Transbronchial biopsy in the presence of profound elevation of the international normalized ratio.

STUDY OBJECTIVE: To identify a level of coagulopathy, reported as the international normalized ratio (INR), that predicts hemorrhage following transbronchial forceps biopsy (TBBx) in an animal model. DESIGN: Crossover blinded study using Yucatan mini-swine (Sus scrofa). SETTING: Tertiary medical center with a dedicated animal research facility. STUDY DESIGN: A two-stage study. In stage 1, flexible fiberoptic bronchoscopy with TBBx was performed to establish the amount of bleeding in animals with normal coagulation systems. Animals then were administered escalating dosages of warfarin to obtain one of several increased INR levels. The endpoint of stage 1 was defined as the INR that resulted in a blood loss of > or = 100 mL in > or = 50% of the study animals. In stage 2, all the animals were to be anticoagulated to the INR level determined in stage 1. Topical and systemic measures would then be administered in an attempt to decrease postprocedure hemorrhage, and the results were recorded. RESULTS: Eighteen animals were enrolled in the study. Despite INR levels > 10, no animals developed a hemorrhagic complication of the transbronchial forceps biopsy (TBBx). Eleven animals had INRs > 7. Four animal deaths were recorded, with three animal deaths attributed to nonpulmonary hemorrhage, each due to a ruptured ovarian cyst. One death was anesthesia related. Stage 2 of the study was not performed due to the extreme INR levels reached in the animals during stage 1 and to the lack of a procedure-related complication. CONCLUSIONS: Our study suggests that INR elevation does not correlate with an increased risk of bleeding following TBBx in this animal model.

Oral anticoagulant therapy and international normalized ratios in swine.

While monitoring coagulation testing in Yucatan miniature swine being given oral anticoagulants, we noticed instances of high international normalized ratios (INR) without clinical complications in our animal model. All pigs (n = 17) weighed approximately 35.2 kg and were dosed daily with 2 to 3 mg of coumadin. Plasma samples were obtained and assayed for prothrombin time (PT), calculated INR, and activated partial thromboplastin time (APTT) at baseline, and after 7 and 14 days of coumadin therapy. Results of initial testing indicated high INR values after anticoagulation and short APTT values at baseline, which led us to consider the activity of vitamin K-dependent coagulation factors in the pig. This information was not available in literature concerning this strain of swine, and was surprising given that pigs are frequently used cardiac research models. Using the same plasma samples, we repeated the PT, INR, and APTT determinations using different reagents and a different analyzer. We also determined activities of coagulation factors II, VII, IX, and X. Large PT and INR differences were seen between the two instrument/reagent combinations, possibly due to the differences in the thromboplastins used and differences in the photo-optic versus manual clot-detection method of the instruments. Vitamin K-dependent factors in all pigs responded to coumadin by decreasing to < 30.0% activity, except for factor IX. The high INR values were not as pronounced when the second instrument/reagent combination was used, and the results seemed more in line with the animals' clinical condition. With this instrument/reagent combination, the pig can be considered a good model for research requiring oral anticoagulant medication.

A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1.

We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.

Comparison of quantitative cultures to semiquantitative loop cultures of bronchoscopic protected specimen brush samples.

STUDY OBJECTIVE: To compare quantitative growth from a calibrated loop to growth from the standard serial dilution technique of culturing bronchoscopic protected specimen brush (PSB) samples and to determine the effect of refrigeration of the PSB sample on subsequent quantitative growth. DESIGN: Laboratory stock cultures were sampled with a PSB and cultured by both standard 100-fold serial dilution as well as 1:100 mL and 1:1,000 mL calibrated loops. Stock cultures were also sampled with a PSB and growth before and after refrigeration for 24 h at 4 degrees C (both serial dilution and calibrated loops) was compared. Clinical PSB samples from seven patients suspected of having lower respiratory tract infections were cultured by both techniques as well. SETTING: Clinical research laboratory and teaching hospital. PATIENTS AND INTERVENTIONS: PSB samples from inpatients and outpatients who had clinically indicated bronchoscopy. No interventions. MEASUREMENTS AND RESULTS: Quantitative growth from the 1:1,000 mL calibrated loop was within 1 log10 of growth from the serial dilution technique for 20 of 21 organisms, including 2 yeasts. Except for Haemophilus influenzae (known to be cold intolerant), there were no important differences in growth of bacteria before and after 24 h at 4 degrees C. For quantitative bacterial growth, there was a significant correlation between serial dilution and the 1:1,000 mL loop cultures (r=0.86, p < 0.0001). CONCLUSION: In this study, quantitative growth from a single 1:1,000 mL loop culture of PSB samples was comparable to growth from the standard serial dilution technique. Our results also suggest that overnight refrigeration of PSB samples may be possible in certain clinical situations.

Isolation and characterization of glycogen synthase in Dictyostelium discoideum.

We have partially purified the protein and isolated the glcS gene for glycogen synthase in Dictyostelium. glcS mRNA is present throughout development and is the product of a single gene coding for 775 amino acids, with a predicted molecular mass of 87 kD. The sequence is highly similar to glycogen synthase from human muscle, yeast, and rat liver, diverging significantly only at the amino and carboxy termini. Phosphorylation and UDPG binding sites are conserved, with K(m) values for UDPG being comparable to those determined for other organisms, but in vitro phosphorylation failing to convert between the G6P-dependent (D) and -independent (I) forms. Enzyme activity is relatively constant throughout the life cycle: the I form of the enzyme isolates with the soluble fraction in amoebae, switches to the D form, becomes pellet-associated during early development, and finally reverts during late development to the I form, which again localizes to the soluble fraction. Deletion analysis of the promoter reveals a GC-rich element which, when deleted, abolishes expression of glcS.

Identification of a Ca2+/calmodulin-dependent protein kinase II regulatory phosphorylation site in non-N-methyl-D-aspartate glutamate receptors.

Glutamate receptor ion channels are colocalized in postsynaptic densities with Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II), which can phosphorylate and strongly enhance non-N-methyl-D-aspartate (NMDA) glutamate receptor current. In this study, CaM-kinase II enhanced kainate currents of expressed glutamate receptor 6 in 293 cells and of wild-type glutamate receptor 1, but not the Ser-627 to Ala mutant, in Xenopus oocytes. A synthetic peptide corresponding to residues 620-638 in GluR1 was phosphorylated in vitro by CaM-kinase II but not by cAMP-dependent protein kinase or protein kinase C. The 32P-labeled peptide map of this synthetic peptide appears to be the same as the two-dimensional peptide map of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptors phosphorylated in cultured hippocampal neurons by CaM-kinase II described elsewhere. This CaM-kinase II regulatory phosphorylation site is conserved in all AMPA/kainate-type glutamate receptors, and its phosphorylation may be important in enhancing postsynaptic responsiveness as occurs during synaptic plasticity.

Obstructed tracheal bronchus as a cause of post-obstructive pneumonia.

This article describes a case of obstructed supernumerary tracheal bronchus with partial atelectasis and pneumonia of the right upper lobe, diagnosed using trispiral tomograms. An obstructing broncholith, a supernumerary tracheal bronchus, and granulation tissue mass extruding from the bronchus were confirmed by resection of the pulmonary segment.

Activation mechanisms for Ca2+/calmodulin-dependent protein kinase IV. Identification of a brain CaM-kinase IV kinase.

This manuscript examines the mechanisms by which Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) is activated through the binding of Ca2+/CaM and by phosphorylation. Studies with the synthetic autoinhibitory domain peptides of CaM-kinase II indicate that CaM-kinase IV has a similarly located autoinhibitory domain, and this was confirmed since site-directed mutagenesis of this region (HMDT308 to DEDD and FN317 to DD) generated fully active Ca2+/CaM-independent kinases. Total activities of purified, baculovirus-expressed wild type and mutant kinases were increased 2-fold by intramolecular autophosphorylation, but this reaction was extremely slow (1-2 h) and probably not physiological. However, CaM-kinase IV can be activated by brain CaM-kinase IV kinase resulting in large increases in both total (5-7-fold) and Ca2+/CaM-independent (> 20-fold) CaM-kinase IV activities. This activation reaction required Mg2+/ATP and Ca2+/CaM, was intermolecularly catalyzed, and was reversed by protein phosphatase 2A. Activation of CaM-kinase IV resulted in a 10-fold decrease in Km for syntide-2 with little effect on Km for ATP or Vmax. CaM-kinase IV kinase was highly purified from rat brain extract and was shown to be a 68-kDa monomer. The results of this study demonstrate that CaM-kinase IV does have an autoinhibitory domain within residues His305-Lys321 that suppresses kinase activity in the absence of Ca2+/CaM. CaM-kinase IV is not significantly activated by autophosphorylation, but it can be activated 10-fold by a CaM-kinase IV kinase. This kinase cascade activation mechanism may be important for the physiological function of CaM-kinase IV such as transcriptional regulation through phosphorylation of cAMP responsive element binding protein (Enslen, H., Sun, P., Brickey, D., Soderling, S. H., Klamo, E., and Soderling, T.R. (1994) J. Biol. Chem. 269, 15520-15527).

Mutational analysis of the autoinhibitory domain of calmodulin kinase II.

Calmodulin (CaM)-kinase II is inactive in the absence of Ca2+/CaM due to interaction of its autoinhibitory domain with its catalytic domain. Previous studies using synthetic autoinhibitory domain peptides (residues 281-302) identified several residues as important for inhibitory potency and suggested that His282 may interact with the ATP-binding motif of the catalytic domain. To further examine the autoinhibitory domain, site-specific mutants were expressed using the baculovirus/Sf9 cell system. The purified mutants had many biochemical properties identical to wild-type kinase, but mutants H282Q, H282R, R283E, and T286D had 10-20% constitutive Ca(2+)-independent activities, indicating that these residues are involved in the autoinhibitory interaction. The Ca(2+)-independent activities of the H282Q, H282R, and R283E mutants exhibited 10-fold lower Km values for ATP than the wild-type kinase. Wild-type and mutant kinases, except T286A and T286D, generated Ca2+ independence upon autophosphorylation in the presence of Ca2+/CaM, and those mutants having constitutive Ca2+ independence also exhibited enhanced Ca2+/CaM-independent autophosphorylation. This Ca(2+)-independent autophosphorylation resulted in a decrease in total kinase activity, but there was little increase in Ca(2+)-independent activity, consistent with autophosphorylation of predominantly Thr306 rather than Thr286. These results are consistent with an inhibitory interaction of His282 and possibly Arg283 with the ATP-binding motif of the catalytic domain, and they indicate that constitutively active CaM-kinase II cannot autophosphorylate on Thr286 in the absence of bound Ca2+/CaM. Based on these and other biochemical characterizations, we propose a molecular model for the interaction of a bisubstrate autoinhibitory domain with the catalytic domain of CaM-kinase II.

Alpha subunit of calcium/calmodulin-dependent protein kinase enhances excitatory amino acid and synaptic responses of rat spinal dorsal horn neurons.

1. Here we report that in acutely isolated rat spinal dorsal horn (DH) neurons, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate and N-methyl-D-aspartate (NMDA) receptors can be regulated by endogenous and exogenous calcium/calmodulin-dependent protein kinase II (CaM-KII). Intracellularly applied, the alpha-subunit of CaM-KII enhanced AMPA/kainate and NMDA currents recorded with the use of the whole cell patch-clamp technique. 2. Microcystin, a nonselective phosphatases inhibitor, also enhances AMPA and NMDA responses. 3. Conventional intracellular recordings were made from substantia gelatinosa neurons in spinal cord slices to determine the effect of intracellular application of CaM-KII on excitatory synaptic potentials evoked by electrical stimulation of primary afferent fibers. Excitatory synaptic transmission was enhanced by CaM-KII, which is consistent with the importance of phosphorylation of the postsynaptic AMPA/kainate and NMDA receptor-ion complexes in the short- and long-term changes in synaptic transmission.

Mutational analysis of secondary structure in the autoinhibitory and autophosphorylation domains of calmodulin kinase II.

A previous study has suggested that the autoinhibitory domain of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) may contain an alpha-helical structure, which is important for the proper orientation of inhibitory residues to interact with the catalytic domain (Smith, M. K., Colbran, R. J., Brickey, D. A., and Soderling, T.R. (1992) J. Biol. Chem. 267, 1761-1768). The present study was designed to test the importance of the secondary structure in the autoinhibitory domain (residues 281-302) by site-specific mutagenesis of selected residues to prolines. Single mutants C289P, C289A, A295P, and the double mutant C289P/A295P were expressed using the baculovirus/Sf9 cell system and purified on CaM-Sepharose. The single mutants had specific activities (7-12 mumol/min/mg) and eluted from gel permeation chromatography (600-650 kDa) identical to wild-type kinase. Since the double mutant had a very low specific activity and eluted as a mixture of a large aggregate and proteolyzed monomer, it was not characterized further. Only the C289P mutant exhibited enhanced Ca(2+)-independent activity (5-12% of total activity) prior to autophosphorylation. When autophosphorylation was performed in the absence of Ca2+/CaM at 5 degrees C, only the C289P mutant showed a significant increase in Ca(2+)-independent activity. This autophosphorylation of Thr286 in the absence of Ca2+/CaM has not been observed with wild-type kinase or any other autoinhibitory domain mutant we have characterized. This result suggests that Thr286, the autophosphorylation site responsible for Ca(2+)-independent activity, may not be available for autophosphorylation in the wild-type kinase or the other mutants because of structural restrictions due to the secondary structure in this region. This structural restraint is presumably disrupted by the binding of Ca2+/CaM or by insertion of a proline residue.

Surgical management of hyperparathyroidism at an Air Force medical center.

A critical determinant of successful neck exploration for primary hyperparathyroidism (HPT) is the experience of the surgeon. Results of 17 patients treated surgically for HPT were reviewed to compare results at our medical center with results of large series reported from established national centers. Preoperative laboratory evaluation of 15 patients with surgically proven HPT (solitary adenoma, 14; diffuse hyperplasia, 1) included: mean serum calcium (Ca) 10.9 mg/dl +/- 0.79 and mean serum chloride/phosphate ratio 39 mg/dl +/- 7.9. Serum parathyroid hormone (PTH) was elevated in all patients. Seven neck ultrasounds were performed with a positive predictive value of 42%. No significant operative complications occurred. Mean postoperative serum Ca was 8.34 mg/dl +/- 0.75. One patient had asymptomatic hypocalcemia (Ca = 6.5 mg/dl). Follow-up in 11 patients (65%) at a mean of 8.6 months revealed no evidence of recurrence in these patients. In our experience, proper patient selection and careful surgical technique within established principles for neck exploration and parathyroidectomy lead to excellent results despite the size of the medical center. Patient selection is based on serum Ca, PTH, and chloride/phosphate ratio. Preoperative imaging studies did not appear to be of benefit in the small study reported here.

Phosphorylation and regulation of glutamate receptors by calcium/calmodulin-dependent protein kinase II.

The major postsynaptic density (PSD) protein at glutaminergic synapses is calcium/calmodulin-dependent protein kinase II (CaM-K II), but its function in the PSD is not known. We have examined glutamate receptors (GluRs) as substrates for CaM-K II because (1) they are colocalized in the PSD, (2) cloned GluRs contain consensus phosphorylation sites for protein kinases including CaM-K II, and (3) several GluRs are regulated by other protein kinases. Regulation of GluRs, which are involved in excitatory synaptic transmission and in mechanisms of learning and memory, by CaM-K II is of interest because of the postulated role of CaM-K II in synaptic plasticity and its known involvement in induction of long-term potentiation. Furthermore, mice lacking the major neural isoform of CaM-K II exhibit deficits in models of learning and memory that require hippocampal input. We report here that CaM-K II phosphorylates GluR in several in vitro systems, including the PSD, and that activated CaM-K II enhances kainate-induced ion current three- to fourfold in cultured hippocampal neurons. These results are consistent with a role for PSD CaM-K II in strengthening postsynaptic GluR responses in synaptic plasticity.

Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.

Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.

Functional determinants in the autoinhibitory domain of calcium/calmodulin-dependent protein kinase II. Role of His282 and multiple basic residues.

Important determinants in the autoinhibitory domain of calcium/calmodulin-dependent protein kinase II (CaMK-II), corresponding to residues 281-302 of the kinase alpha-subunit sequence, were identified. Replacement of Thr286 with Ala (CaMK-(281-302 Ala286)) had no effect on either the potency (IC50 = 2 MicroM) or inhibitory mechanism (competitive with ATP) using the catalytic fragment of CaMK-II. Single replacement of charged residues in CaMK-(281-302, Ala286) identified His282, Arg283, Lys291, Arg297, and Lys298 as important determinants (greater than 10-fold increase in IC50) for potent inhibition of CaMK-II. Glu285, Asp288, Lys291, Arg296, and Lys300 were not as essential (less than 4-fold change in IC50) for potent CaMK-II inhibition. Replacement of either Arg283, Lys291, or Arg297, and Lys298 with Ala did not alter the ATP-competitive mechanism of inhibition although the Ki values increased 16-530-fold. However, replacement of His282 with Ala decreased the IC50 by 20-fold and altered the mechanism of inhibition to noncompetitive with respect to ATP. The non-protonated form of His282 was functionally active since decreasing the pH from 7.5 to 5.5 increased the IC50 of CaMK-(281-302, Ala286) almost 20-fold. Histidine protonation also appeared to disrupt the autoinhibitory domain of intact forms of CaMK-II since preincubation of non-proteolyzed rat brain CaMK-II with calcium/calmodulin (in the absence of ATP) at pH 5.5 generated up to 16% calcium-independent activity when assayed at pH 5.5. Similarly, the level of calcium-independent activity of a baculovirus-expressed Asp286 mutant CaMK-II ((D286)mCaMK alpha) increased to almost 80% calcium independence when assayed at pH 5.5 compared to only 20% when assayed at pH 7.5. The levels of calcium-independent activity of both the (D286)mCaMK alpha (at pH 5.5 and 7.5) and the rat brain CaMK-II (at pH 5.5) were sensitive to the concentrations of both ATP and peptide substrate (syntide-2) in the assays. These data suggest that the basic residues Arg283, Lys291, Arg297, and Lys298 are important for potent inhibition of CaMK-II and that the non-protonated form of His282 may play a unique role in the ATP-directed mechanism of inhibition by the CaMK-II autoinhibitory domain.

Expression and characterization of the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II using the baculovirus expression system.

Sf9 cells infected with the recombinant mouse CaMKII-alpha (Ca2+/calmodulin dependent kinase II) baculovirus expressed 12-15 mg of MCaMKII-alpha per liter of cells. Approximately 50% of the MCaMKII-alpha activity could be purified using a CaM-Sepharose affinity column. The purified MCaMKII-alpha had a M(rapp) of 50 kDa by SDS-PAGE and a native Mr of 600 kDa. MCaMKII-alpha, like rat brain CaMKII, had an A0.5 for CaM of 100 nM, a Km for syntide-2 of 8 microM, and was able to generate Ca2(+)-independent activity by autophosphorylation. The baculovirus system expressed large quantities of MCaMKII-alpha with characteristics similar to the rat brain CaMKII, thus providing an expression system for the detailed biochemical analysis of MCaMKII-alpha.

Regulation of the two forms of glycogen phosphorylase by cAMP and its analogs in Dictyostelium discoideum.

We have recently reported the existence of two forms of glycogen phosphorylase (1,4-alpha-D-glucan: orthophosphate-alpha-glucosyltransferase; EC 2.4.1.1) in Dictyostelium discoideum. During development the activity of the glycogen phosphorylase b form decreased as the activity of the a form increased. The total phosphorylase activity remained constant. The physical and kinetic properties of the Dictyostelium enzyme were similar to those of the mammalian enzyme. In mammals, cAMP regulates the conversion of the two forms by a cAMP dependent protein kinase (cAMPdPK). We report here that if cAMP is added to a single cell suspension, the Dictyostelium phosphorylase activity becomes independent of 5'AMP and a 104 kd peptide appears. We also show the effect of several cAMP analogs on the phosphorylase activity in these single-cell suspensions. The cAMP analogs were selected on the basis of their affinities for the membrane-bound cAMP receptor or the cytoplasmic cAMPdPK. We found that relatively low levels, 100 microM, of cAMP or 2'd-cAMP added to aggregation-competent cells in shaking culture caused a loss of phosphorylase b activity and the appearance of phosphorylase a activity. The analog, 2'd-cAMP, has a high affinity for the cAMP receptor but a low affinity for the cAMPdPK. Two other analogs, Bt2-cAMP and 8-Br-cAMP, which have low affinities for the cAMP receptor but high affinities for the cAMPdPK, required high levels (500 microM) for 'b' to 'a' conversion. cDNAs to three cAMP-regulated genes--PL3, D11, and D3--were used as controls in the above experiments. In order to determine if intracellular levels of cAMP were involved in the regulation of phosphorylase activity, both the phosphorylase and the PL3, D11 and D3 mRNA levels were examined in cells suspended in a glucose/albumin mixture--a medium in which adenylate cyclase is inhibited. Under these conditions, neither gene regulation nor a change in the phosphorylase b to a activity occurred in response to added extra cellular cAMP. The results suggest that an intracellular increase in cAMP is involved in the regulation of the two forms of glycogen phosphorylase in Dictyostelium.

Glycogen phosphorylase 'b' in Dictyostelium: stability and endogenous phosphorylation.

The slime mold Dictyostelium discoideum has two forms of the enzyme glycogen phosphorylase. The inactive phosphorylase 'b' form requires 5' AMP for activity and is present in early development. The active phosphorylase 'a' form is 5' AMP independent and occurs during later development. We here show that the 92 kd 'b' enzyme subunit exists either as a singlet or a doublet upon SDS-PAGE, depending on the method of sample extraction. In the presence of exogenously added Mn2+ and ATP, the phosphorylase 'b' shows apparent conversion into a 5' AMP independent form as measured by enzyme activity. In addition, Mn2+ and ATP also support an in vitro phosphorylation of the 92 kd phosphorylase 'b' subunit. We also demonstrate phosphorylation of the 'b' enzyme subunit in vivo by 32-P incorporation into the enzyme protein. A protein kinase responsible for the observed in vitro phosphorylation of the phosphorylase 'b' subunit is characterized.