Two-dimensional Isoelectric Focusing Research Articles

Hormonal stimulation in the exocrine pancreas results in coordinate and anticoordinate regulation of protein synthesis.

24-h intravenous caerulein infusion studies in the rat were combined with in vitro amino acid incorporation studies followed by high-resolution separation of proteins by two-dimensional isoelectric focusing and SDS gel electrophoresis to study the extent to which persistent changes in the biosynthesis of exocrine pancreatic proteins are regulated by cholecystokinin-like peptides. Beginning in the third hour of optimal hormone infusion at 0.25 microgram kg-1 h-1, changes were observed in the synthetic rates of 12 proteins, which progressed over the course of the 24-h study. Based on coordinate response patterns, exocrine proteins could be classified into four distinct groups. Group I (trypsinogen forms 1 and 2) showed progressive increases in synthetic rates reaching a combined 4.3-fold increase over control levels. Group II (amylase forms 1 and 2) showed progressive decreases in synthesis to levels 7.1- and 14.3-fold lower than control levels, respectively. Group III proteins (ribonuclease, chymotrypsinogen forms 1 and 2, procarboxypeptidase forms A and B, and proelastase 1) showed moderate increases in synthesis, 1.4-2.8-fold, and group IV proteins (trypsinogen 3, lipase, proelastase 2, and unidentified proteins 1-4) did not show changes in synthesis with hormone stimulation. Regulation of protein synthesis in response to caerulein infusion was specific for individual isoenzymic forms in the case of both trypsinogen and proelastase. The ratio of biosynthetic rates of trypsinogen forms 1 + 2 to amylase forms 1 + 2 increased from a control value of 0.56 to 24.4 after 24 h of hormonal stimulation (43.5-fold increase). Biosynthetic rates for an unidentified protein (P23) with an Mr = 23,000 and isoelectric point of 6.2 increased 14.2-fold, and the ratio of synthesis of P23 to amylase 2 increased 200-fold during caerulein infusion. During hormone stimulation the anticoordinate response in the synthesis of pancreatic glycosidases (decreased synthesis) and serine protease zymogens (increased synthesis) explain previous observations that showed little change in rates of total protein synthesis under similar conditions.

Effect of amino acid residues on conformational stability in eight mutant proteins variously substituted at a unique position of the tryptophan synthase alpha-subunit.

To elucidate the role of individual amino acid residues in stabilizing the conformation of a protein, the stabilities of wild-type tryptophan synthase alpha-subunit from Escherichia coli and seven mutant proteins substituted by single amino acid residues at position 49, which is buried in the interior of the protein, were compared. The mutant proteins have Gln, Met, Val, Tyr, Leu, Ser, or Lys in place of Glu in the wild-type protein. The dissociation constant, pK, of the Glu residue at position 49 for the wild-type protein was determined to be 7.5 from a titration curve obtained by comparison of two-dimensional isoelectric focusing electrophoresis of the wild-type and mutant proteins. Our results indicate that 1) the conformational stabilities of the proteins studied increase linearly with hydrophobicity of the substituting residues (except Tyr), with the coefficient of this linear dependence being 2.0, 3.4, or 1.3 at pH 5.5, 7.0, or 9.0, respectively; and 2) Lys or Glu at position 49 serve as a destabilizing factor when ionized.

Further characterization of a lectin and its in vivo receptor from Geodia cydonium

From the results of two-dimensional isoelectric focusing, SDS-gel electrophoresis and from immunochemical data it became evident that lectin I and lectin II (corresponding to fractions Geodia I and Geodia II isolated on immobilized lactose) from the sponge Geodia cydonium are apparently identical mixtures of several isolectins, the pI values of their subunits ranging, in contrast to our previous report, from 4.8–7.5. The hypothetical concept of sugar-mediated, specific lectin-lectin interactions (self-recognition) could not be verified by binding of FITC-labelled isolectins (Geodia I) to the lectin subunits, which had been purified by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The concept should also be dismissed on the basis of carbohydrate analyses revealing in contradiction with previous results the exclusive presence of alkali-labile bound tetraglucose on the purified isolectins (1 mol/mol lectin protein). The combining site of the isolectins was shown by a quantitative microprecipitation inhibition assay to be most complementar to oligosaccharides of the β-galactoside series and to interact specifically with particular structural elements of the subterminal sugar(s). Carbohydrates of the anti aggregation receptor, which are assumed to represent the functional ligand of the Geodia-isolectins in vivo, could be demonstrated to have a high affinity for the lectin combining site, exceeding that of the best disaccharide inhibitor, lactose, by five orders of magnitude. A preliminary chemical characterization of the receptor carbohydrate revealed that D-galactose and D-glucose (each approx. 200 mol/mol receptor) are organized in an oligosaccharide, which could be cleaved from the protein by trifluoroacetolysis.

Apolipoproteins as the basis for heterogeneity in high-density lipoprotein2 and high-density lipoprotein3. Studies by isoelectric focusing on agarose films.

A method is described for the isoelectric focusing (IEF) of lipoproteins on thin films of agarose. Within a pH gradient of 4.60-5.30 both high-density lipoproteins 2 and 3 (HDL2 and HDL3) are resolved into more than 10 fractions which could be stained either for protein or for lipids. The isoelectric focusing patterns for HDL2 and HDL3 are similar although HDL2 appears richer in the more alkaline bands. Narrow film strips from the IEF separation of HDL2 and HDL3 were interfaced with various agarose plates containing antisera against apolipoproteins apoAI, apoAII and apoCIII either alone or in combination, to provide two-dimensional IEF immunoelectrophoresis patterns. This technique demonstrated that apoAI and apoAII were present throughout the IEF gel for both subclasses of HDL. It also provided evidence for the existence of lipoproteins containing both apoAI and apoAII and other lipoproteins present in the alkaline region of the gel which contained apoAI but no apoAII. ApoCIII was found mostly in acidic lipoproteins and was not distributed identically in HDL2 and HDL3. The lipoproteins separated by IEF on agarose were also analysed by two-dimensional IEF-SDS electrophoresis and the individual apolipoproteins were identified by reaction with antibodies to apolipoproteins AI, AII, CI, CII, CIII, D, and E. This technique confirmed that in IEF of HDL, apoAI extended throughout the spectrum of lipoproteins whereas apoE was only present in alkaline lipoproteins and apoD was only present in acidic lipoproteins. IEF on agarose of either HDL2 or HDL3 allowed us to collect eight different fractions, which have the same pI in either lipoprotein class. The apolipoprotein composition of each isolated band was analysed by electroimmuno-assays for apolipoproteins AI, AII, CI, CII, CIII, D, and E and the results expressed as the ratio of the measured apolipoprotein to measured apoAI. In both HDL2 and HDL3, acidic lipoprotein fractions were enriched in apoAII, apoCIII and apoD. ApoCII and apoCII were not similarly distributed in HDL2 and HDL3 subfractions whereas the apoCI distribution was similar in both classes. Noteworthy in all experiments was the difference in the distributions of apoCI, apoCII, and apoCIII in HDL2 and HDL3, which indicated that the existence of a lipoprotein containing simultaneously CI, CII and CIII can only account for a small fraction of these apolipoproteins. Therefore these experiments substantiate the theory of the protein basis of HDL heterogeneity and suggest that the majority of apolipoproteins are present in complexes which upon IEF result in lipoprotein fractions of identical pI for both HDL2 or HDL3.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of polypeptides in Ustilago spp. pathogenic on wheat, barley, and oats: a chemotaxonomic study

Detergent-soluble polypeptides were extracted from spores of several isolates of Ustilago spp. pathogenic on wheat, barley, and oats: U. tritici, U. nuda, U. nigra, U. hordei, U. avenae, and U. kolleri. These polypeptides were then separated by two-dimensional isoelectric focusing – polyacrylamide gel electrophoresis. Despite large differences in virulence, the three isolates of U. tritici virulent on wheat gave almost identical polypeptide patterns; an isolate virulent only on Aegilops spp. differed from these three by nine polypeptides. In U. nuda, two isolates had identical patterns, and four polypeptides varied between two other isolates. There were five or fewer differences in polypeptides between two isolates of each of U. nigra, U. hordei, U. avenae, and U. kolleri. This low level of intraspecific variation and the variability between closely related isolates preclude use of the present technique to differentiate isolates of unlike virulence of these six fungi. However, the species U. tritici and U. nuda differed by 47 polypeptides; this high level of interspecific variation supports the concept of treating these two pathogens as separate taxa. In contrast, the seedling-infecting smuts of barley and oats, U. nigra, U. hordei, U. avenae, and U. kolleri, showed only a difference in 13 polypeptides overall, with the last two differing by only one polypeptide. This limited interspecific variation supports the view that these four pathogens are very closely related and should be united under one taxon.

Solubilization of plasma membranes in anionic, non-ionic and zwitterionic surfactants for iso-dalt analysis: a critical evaluation

A critical study has been made of the solubilizing properties of anionic, non-ionic and zwitterionic surfactants to be used in the first dimension of two-dimensional isoelectric focusing—sodium dodecyl sulphate (IF—SDS) maps. Excess of SDS is a powerful solubilizing agent, but prevents proteins from entering the IF gel. Nonidet P-40 (NP-40)—urea mixtures are mediocre solubilizing agents, but are compatible with the IF dimension. Zwitterionic detergents (zwittergents) appear to exhibit a well balanced solubilizing power and are able to generate sharp two-dimensional maps, with round spots and minimal vertical and horizontal streaking. SB3-12 and SB3-14 appear to have the best solubilizing properties; shorter surfactants (SB3-8) exhibit a poor solubilization efficiency, while longer detergents (SB3-16) bind too strongly to hydrophobic regions in proteins. The random performance of non-ionic detergents has been attributed to their ability to form mixed micelles with the carrier ampholytes used in the IF step: depending on the relative ratio of NP-40 to Ampholines, different types of micelles would be formed, which, when reaching appropriate stoichiometries and charge densities, would mimic the behaviour of natural zwittergents. Acceptable two-dimensional maps can be obtained when the sample is lysed in limiting amounts of SDS (SDS:protein  1:3), suggesting that excess of free SDS in solution is deleterious to the IF process.

Investigation of the heterogeneity of the MM isozyme of creatine kinase

MM creatine kinase (MM CK) from human skeletal muscle was resolved into 21 subspecies by isoelectric focusing. Rabbit and bovine MM CK's were also shown to have patterns of 21 bands. These results are compatible with the presence of six different M subunit forms in skeletal muscle, which by random association into dimers, could produce 21 different subspecies of creatine kinase. Two-dimensional isoelectric focusing of human MM CK yielded a diagonal pattern, indicating that the various enzyme subspecies were not induced by ampholyte-protein interaction. Heat inactivated serum appeared to stabilize various CK subspecies and also produced one further anodally migrating MM CK form. Fresh serum had combined inhibitory and stabilizing effects on the MM CK subforms, and allowed for the identification of four anodally migrating CK subspecies not present in tissue extract. These findings are of critical importance because of the widespread use of CK isoenzymes in serum for diagnostic purposes.

Comparisons of highly purified hepatic microsomal cytochromes P-450 from holtzman and long-evans rats

The present study describes the purification and characterization of strain variant forms of a major phenobarbital-inducible microsomal hemoprotein, cytochrome P-450b, from Holtzman and Long-Evans rats. The strain variant hemoproteins cannot be resolved by sodium dodecyl sulfate gel electrophoresis, but can be partially separated in two-dimensional isoelectric focusing SDS gels. If, however, sodium tetradecyl sulfate is incorporated into the one-dimensional gel system, separation of the cytochromes P-450b is achieved. Minor structural differences are detected in the peptides of the cytochromes P-450b following limited proteolysis by Staphylococcus aureus V8 protease, cleavage by cyanogen bromide, or reverse-phase high-pressure liquid chromatography of tryptic peptides. The strain variant cytochromes P-450b are immunochemically and spectrally indistinguishable. The optical spectra of the ferric and ferrous hemoproteins are identical, as are the CO- and ethylisocyanide-reduced difference spectra. Ferrous cytochromes P-450b from both rat strains effectively bind metyrapone with equivalent affinities. In addition, the cytochromes P-450b do not differ in their catalytic activities toward benzphetamine, hexobarbital, benzo[a]pyrene, zoxazolamine, 7-ethoxycoumarin, estradiol-17β and testosterone. Cytochrome P-450c, the predominant isozyme inducible in rat liver by 3-methylcholanthrene, was purified from Holtzman and Long-Evans rats. Cytochromes P-450c from both rat strains are indistinguishable based on electrophoretic, immunological, spectral and catalytic properties. Minor structural differences in the cytochromes P-450c were revealed in the reverse-phase high-pressure liquid chromatographic profiles of the tryptic peptides of these hemoproteins, but not in the peptides generated by limited proteolysis or cleavage with cyanogen bromide.

Structural characterization of developmentally expressed antigenic markers on chicken erythrocytes using monoclonal antibodies

Monoclonal antibodies selected for embryonic and adult erythrocyte specificity have been used to characterize developmentally expressed markers on the surfaces of mature circulating erythrocytes of young and adult chickens. The data presented demonstrate that the antigenic changes which occur on the avian erythrocyte membrane with organismic maturation can be accounted for, at least in part, by changes in the expression of structurally different, but possibly related polypeptides. Monoclonal antibodies selected for specific reactivity with the erythrocytes of newly hatched chicks recognize a glycoprotein of 48,000 daltons apparent molecular weight. On two-dimensional isoelectric focusing gels, this antigen, which appears identical in all strains studied, displays microheterogeneity; consisting of eight to nine closely spaced spots with an isoelectric midpoint of approximately 5.5. This antigen is not expressed on the circulating erythrocytes of mature birds; however, an antigen with similar, but perhaps not completely identical structure, can be detected within the adult bone marrow. The monoclonal antibodies which show preferential binding to the circulating erythrocytes of adult birds also immune precipitate an antigen of 48,000 daltons apparent molecular weight, but this antigen has a more basic isoelectric point. The adult antigen is polymorphic. Slightly different patterns were obtained on two-dimensional gels with erythrocytes from inbred birds having different major histocompatibility genotypes. It has a major component near pH 7.0 and additional focusing spots usually occurring at a slightly lower molecular weight near pH 6.8 or 6.6 depending upon strain. Competitive radiobinding assays with B-system-specific alloantisers suggest that these antigens may in fact be antigens of the polymorphic BG locus of the chicken major histocompatibility complex. One-dimensional peptide mapping of the immune precipitated embryonic and adult erythrocyte polypeptides demonstrate that the antigens are borne on distinct but possibly related polypeptides. Both common and unique peptide fragments are found in the digestion products. Selective solubilization of the chicken erythrocyte membrane suggests that the antigens are integral membrane proteins extractable with nonionic detergent but not with reagents which remove peripheral proteins.

Effects of cis-diamminedichloroplatinum (CDDP) on HeLa cell non-histone nuclear proteins.

Two-dimensional isoelectric focusing and sodium dodecyl sulfate (SDS) gel electrophoretic analyses of successive salt extracts of purified HeLa cell nuclei were used to study the effects of cis-diamminedichloroplatinum (CDDP) (cisplatin) on the synthesis and extractability of nuclear non-histone proteins. Nuclei were extracted sequentially with 0.025 M NaCl-0.075 M EDTA, 0.01 M Tris, and 0.6 M NaCl. Each fraction contained 100-400 polypeptide spots, only a few of which were affected by a 3.5-h CDDP pretreatment of the cells. The biosynthesis and/or metabolism of four polypeptide spots was significantly affected by the CDDP treatment. These polypeptides included: (a) 36K/5.8 (designated by MW/PI) in the 0.025 M NaCl-0.075 M EDTA extract, which decreased in intensity with treatment at 10 micrograms CDDP/ml; (b) polypeptide spots 50K/6.0 and 45K/5.3 in the Tris extract, which increased in intensity over a range of CDDP concentrations of 0-5 microgram CDDP/ml; and (c) a polypeptide complex at 110K/7.7 in the 0.6 M Na Cl extract, which decreased in intensity at CDDP concentrations of 0-5 microgram CDDP/ml. Scanning densitometry of the protein spots of the 0.6 M NaCl extract demonstrated that the 110K/7.7 complex decreased to half its intensity compared with non-drug-treated controls at a CDDP concentration of 0.9 microgram CDDP/ml. We have found that high-resolution two-dimensional gel electrophoretic analysis of nuclear proteins is a valuable technique for studying the effects of cytotoxic agents on the synthesis and/or extractability of specific cellular proteins.

Role of sialic acid in the microheterogeneity of serum thyroxine-binding globulin: Study by two-dimensional isoelectric focusing

A new technique combining a neuraminidase treatment of thyroxine-binding giobulin after serum isoelectric focusing and a second-dimensional isocelectric focusing was developed to study the fole of sialic acid in the microheterogeneity of native thyroxine-binding giobulin. By showing the change of p1 occeasioned by the desialylation for each of the bands constimting the thyroxine-binding gcobulin pattern separately, this procedure clearly demonstrated that the microheterogeneity of the native protein could not be imputed to the varying sialic acid content of the bands only. We suggest that at least three molecules of thyroxine-binding globulin, with probably slight differences in their amino acid composition, are present in the serum, and that different degrees of sialylation ensure greater mieroheterogeneity of this protein.

Chapter 16 Biochemical and Immunocytological Characterization of Intermediate Filaments in Muscle Cells

Publisher Summary This chapter describes the biochemical and immunocytological characterization of intermediate filaments in muscles cells. An indispensable tool for the unambiguous characterization of the subunits of intermediate filaments in both muscle and nonmuscle cells has been the technique of two-dimensional isoelectric focusing (IEF)/sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The comparative one-dimensional peptide mapping of total smooth- and skeletal-muscle desmin has revealed the presence of peptide differences in desmin within a given species and among species, indicating that although desmin is a conserved molecule, it may be coded by different genes in the different muscle types and species. The two desmin variants co-purify with desmin purified from chicken gizzard through a number of cycles of polymerization–depolymerization, indicating that they are both integral components of desmin filaments and of the desmin molecule. It is found that proteolysis of vimentin can be avoided if the cells are first cross-linked with the reversible cross-linker dimethyl-3,3-dithiobispropionimidate, then lysed with 0.5% NP-40, and finally denatured and reduced with 9.2 M urea-0.5% P-mercaptoethanol prior to IEF.

Labeling of pigeon erythrocyte membrane proteins by low-level reductive methylation

The reductive methylation procedure of G.E. Means and R. E. Feeney (1968) Biochemistry 7, 2192–2201) was adapted for 3H-labeling of membrane proteins using pigeon erythrocyte membrane. Usably high 3H incorporation into protein was obtained, e.g., 28 μCi/mg protein with 83 nmol (input) H 2CO/mg protein, B 3H 4 − at 10 Ci/mmol, and a B 3H 4 −/H 2CO ratio of 0.34. With this low H 2CO/protein ratio, methylation did not perturb ATP-dependent 45Ca 2+ uptake, Na +-dependent [ 14C]glycine uptake, membrane vesicle sealing, or isoelectric focusing patterns of methylated membrane proteins. The labeled membrane proteins were shown to be good tracers for the unlabeled proteins by using two-dimensional isoelectric focusing x sodium dodecyl sulfate gel electrophoresis.

Highly conserved glucose-regulated protein in hamster and chicken cells: preliminary characterization of its cDNA clone.

A temperature-sensitive mutant K12 derived from a Chinese hamster fibroblast has been shown to overproduce three specific proteins of Mr 94,000, 78,000, and 58,000 when incubated at the nonpermissive temperature (40.5 degrees C). We previously identified these proteins as glucose-regulated proteins similar to those observed in chicken embryo fibroblasts when the cells are starved of glucose. In this report, we show that the Mr 78,000 proteins isolated from the hamster K12 cell line and from chicken embryo fibroblasts have identical electrophoretic mobilities in two-dimensional isoelectric focusing gels and nearly identical peptide maps. However, these proteins are different from heat-shock proteins previously described for animal cells. We have constructed a library of cDNA clones by using the RNA extracted from the hamster K12 cells incubated at 40.5 degrees C. Clones that hybridize preferentially with cDNA made from RNA at 40.5 degrees C were selected. By using the hybrid-selection technique, followed by in vitro translation, a cDNA clone containing a 2550-nucleotide insert coding for the hamster Mr 78,000 protein has been identified.

Polymorphism of brain tubulin.

Calf brain tubulin was subjected to isoelectric focusing and tryptic peptide map analysis. Results from isoelectric focusing experiments showed a total number of 17 well-resolved protein peaks. The number of peaks and the mass distribution under each peak remained the same when the concentration of protein or ampholyte was altered. When the protein was subjected to two-dimensional isoelectric focusing, a diagonal pattern was observed, indicating that the multiple peaks observed are not a manifestation of tubulin- ampholyte interaction. Further investigation by isolating these individual subspecies and subjecting them to isoelectric focusing yielded single peaks corresponding to the original ones without generating the initial pattern of multiple peaks. Tryptic peptide maps showed that among the subspecies of the alpha subunit there are 26 spots that are common among them. There are, however, 7 +/- 1 spots that are unique in each subspecies. Similar observations were obtained for the subspecies of the beta subunit although there are only 2 +/- 1 unique spots in each subspecies. These results suggest that tubulin subunits probably consist of polypeptides with both constant and variable regions in their sequences. Identical results were obtained for canine and rabbit brain tubulin, indicating that tubulin polymorphism is common among brain tissues. Tubulin isolated by either the polymerization-depolymerization or the modified Weisenberg procedures yielded identical results. These results show that the same subspecies of tubulin are extracted by both isolation procedures.

Enzymatic methylation of calmodulin in rat brain cytosol.

The methylation of brain peptides was investigated by incubating brain high speed supernatant fractions with S-adenosyl-L-[methyl-3H]methionine and resolving the radioactive peptide products by polyacrylamide gradient gel electrophoresis. Peptides of 17,000, 21,000, 29,000, and 34,000 daltons were methylated in vitro; the 17,000-dalton peptide had the same electrophoretic mobility as authentic calmodulin and showed the shift in electrophoretic mobility in the presence of EGTA which is characteristic of calmodulin. The identification of the 17,000-dalton peptide as calmodulin was confirmed by electrophoresis under nondenaturing conditions and by two-dimensional isoelectric focusing electrophoresis. The enzyme responsible for calmodulin methylation and its substrate, the putative desmethylcalmodulin, were separated from one another by chromatography on DEAE-cellulose. When this partially purified enzyme was incubated with the substrate-containing fraction, calmodulin was the only methylated product. A test tube assay for calmodulin N-methyltransferase was developed and validated by comparison with calmodulin methylation assessed by gel electrophoresis. Both procedures showed that calmodulin methyltransferase is inhibited by EGTA and stimulated by divalent cations, with manganese giving the highest activity. Similar values for ph optimum and kinetic constants were obtained when the electrophoresis and test tube assays were compared. A rapid chromatographic procedure for the separation of methylated lysines and arginines was developed and used to demonstrate that epsilon-trimethyllysine is the radioactive amino acid formed when calmodulin is methylated in vitro. The methyltransferse, which is distinguished from previously described methylase III enzymes by its divalent cation requirement and substrate specificity, is thus designated as S-adenosyl-L-methionine calmodulin lysine methyltransfease.

A mutation which bypasses the requirement for p24 in bacteriophage T4 capsid morphogenesis

A mutation ( byp24) affecting the N-terminal region of p23 will suppress the lethal effects of am and ts mutations in gene 24. In the presence of normal p24, the byp24 alteration causes a delay in the cleavage of capsid proteins and the assembly of a high percentage of isometric, short-headed particles; therefore, the byp24 mutation can affect the length of the T4 capsid. In the absence of p24, 24 − byp24 double mutants show a reduced rate of cleavage of capsid precursor proteins, and a reduced rate of virus assembly. Iminunoprecipitation with anti-p24 serum has shown the presence of both p24 and p24 c in wild-type phage particles. The 24 − byp24 particles contain no p24 or p24 c, as determined by immunoprecipitation, urea/acrylamide gel electrophoresis, and two-dimensional isoelectric focusing, urea/acrylamide gradient gel electrophoresis. They have a normal electron microscopic appearance, pH stability, and heat stability; but they are more resistant to osmotic shock than wild-type T4. We suggest that p24 normally functions in the initiation of phage T4 capsid protein cleavage reactions.

Identification and properties of bacteriophage T4 capsid-formation gene products

The previously unidentified products of the remainder of those genes ( Y -genes) known to be essential for bacteriophage T4 capsid formation (genes 21, 31 and 40 ) have been identified by sodium dodecyl sulfate/urea/acrylamide gradient gel electrophoresis. Characteristics of this gel system, which differs from the standard gel system and has superior resolution of low molecular weight proteins, are described. Protein identifications have been confirmed by two-dimensional isoelectric focusing sodium dodecyl sulfate/urea/acrylamide gradient gel electrophoresis. P31 ( M r approx. 16,000) and P40 ( M r approx. 14,000) are early proteins which are apparently not cleaved during head assembly, nor incorporated into either the mature phage head or prohead (τ-particles); P31 appears to be greatly overproduced by certain T4 mutants. P21 ( M r approx. 26,000) is a late protein incorporated in large amounts into τ-particles, but is cleaved during head maturation. Properties of these three proteins which appear to have morphogenetic rather than structural roles in capsid formation, are correlated with known properties of their structural genes. It appears that essential T4 head formation gene products in the capsid are limited to P20, P23 * , and P24 * .