Correlation between protein kinase-mediated stimulation of calcium transport by cardiac sarcoplasmic reticulum and phosphorylation of a 22000 dalton protein.

Increases in protein kinase-catalyzed phosphorylation of a 22000 dalton protein correlated closely with increases in phosphate-facilitated calcium transport measured concurrently in canine cardiac sarcoplasmic reticulum under similar conditions in the presence of varying concentrations of bovine cardiac protein kinase. A correlation coefficient of 0.93 and a P value of less than 0.001 were obtained. Protein kinase-catalyzed phosphorylation of the 22000 dalton microsomal protein may mediate the abbreviation of systole seen in the mammalian heart in response to inotropic agents like catecholamines.     

Adenosine 3′,5′-monophosphate-dependent phosphorylation of a 6000 and A 22000 dalton protein from cardiac sarcoplasmic reticulum

In canine cardiac sarcoplasmic reticulum, adenosine 3′,5′-monophosphate (cyclic AMP)-dependent protein kinase specifically phosphorylates two proteins, as seen by sodium dodecyl sulfate-slab gel electrophoresis and autoradiography. One protein has a molecular weight ranging between 22 000 and 24 000 daltons and has previously been identified and named phospholamban (Tada, M., Kirchberger, M.A. and Katz, A.M. (1975) J. Biol. Chem. 250, 2640–2647). The other protein that the ³²P label incorporates into has a molecular weight of approximately 6000. Like the 22 000 dalton protein, the 6000 dalton protein has characteristic of phosphoester bonding. The time-dependent course of phosphorylation shows that initially the ³²P label is incorporated more rapidly into the 22 000 dalton protein than the 6000 dalton protein, with both proteins reaching a steady-state level of phosphorylation after 10 min of incubation. When both protein kinase and cyclic AMP are eliminated from the incubation medium, both the 22 000 and the 6000 dalton protein are still phosphorylated but only to about a quarter of the activity found when cyclic AMP and protein kinase are included in the incubation mixture. The addition of phosphodiesterase completely eliminates the phosphorylation of both proteins. Treating the microsomes with trypsin prevents subsequent phosphorylation of either protein. Phosphorylating the microsomes first, then treating with trypsin, renders both the 22 000 and the 6000 dalton proteins resistant to even prolonged trypsin attack. Unphosphorylated, both proteins are solubilized by a very low concentration of deoxycholate. After phosphorylation the proteins cannot be solubilized by deoxycholate. Phosphorylation appears to alter greatly the physical properties of these proteins.Control experiments exclude the possibility that a lipid is being phosphorylated. After phosphorylation, the phosphorylated 22 000 dalton protein is separated from the 6000 dalton protein by proteolipid extraction. After first treating the microsomes with methanol, the 22 000 dalton protein is then soluble in acidified chloroform/methanol, while the 6000 dalton protein remains insoluble. The finding that both proteins have much different biochemical properties when phosphorylated than when not, may be relevant in how they regulate calcium transport in the sarcoplasmic reticulum.     

Partial characterization of protein kinase-catalyzed phosphorylation of low molecular weight proteins in purified preparations of pigeon heart sarcolemma and sarcoplasmic reticulum

Pigeon heart microsomes contain three minor size protein kinase substrates of minimal molecular weights of 22 000, 15 000, and 11 500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the microsomes were partially loaded with calcium oxalate and subjected to rate zonal and isopynic centrifugations in sucrose density gradient columns, the 22 000 and the 15 000 dalton proteins settled in the heaviest fraction, which was composed mainly of vesicles of sarcoplasmic reticular membranes; the 11 500 dalton protein was concentrated in the lightest fractions, which consisted chiefly of vesicles of sarcolemmal origin. During incubation of the membrane fractions with Mg[γ-³²P]ATP significant amounts of ³²P were incorporated into all these proteins. Incorporation of ³²P into the 15 000 dalton protein was moderately and ³²P incorporation into the 22 000 dalton protein was markedly enhanced in the presence of exogenous soluble cyclic AMP-dependent protein kinase and cyclic AMP. The phosphorylation of the three proteins was virtually unaffected by CA²⁺ concentrations up to 0.1 mM and by ethyleneglycol-bis(β-aminoethylether)-N,N′-tetraacetic acid in the absence of added Ca²⁺.Phosphorylation of the 22 000 and the 11 500 dalton proteins occurred mainly at serine residues. In the 15 000 dalton protein threonine residues were the main site of endogenous phosphorylation. Nearly equal amounts of [³²P]-phosphate were incorporated into threonine and serine residues of this protein when phosphorylation was supported by exogenous cyclic AMP-dependent protein kinase and cyclic AMP.The 15 000 dalton protein could be removed from its membrane attachment by extraction with an acidic chloroform/methanol mixture. This step opens the way for the purification of this membrane-bound protein kinase substrate.     

Partial characterization of protein kinase-catalyzed phosphorylation of low molecular weight proteins in purified preparations of pigeon heart sarcolemma and sarcoplasmic reticulum.

Pigeon heart microsomes contain three minor size protein kinase substrates of minimal molecular weights of 22 000, 15 000, and 11500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the microsomes were partially loaded with calcium oxalate and subjected to rate zonal and isopycnic centrifugations in sucrose density gradient columns, the 22 000 and the 15 000 dalton proteins settled in the heaviest fraction, which was composed mainly of vesicles of sarcoplasmic reticular membranes; the 11 500 dalton protein was concentrated in the lightest fractions, which consisted chiefly of vesicles of sarcolemmal origin. During incubation of the membrane fractions with Mg [gamma-32P]ATP significant amounts of 32P were incorporated into all these proteins. Incorporation of 32P into the 15 000 dalton protein was moderately and 32P incorporation into the 22 000 dalton protein was markedly enhanced in the presence of exogenous soluble cyclic AMP-dependent protein kinase and cyclic AMP. The phosphorylation of the three proteins was virtually unaffected by Ca2+ concentrations up to 0.1 mM and by ethyleneglycol-bis-(beta-aminoethyl-ether)-N,N'-tetraacetic acid in the absence of added Ca2+. Phosphorylation of the 22 000 and the 11 500 dalton proteins occurred mainly at serine residues. In the 15 000 dalton protein threonine residues were the main site of endogenous phosphorylation. Nearly equal amounts of [32P]-phosphate were incorporated into threonine and serine residues of this protein, when phosphorylation was supported by exogenous cyclic AMP-dependent protein kinase and cyclic AMP. The 15 000 dalton protein could be removed from its membrane attachment by extraction with an acidic chloroform/methanol mixture. This step opens the way for the purification of this membrane-bound protein kinase substrate.     

Studies on the mechanism of insulin-stimulated protein phosphorylation in adipocytes

Insulin exerts two types of effects on protein phosphorylation in adipocytes. First, insulin stimulates phosphorylation of a 123,000 dalton peptide (ATP citrate lyase); second, insulin inhibits the epinephrine-stimulated phosphorylation of a 69,000 dalton peptide.Propranolol, nicotinic acid and concanavalin A, agents which, like insulin, inhibit epinephrine-stimulated cAMP accumulation, also inhibit epinephrine-stimulated phosphorylation of the 69,000 dalton peptide. These agents do not, however, stimulate the phosphorylation of the 123,000 dalton peptide. Carbamylcholine and a variety of cyclic nucleotides (other than cyclic AMP and dibutyryl cAMP) do not alter protein phosphorylation in intact adipocytes. Finally, under conditions wherein insulin fails to inhibit dibutyryl cAMP-stimulated phosphorylation of the 69,000 dalton peptide, insulin-stimulated phosphorylation persists.Thus, while insulin $\text{inhibition}$ of epinephrine-stimulated phosphorylation may be mediated by insulin-induced alterations in cAMP accumulation or action, insulin-$\text{stimulated}$ phosphorylation is $\text{not}$ due to alterations in cyclic nucleotide accumulation or action.     

Phosphorylation of the α-subunit of (Na+ + K+)-ATPase by carbachol in tissue slices and the role of phosphoproteins in stimulus-secretion coupling

This study examined the changes in protein phosphorylation in response to cholinergic (muscarinic) stimulation of salivary secretion in the rat submandibular gland. Carbachol stimulation was associated with phosphorylation in a number of protein bands as detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and autoradiography. The molecular masses (M_r) of two proteins, in which the amount of phosphorylation more than doubled in response to carbachol, were 22 000 and 96 000. The M_r 96 000 protein precipitated at 120 000 × g while most of the M_r 22 000 protein remained in the supernatant at this speed. The effect of carbachol on the phosphorylation of the M_r 22 000 and 96 000 proteins was blocked by atropine, indicating that the cholinergic receptor involved is muscarinic. The time course of phosphorylation of the M_r 22 000 protein consisted of a rapid incrase in phosphorylation within the first min of carbachol stimulation. This increased phosphorylation persisted for less than 1 min. The increased phosphoryaltion of the M_r 96 000 protein also occurred within the first min but it persisted for at least 10 min. However, removal of the muscarinic agonist, carbachol, resulted in the rapid dephosphorylation of this protein. When the plasma membranes were purified, the M_r 96 000 protein was phosphorylated by ATP in the presence of Na⁺ and Mg²⁺. It was dephosphorylated by K⁺. This proves that the M_r 96 000 dalton protein is the α-subunit of the (Na⁺ + K⁺)-ATPase.     

Effect of parathyroid hormone and cyclic AMP on protein phosphorylation in rabbit kidney cortex

Suspensions of renal cortical tubules were incubated with ³³P_i and exposed to parathyroid hormone (40 μg/ml) or 1 mM dibutyryl cyclic AMP. In other experiments homogenates of renal cortex were assayed for protein kinase and phosphoprotein phosphatase activity using [γ-³²P]ATP with or without 5 mM cyclic AMP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorylation of proteins measured by liquid scintillation counting of gel slices. The pattern of protein phosphorylation was similar in control tissue from both tubule suspensions and homogenates. In intact tubules, parathyroid hormone stimulated the phosphorylation of four proteins with molecular weights of approx. 1500 000, 125 000, 100 000 and 50 000 by 28%, 24%, 13%, and 20%, respectively. Results with dibutyryl cyclic AMP were comparable but more variable. Stimulation of phosphorylation by cyclic AMP in homogenates was more generalized with the major effect on a 50 000 dalton protein (50% stimulation). No effect of cyclic AMP on dephosphorylation of proteins was observed. The results are interpreted as indicating that increased phosphorylation of cell proteins is part of the cyclic AMP-mediated response of the renal cortex to parathyroid hormone.     

Electron microscopic visualization of the arrangement of the two protein components of (Na + K)-ATPase

The information obtained by electron microscopic examination of highly purified membrane preparations of (Na⁺ + K⁺)-ATPase after freeze-fracturing or negative staining suggests the following conclusions. The catalytic 100 000 dalton protein component penetrates with its greater ‘globular’ mass the plasma membrane and protudes with its smaller mass from the protoplasmic surface by a stalked knob carrying the catalytic centre. The 40 000 dalton glycoprotein component is anchored in the membrane interior by a non-polar ‘fibrous’ side chain, whereas its major polar mass projects from the outer membrane surface forming a surface coat of ill-definable substructure.     

Resolution and properties of a protein kinase catalyzing the phosphorylation of a wheat germ cytokinin-binding protein

The major cytokinin binding protein of wheat germ (CBP) was extensively purified employing chromatography on Cibacron F3GA-Sepharose CL6B and concanavalin A-agarose as key purification steps. The major polypeptides present in the purified CBP preparations have molecular weights of 60,000 ± 4,000, 42,000 ± 3,000, and 37,000 ± 3,000, respectively. A protein kinase that catalyzes the phosphorylation of CBP (CBP kinase) was extensively purified from wheat germ by affinity chromatography on casein-Sepharose 4B and CBP-Sepharose 4B. The purification procedure resolves CBP kinase from an abundant casein kinase that does not phosphorylate CBP. CBP kinase catalyzes the phosphorylation of casein, phosvitin, CBP, and the wheat germ cyclic AMP-binding protein cABPII. CBP kinase phosphorylates the major 60,000 dalton subunit of CBP as well as 16,000 to 18,000 dalton polypeptides present in CBP preparations. CBP fractions with differing activities as substrates for CBP kinase were partly resolved by gel filtration and by chromatography on DEAE-Sephacel.     

A novel stimulator of protein phosphorylation in Neurospora crassa

Summary An endogenous thermostable activator of Protein kinase III (PKIII) was purified from 100000 × g supernatants of Neurospora crassa mycelial extracts. This 38 000 dalton polypeptide, clearly separable from calmodulin on P-60 gel filtration, specifically stimulated N. crassa PKIII activity on casein or phosvitin in vitro phosphorylation.The factor was only present in the initial growth phase of the fungus. The mechanism of PKIII activation and its possible regulatory role are discussed.Abbreviations PK protein kinase - MES 2-N-Morpholino ethane-sulfonic acid - PMSF phenylmethylsulfonyl fluoride - S₁₀₀ 100000 × g Supernatant     

Adenosine 3',5'-monophosphate-dependent phosphorylation of a 6000 and a 22,000 dalton protein from cardiac sarcoplasmic reticulum

In canine cardiac sarcoplasmic reticulum, adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase specifically phosphorylates two proteins, as seen by sodium dodecyl sulfate-slab gel electrophoresis and autoradiography. One protein has a molecular weight ranging between 22,000 and 24,000 daltons and has previously been identified and named phospholamban (Tada, M., Kirchberger, M.A. and Katz, A.M. (1975) J. Biol. Chem. 250, 2640-2647). The other protein that the 32P label incorporates into has a molecular weight of approximately 6000. Like the 22,000 dalton protein, the 6000 dalton protein has characteristics of phosphoester bonding. The time-dependent course of phosphorylation shows that initially the 32P label is incorporated more rapidly into the 22,000 dalton protein than the 6000 dalton protein, with both proteins reaching a steady-state level of phosphorylation after 10 min of incubation. When both protein kinase and cyclic AMP are eliminated from the incubation medium, both the 22,000 and the 6000 dalton protein are still phosphorylated, but only to about a quarter of the activity found when cyclic AMP and protein kinases are included in the incubation mixture. The addition of phosphodiesterase completely eliminates the phosphorylation of both proteins. Treating the microsomes with trypsin prevents subsequent phosphorylation of either protein. Phosphorylating the microsomes first, then treating with trypsin, renders both the 22,000 and the 6000 dalton proteins resistant to even prolonged trypsin attack. Unphosphorylated, both proteins are solubilized by a very low concentration of deoxycholate. After phosphorylation the proteins cannot be solubilized by deoxycholate. Phosphorylation appears to alter greatly the physical properties of these proteins. Control experiments exclude the possibility that a lipid is being phosphorylated. After phosphorylation the phosphorylated 22,000 dalton protein is separated from the 6000 dalton protein by proteolipid extraction. After first treating the microsomes with methanol, the 22,000 dalton protein is then soluble in acidified chloroform/methanol, while the 6000 dalton protein remains insoluble. The finding that both proteins have much different biochemical properties when phosphorylated than when not, may be relevant in how they regulate calcium transport in the sarcoplasmic reticulum.     

Filament formation in vitro of a sieve tube protein from Cucurbita maxima and Cucurbita pepo

Summary Phloem proteins of the sieve tube exudate from Cucurbita maxima Duch. and Cucurbita pepo L. were investigated as to their filament forming ability in vitro. From the two main proteins (116000 dalton, 30000 dalton) only the 116000 dalton protein was found to form reversibly distinct filaments of 6–7 nm diameter upon removal of SH-protecting agents from the buffer, whereas the 30000 dalton protein was precipitated as amorphous material under these conditions. The protein filaments were similar to the filaments ocurring within the sieve tube cells in vivo.Abbreviations SDS sodium dodecyl sulfate - TCA trichloroacetic acid     

Phosphorylation of a 100 000 dalton component and its relationship to calcium transport in sarcoplasmic reticulum from rabbit skeletal muscle

Sarcomplasmic reticulum from rabbit fast skeletal muscle contains intrinsic protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) and a substrate. The protein kinase activity was Mg2+ dependent and could also phosphorylate exogenous protein substrates. Autophosphorylation of sarcoplasmic reticulum vesicles was not stimulated by cyclic AMP, neither was it inhibited by the heat-stable protein kinase inhibitor protein. The phosphorylated membranes had the characteristics of a protein with a phosphoester bond. An average of 73 pmol Pi/mg protein were incorporated in 10 min at 30 degrees C. Addition of exogenous cyclic AMP-dependent protein kinase increased the endogenous level of phosphorylation by 25-100%. Sarcoplasmic reticulum membrane phosphorylation, mediated by either endogenous cyclic AMP-independent or exogenous cyclic AMP-dependent protein kinase, occurred on a 100 000 dalton protein and both enzyme activities resulted in enhanced calcium uptake and Ca2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3), in a manner similar to cardiac microsomal preparations. Regulation of Ca2+ transport in skeletal sarcoplasmic reticulum may be mediated by phosphorylation of a 100 000 dalton component of these membranes.     

Alteration of protein metabolism in individual,identified neurons from Aplysia

A search for control mechanisms governing protein metabolism in neurons from Aplysia californica has uncovered two examples of altered patterns of newly synthesized proteins: (1) The pattern of newly synthesized proteins in the R2 neuron is altered when protein synthesis occurs at elevated temperatures (22–30°C as compared with 13–15°C). (2) The processing of newly synthesized 12,000 dalton (12k) material to 6–9,000 dalton (6–9k) size in the R15 neuron (Strumwasser, F. and Wilson, D. F. [1976], J. Gen. Physiol., in press) can be blocked by certain ion replacements. If acetate replaces chloride in the incubation medium during the synthesis of 12k material, an early step in the processing, prior to the actual breakdown of 12k material, is blocked. Experiments with RNA-synthesis inhibitors indicate that none of the mRNAs which code for abundantly synthesized protein species in the R2 or R15 neurons have short (less than 4 hr) half-lives. This result has implications for an earlier report of regulation of protein synthesis in the R15 neuron.     

Purification and characterization of a latent precursor of a double-stranded RNA dependent protein kinase from reticulocyte lysates

Double-stranded RNA (dsRNA) activates a cyclic 3′: 5′-AMP independent protein kinase (dsI) in reticulocyte lysates which inhibits protein synthesis by phosphorylating the 38, 000 dalton (38K) subunit of the initiation factor eIF-2 (eIF-2α). A latent precursor form of dsI (latent dsI) has been partially purified (1000–2000 fold) from lysates. Activation of dsI at all stages in the purification of latent dsI requires ATP and low levels of dsRNA (1–20 ng/ml), and is accompanied by the phosphorylation of a broad 67,000 dalton (67K) band. However, as purification proceeds the 67K band is resolved into two phosphorylated polypeptides of 68,500 and 67,000 daltons ($\text{68.5K}\text{67K}$). Although latent dsI and activated dsI have distinctly different chromatographic properties, both forms have similar molecular weights (～120,000) and similar sedimentation coefficients (～3.8S) in glycerol gradients. The data support the view that one or both components of the $\text{68.5K}\text{67K}$ doublet are associated with the dsRNA-dependent protein kinase activity.     

Synthesis,glycosylation and rapid secretion of a glycoprotein by Achlya a primitive eucaryote

Achlya ambisexualis, a water mold, secretes several glycoproteins during exponential growth. Among these is a major protein of 39 000 daltons (protein A-39) which is secreted very rapidly. Protein A-39 is detected among the soluble cellular proteins labeled for 5 min. However, after longer labeling times, an additional 95 000 dalton glycoprotein was immunoprecipitated from among the cytoplasmic proteins by antiserum against protein A-39. This antiserum reacted with a single 37 000 dalton protein from the in vitro translation products of poly(A)-containing RNA in a wheat germ cell-free system which is cleaved to a faster moving component in the presence of dog pancreatic membranes. Immunoprecipitated, chain-completion products of polysomes also show a 37 000 dalton peptide which does not bind to lectins, indicating absence of co-translational cleavage and glycosylation.Tunicamycin inhibits the appearance of the 95 000 dalton protein. Several immunoprecipitable proteins, including protein A-39, having sizes identical to the secretory proteins accumulate in the cytoplasm in the presence of this inhibitor. A short pulse with [³H]glucosamine followed by a chase showed that incorporation in protein A-39 increases while that in 95 000 dalton protein is decreasing. These results suggest that the 95 000 dalton glycoprotein may serve as a glycosyl donor to secretory protein A-39.     

Stimulation of platelet protein phosphorylation by arachidonic acid and endoperoxide analogs

The present study has investigated the influence of arachidonate, endoperoxide analogs, and the calcium ionophore A23187 on platelet aggregation and on the phosphorylation of platelet proteins. Following stimulation of platelets by these agents a rapid increase in phosphorylation of three proteins was observed which began at the same time as the initial formation of platelet aggregates. These three proteins were the 260,000 dalton actin-binding protein, a 40,000 dalton protein of unknown function, and the 20,000 dalton myosin light chain. When extensive aggregation was reached, the extent of phosphorylation returned toward baseline. Pretreatment of platelets with aspirin completely inhibited both aggregation and protein phosphorylations induced by arachidonate, but had only partial inhibitory effects on endoperoxide analogs or A23187. Since endoperoxide analogs and A23187 may trigger endogenous production of prostaglandin endoperoxides and thromboxane A₂, in addition to having a direct effect of their own, it is probable that the partial inhibition seen was due to inhibition of that component of their effect due to this endogenous production, through other effects of aspirin can not be entirely ruled out. Since recent evidence shows that phosphorylation of myosin light chain results from calcium stimulation of a protein kinase in the presence of calmodulin, the results are consistent with mobilization of calcium as the primary role of the arachidonate-endoperoxide-thromboxane pathway.     

On the role of protein phosphorylation in the ATP-dependent permeabilization of transformed cells

Incubation of transformed mouse fibroblasts with external ATP in alkaline medium low in divalent cations causes an increase in the permeability of the plasma membrane to nucleotides and other small molecules. Previous suggestions that the phosphorylation of a 44,000 dalton membrane protein is involved in this permeabilization process have been pursued. Fractionation of cells that had been incubated with [γ-³²P] ATP revealed that the labeled 44K phosphoprotein was found in both the membrane and mitochondrial fractions. Incubation of fractions isolated from unlabeled cells with [γ-³²P] ATP resulted in substantial formation of ³²P-44K in the mitochondrial fraction and less incorporation in the membrane fraction. The 44,000 dalton protein was identified as the α-subunit of mitochondrial pyruvate dehydrogenase by partial proteolytic mapping and immunological cross-reactivity with antibodies prepared against bovine pyruvate dehydrogenase. The phosphorylation of this protein in whole cells by externally added ATP is suppressed by inclusion in the incubation medium of carboxyatractyloside (CAT) and EDTA. These substances have no effect on ATP-dependent permeabilization, indicating that the phosphorylation of pyruvate dehydrogenase is not involved in this process.     

Hormone-induced phosphorylation of a 16000 Dalton polypeptide following meiosis reinitiation in starfish oocytes

In vitro phosphorylation of endogenous proteins is increased in homogenates prepared from 1-methyladenine-treated starfish oocytes when compared with control oocytes, although addition of the hormone to homogenates from control oocytes has no such effect. Following hormonal stimulation the best endogenous substrate is by far a 16 000 dalton (D) protein, the content of which also seems to increase, perhaps through proteolysis of a 21 000 D precursor. cAMP-dependent protein kinases are not involved in either basal or hormone-stimulated phosphorylations, as demonstrated by the lack of effect of either cAMP or of the heat-stable inhibitor of cAMP-dependent protein kinase on the extent of phosphorylation of individual endogenous substrates. Addition of 0.1 mM Ca²⁺ decreases to some extent the protein kinase activity in starfish homogenates and specifically suppresses the phosphorylation of a 40 000 D membrane protein. Starfish oocytes appear to contain myosin light chain kinase activity, as shown by the ability of homogenates to catalyse phosphorylation of exogenous 20 000 D myosin light chains.     

Genetic control of the serum concentration of an H-2-antigen-like polypeptide chain

Mouse serum contains a protein complex consisting of at least three polypeptide chains. One of the chains with an approximate molecular weight of 40 000 is similar to the heavy chain of normal H-2 antigens. The serum concentration of this 40 000 dalton chain is under genetic control. Formal genetic analyses in B10.M and B10.S mice, their F₁ progeny and in backcrosses show that there are two codominantly expressed alleles at a single locus regulating the serum concentration. Measurements of the 40 000 dalton chain in recombinant mice suggest that the controlling locus is situated to the right of theS region.