the affinity of Ca(2+)-ATPase for Ca(2+). An analysis of enzyme kinetics reveals a reversible type of binding of the stimulator to the ATPase and non-competitive type of stimulation with respect to the substrate. Stimulation seems due to binding of the protein at a single site following Michaelis-Menten model. The protein can also counter the effect of calcium antagonists exerted on the ATPase. The pI of the protein is 6.2 and its molecular mass has been determined to be 13, 961 by Q-TOF-MS.

Regulation of Mg2+-independent Ca2+-ATPase by a low molecular mass protein purified from bovine brain.

The goat sperm microsomal membranes have been found to contain an Mg2+-independent Ca2+-ATPase, a low affinity but highly active enzyme sharing similarities with the SERCA family of ATPases. The present study reports the identification and characterization of a 14 kilodalton cytosolic protein from bovine brain which can act as an endogenous stimulator of the enzyme with an S50 (concentration producing 50% stimulation) of 0.8 mu molar. Kinetic analysis suggests that the stimulation is noncompetitive with respect to the substrate, and the binding site(s) of the stimulator and substrate are distinct. Binding of the stimulator to the enzyme is reversible. The stimulator increases the affinity of the enzyme for calcium as evident from a decrease in K0.5 of the enzyme for calcium in presence of the stimulator. Radioactive labeling of the enzyme with [gamma-32P]-ATP suggests that the stimulator enhances the rate of dephosphorylation of the phosphoenzyme intermediate without altering the phosphorylation reaction step. The stimulatory effect of the protein has been observed only for the Mg2+-independent form of the enzyme, the Mg2+-dependent form being unaffected.