Degradation and regurgitation of extracellular proteins by cultured mouse peritoneal macrophages and baby hamster kidney fibroblasts. Kinetic evidence that the transfer of proteins to lysosomes is not irreversible

The uptake and degradation of bovine serum albumin (BSA), bovine liver catalase, and rabbit muscle enolase have been studied in cultured mouse peritoneal macrophages (MPM) and baby hamster kidney fibroblasts (BHK cells). Rates constant for the uptake of the three proteins by MPM were similar. In addition, BSA accumulation was independent of BSA concentration in the uptake medium and was not inhibited by a large excess of serum, suggesting that protein accumulation was by fluid phase pinocytosis. Following an overnight uptake, 20-30% of the accumulated protein was subsequently regurgitated into the medium in a trichloroacetic acid/phosphotungstic acid-precipitable form. This material co-migrated with the authentic protein during molecular sieve chromatography on Sephadex G-50. The rates of appearance of trichloroacetic acid/phosphotungstic acid-insoluble products were greater than expected for cell death and leakage. The observed first order rate constants, kobs, for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and trichloroacetic acid/phosphotungstic acid-insoluble products in the culture medium were identical, indicating that both products were released in parallel from MPM and BHK cells. The kobs for intracellular BSA degradation and regurgitation were independent of the initial BSA concentration in the uptake medium, but were decreased about 35% when degradation was allowed to proceed in the presence of high concentrations of serum. Degradation was also inhibited by chloroquine and pepstatin. Inhibition of degradation was accompanied by an increase in the total amount of regurgitated protein appearing in the medium. Remarkably, however, these inhibitors also decreased kobs for regurgitation, thereby preserving the similarity in the observed rate constants for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and trichloroacetic acid/phosphotungstic acid-insoluble products. These and other results were inconsistent with desorption of proteins from the surface of the culture dish or the surface of cells as the source of the trichloroacetic acid/phosphotungstic acid-insoluble label appearing in the medium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the proteoglycogen fraction non-extractable from retina by trichloroacetic acid.

The trichloroacetic acid-insoluble 1,4-alpha-glucan fraction from bovine retina was purified and characterized. It is a proteoglycogen fraction containing a 42 kDa protein moiety similar in size to the protein moiety of the trichloroacetic acid-soluble proteoglycogen fraction. The apparent weight-average Mr of acid-insoluble and acid-soluble proteoglycogens are 4.7 x 10(5) and 7.0 x 10(5) respectively. The present results support suggestions from earlier studies indicating that acid-insoluble proteoglycogen is the precursor of the acid-soluble form.     

Peptidoglycan turnover during growth of a Bacillus megaterium Dap- Lys- mutant.

The aim of this study was to ascertain whether or not the absence of cell wall growth zones, deduced from the analysis of autoradiographs of DL-[3H]mesodiaminopimelic acid pulse-labeled cells of a Dap- Lys- mutant of Bacillus megaterium, was due to a high peptidoglycan turnover. Turnover was determined in very precise experimental conditions because two kinds of turnover occurred: a low, acid-soluble turnover and a high, acid-insoluble one. The latter was detected during a chase in the culture medium when bacteria were centrifuged before treatment with trichloroacetic acid. Otherwise the acid-insoluble released material precipitated with the bacteria. In the electron microscope this material presented a globular structure and contained both peptidoglycan and teichoic acid. The acid-insoluble turnover was mainly produced by a lytic acitivity that was released into the culture medium. This thermolabile activity was not due to cell lysis. It was implicated in septum cleavage and in the detachment of wall fragments from the cell surface, but did not seem indispensable for cell elongation. The acid-soluble turnover was much weaker and seemed to be indispensable for cell elongation.     

Biosynthesis of hyaluronic acid by Streptococcus.

Synthesis of hyaluronic acid was investigated in a cell-free system derived from a strain of Group A streptococci. Preparative procedures were improved so that an enzyme system 70 times more active than that previously reported was obtained. The hyaluronic acid synthesized could be separated into trichloroacetic acid-soluble and -insoluble fractions. On the basis of pulse-chase experiments, it was shown that the trichloroacetic acid-insoluble fraction is a precursor of the soluble fraction. The release of the trichloroacetic acid-insoluble hyaluronic acid is specifically blocked with p-chloromercuribenzoate, without inhibition of chain elongation. The addition of butanol to trichloroacetic acid resulted in solubilization of all of the hyaluronic acid. No detectable difference in molecular size was observed between the two hyaluronic acid fractions, both of which were estimated to be more than one million daltons in size. Testicular hyaluronidase digestion of either one of the two types of hyaluronic acid yielded no high molecular weight fragments, indicating that hyaluronic acid is not bound covalently to protein. However, following incubation of enzyme assay mixtures with UDP-[14C]GlcUA, even in the absence of UDP-GlcNAc, radioactive high molecular weight hyaluronic acid was obtained which suggests that the enzyme system elongates rather than initiates hyaluronic acid chains. Tunicamycin did not inhibit hyaluronic acid synthesis, indicating lack of participation of an intermediate of pyrophosphorylpolyisoprenol type. The results obtained are consistent with the hypothesis that chain elongation of hyaluronic acid proceeds by alternate addition of monosaccharides from UDP-sugars by a membrane-bound synthesizing system followed by release of completed hyaluronic acid chains.     

Interactions of soya-bean agglutinin with purified glycoconjugates and soya-bean seed components.

A radioaffinity assay for lectin binding to receptors was developed and characterized by using the interactions between soya-bean agglutinin and four glycoconjugates, namely thyroglobulin, galactomannan, fetuin and asialofetuin. On application of the assay to soya-bean extracts a wide range of seed components were found to have the capacity to interact with soya-bean agglutinin. These included both trichloroacetic acid-soluble and trichloroacetic acid-insoluble glycoconjugates and two classes of particulate matter distinguished by their differential solubility in Triton X-100.     

A cell-free assay for the insertion of a viral glycoprotein into the plasma membrane

A cell-free assay has been developed for the delivery of influenza virus neuraminidase to the plasma membrane. Two types of postnuclear supernatant, which acted as donor and acceptor of the enzyme, were prepared from baby hamster kidney cells. Donor preparations were obtained from cells infected with influenza virus and containing neuraminidase en route to the plasma membrane. Acceptor preparations were obtained from cells containing, bound to their plasma membranes, Semliki Forest virus with envelope glycoproteins bearing [3H]N-acetylneuraminic acid. Fusion between vesicles from these two preparations permits access of the enzyme to its substrate, which results in the release of free [3H]N-acetylneuraminic acid. This release was detected through the transfer of radioactivity from a trichloroacetic acid-insoluble to a trichloroacetic acid-soluble fraction. An ATP-dependent component of release was found, which appears to be a consequence of vesicle fusion. This component was enhanced when the donor was prepared from cells in which the enzyme had been concentrated in a compartment between the Golgi complex and the plasma membrane, which indicates that a specific exocytic fusion event has been reconstituted. The extent of fusion is greatly reduced by pre-treatment of donor and acceptor preparations with trypsin, which points to the involvement of proteins in the fusion reaction.     

Metabolism of Ethionine in Ethionine-Sensitive and Ethionine-Resistant Cells of the Enteric Yeast Candida slooffii

In a defined medium with added ethionine plus low methionine, phenylalanine, tryptophan, tyrosine, adenine, and additional methionine reversed inhibition of the enteric yeast Candida slooffii by ethionine. Isoleucine and 7-methylguanine restored half-maximal growth. Choline but not triethylcholine inhibited C. slooffii. 6-Mercaptopurine reversed ethionine inhibition and also synergistic inhibition by ethionine plus choline. Protection against ethionine by adenine plus aromatics was also evident with log-phase cells in the absence of methionine. Incorporation of ethionine-ethyl-1-(14)C by resting cells was partially inhibited by aromatic amino acids and methionine. Ethionine depressed incorporation of (3)H-phenylalanine but not of (3)H-adenine. Ethionine-resistant mutants were isolated which incorporated ethionine efficiently and degraded it to yet unidentified substances not including 5'-ethylthioadenosine. Ethionine-sensitive cells accumulated more S-adenosylethionine (SAE) than resistant mutants. Adenine was a good precursor of SAE. Radioactivity from ethionine-ethyl-1-(14)C was recovered from cell fractions of ethionine-sensitive cells with the following distribution: cold trichloroacetic acid-soluble > hot trichloroacetic acid-insoluble > lipids > deoxyribonucleic acid > ribonucleic acid. Total radioactivity recovered from ethionine-sensitive cells was twice as much as that from ethionine-resistant mutants.     

Two Glycogen Synthase Activities Associated with Proteoglycogen in Retina

Glycogen synthase of bovine retina was found associated with the acid-insoluble and acid-soluble proteoglycogen fractions. The synthase associated with the acid-insoluble proteoglycogen precursor showed an 8-fold lower Km for UDP-glucose than the synthase associated with the acid-soluble fraction, and was inhibited by detergent. A short digestion with pronase resulted in conversion of the acid insoluble fraction into acid-soluble. The results lead us to postulate that the acid-insolubility of the proteoglycogen fraction and the association with retina membrane proposed before, is caused by glycogen synthase strongly associated to its polysaccharide moiety. The enlargement of the polysaccharide moiety during proteoglycogen biosynthesis, from glycogenin linked to a few 11 to 12 glucose units to the acid-insoluble proteoglycogen precursor (Mr 470,000) would be carried out, together with the branching enzyme, by the glycogen synthase showing a low Km for UDP-glucose. The glycogen synthase with the highest Km for UDP-glucose would participate in conversion of the precursor into mature acid-soluble proteoglycogen.     

Testosterone and 6-N,2''-O-dibutyryladenosine 3'':5''-cyclic monophosphate stimulate protein and lysosomal enzyme secretion in rat seminal vesicle.

Rat seminal-vesicle secretion was studied in vitro in a slice-incubation system. Seminal-vesicle slices were preincubated with 32Pi for 15 min, rinsed, and incubated in an isotope-free 'chase' medium for up to 4h. Gland slices spontaneously discharged protein, three lysosomal hydrolases and trichloroacetic acid-insoluble 32P into the medium in a time- and temperature-dependent manner. Testosterone (10 muM) and dibutyryl cyclic AMP (1 mM) stimulated the discharge of protein, acid hydrolases and trichloroacetic acid-insoluble 32P, and also stimulated the incorporation of 32Pi into trichloroacetic acid-insoluble components. The acid phosphatase and beta-N-acetylhexosaminidase isoenzymes were separated by isoelectric focusing. These hydrolases were secreted into the medium as acidic isoenzymes, presumably contained within primary lysosomes, whereas they occurred largely as less acidic and basic isoenzymes in the glandular tissue.     

Correlation of Carbohydrate Catabolism and Synthesis of Macromolecules During Enzyme Synthesis in Pseudomonas fluorescens.

Kirkland, Jerry J. (Oklahoma State University, Stillwater), and Norman N. Durham. Correlation of carbohydrate catabolism and synthesis of macromolecules during enzyme synthesis in Pseudomonas fluorescens. J. Bacteriol. 90: 23-28. 1965.-Glucose, ribose, and fructose shorten the lag period required for synthesis of protocatechuate oxygenase. Radioactivity from uracil-2-C(14) is incorporated into the hot trichloroacetic acid-soluble fraction after a lag period of approximately 20 min after addition of protocatechuic acid. Addition of glucose or ribose simultaneously with the inducer shortens the lag period to approximately 5 min and increases the rate of uracil incorporation. The inducer must be present to initiate incorporation of radioactivity, and the exogenous carbon source accelerates incorporation but is not sufficient to initiate synthesis by itself. The addition of protocatechuic acid increases the rate and total incorporation of radioactivity from uniformly labeled glucose or ribose-1-C(14) into the hot trichloroacetic acid-soluble fraction. Ribose decreases the incorporation of radioactivity from uniformly labeled glucose into the hot trichloroacetic acid-soluble fraction, and glucose shows a similar effect on incorporation of radioactivity from ribose-1-C(14), indicating the two sugars are serving in the same capacity to enhance enzyme synthesis. Radioactivity from glucose-1-C(14) is not incorporated into the hot trichloroacetic acid-soluble fraction. The results suggest that glucose and ribose shorten the lag period for inducible enzyme formation by serving as a "specific" carbon source for synthesis of macromolecules such as ribonucleic acid.     

The control of fatty acid and triglyceride synthesis in rat epididymal adipose tissue. Roles of coenzyme A derivatives, citrate and l-glycerol 3-phosphate

1. Methods are described for the extraction and assay of acetyl-CoA and of total acid-soluble and total acid-insoluble CoA derivatives in rat epididymal adipose tissue. 2. The concentration ranges of the CoA derivatives in fat pads incubated in vitro under various conditions were: total acid-soluble CoA, 0.20-0.59mm; total acid-insoluble CoA, 0.08-0.23mm; acetyl-CoA, 0.03-0.14mm. 3. An investigation was made of some postulated mechanisms of control of fatty acid and triglyceride synthesis in rat epididymal fat pads incubated in vitro. The concentrations of intermediates of possible regulatory significance were measured at various rates of fatty acid and triglyceride synthesis produced by the addition to the incubation medium (Krebs bicarbonate buffer containing glucose) of insulin, adrenaline, albumin, palmitate or acetate. 4. The whole-tissue concentrations of glucose 6-phosphate, l-glycerol 3-phosphate, citrate, acetyl-CoA, total acid-soluble CoA and total acid-insoluble CoA were assayed after 30 or 60min. incubation. The rates of fatty acid and triglyceride synthesis, calculated from the incorporation of [U-(14)C]glucose into fatty acids and glyceride glycerol respectively, and the rates of glucose uptake, lactate plus pyruvate output and glycerol output were measured over a 60min. incubation. 5. The rate of triglyceride synthesis could not be correlated with the concentrations of either l-glycerol 3-phosphate or long-chain fatty acyl-CoA (measured as total acid-insoluble CoA). Factor(s) other than the whole-tissue concentrations of these recognized precursors appear to be involved in the determination of the rate of triglyceride synthesis. 6. No relationship was found between the rate of fatty acid synthesis and the whole-tissue concentrations of the intermediates, citrate or acetyl-CoA, or with the two proposed effectors of acetyl-CoA carboxylase, citrate (as activator) or long-chain fatty acyl-CoA (as inhibitor). The control of fatty acid synthesis appears to reside in additional or alternative factors.     

Mediation of growth hormone-enhanced expression of the cartilage phenotype in vitro by the availability of the essential amino acid valine

Without increasing cell number, ovine growth hormone was shown to stimulate the incorporation of ²⁵SO₄ by cultured chick embryo chondrocytes into chondroitin sulfate. Since the stimulation of sulfation by growth hormone was abolished when the amino acid concentrations in the medium were doubled, the relationship between amino acids and growth hormone in promoting the synthesis of acid mucopolysaccharides was investigated. Comparison of the incorporation of various labeled amino acids into trichloroacetic acid-soluble and insoluble material revealed that growth hormone promoted the incorporation of only valine into trichloroacetic acid-insoluble material. Furthermore, growth hormone stimulated valine incorporation into both extracellular and intracellular protein, rather than preferentially into extracellular chondromucoprotein. Growth hormone gave a 4-fold stimulation of valine incorporation into collagen without stimulating collagen synthesis. That growth hormone enhances sulfation by stimulating valine availability was further supported by the observations: (a) doubling only the valine concentration in the medium enhanced sulfation; (b) in medium with twice the normal valine concentration, sulfation failed to be further stimulated with the addition of growth hormone; and (c) in medium with all the other amino acids except valine at twice normal concentrations, growth hormone enhanced sulfation. In addition the temporal relationships and synthetic events occurring between growth hormonealtered valine availability and enhanced chondromucoprotein synthesis were studied. It was found that growth hormone-promoted valine incorporation into acid-insoluble material is a rapid effect that can be detected by 10 min after hormone addition and does not require RNA synthesis. Increased valine availability is rapidly reversed after growth hormone removal ( ). On the other hand, growth hormone- and valine-enhanced chondromucoprotein synthesis are slower responses, taking over 24 hr of treatment for a maximal stimulation, and are mediated by RNA synthesis, as indicated by actinomycin D sensitivity. Enhanced chondromucoprotein synthesis is also relatively stable after removal of growth hormone or valine ( ).The evidence suggests that the availability of a single amino acid, valine, plays a regulatory role in the synthesis of a specialized cellular product and that growth hormone acts at some level to alter the availability of this essential amino acid.     

Effect of subunit size and conformation on the rate of lysosomal degradation of extracellular proteins in cultured mouse peritoneal macrophages

The degradation of nine well-defined proteins was studied in cultured mouse peritoneal macrophages following their uptake by fluid phase pinocytosis. After uptake, approximately one-third of the radioactivity was released into the medium in the form of trichloroacetic acid/phosphotungstic acid-insoluble material. When the time courses for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and -insoluble radioactivities were independently analyzed, identical observed rate constants (kobs) were obtained. This is in agreement with an earlier claim that regurgitated protein and low molecular weight products arise from a common intracellular pool of radiolabeled substrates, presumably within lysosomes, and that the traffic of substrates between the plasma membrane and the lysosome is probably bidirectional (Buktenica, S., Olenick, S. J., Salgia, R., and Frankfater, A. (1987) J. Biol. Chem. 262, 9469-9476). When intrinsic degradation rate constants (kd) were calculated, these were found to vary inversely with protein subunit molecular weights, from 0.0347 h-1 for horse heart cytochrome c to 0.0104 h-1 for rabbit muscle phosphorylase b. The proportion of peptide bonds in a protein which are initially available to the action of lysosomal proteases should be proportional to the fraction of the total potential surface of a protein which remains accessible to solvent after polypeptide folding (AS/AT). In agreement, lysosomal degradation rates were observed to correlate well with known or estimated values of AS/AT, and thermal denaturation, which may expose previously buried amino acid residues, increased the rate of degradation of bovine serum albumin.     

Oligopeptide uptake by Bacteroides ruminicola

Bacteroides ruminicola did not take up (14)C from exogenous (14)C-labeled l-proline or (14)C-labeled l-glutamic acid and took up very little (14)C from exogenous (14)C-labeled l-valine. Growing cultures of B. ruminicola rapidly took up (14)C from (14)C-proline-labeled peptides of molecular weights up to 2,000 and incorporated it into trichloroacetic acid-insoluble cell material. Uptake and incorporation did not occur at 0 C and were reduced or eliminated in glucose-starved cells, depending upon the length of time the cells were starved. The initial rate of uptake of peptides seemed to exhibit saturation kinetics, but it was impossible to establish this conclusively. The initial uptake of (14)C from peptides was not affected by chloramphenicol but the incorporation of it into trichloroacetic acid-insoluble cell material was virtually eliminated. Only moderate amounts of trichloroacetic acid-extractable, labeled material were present in cells during peptide uptake, whether or not chloramphenicol was present. (14)C-proline was rapidly released from labeled peptides during uptake, whether or not chloramphenicol was present. The amount of (14)C fixed into trichloroacetic acid-insoluble cell material was directly related to the size of peptides originally supplied in the medium. It is concluded that B. ruminicola possesses a general system for the uptake of peptides, that peptides are rapidly hydrolyzed during or after uptake, and that oligopeptides function only to supply amino acids in a form available to the organism.     

Role of Pectinesterase in pH-Dependent Interactions between Pea Cell Wall Polymers

Extracts of etiolated pea (Pisum sativum L.) shoots converted soluble pectin from the seedlings to a trichloroacetic acid-insoluble form. This activity coincided with pectinesterase peaks separated from the extracts by gel filtration and ion exchange. The conversion of pectin to the trichloroacetic acid-insoluble form and pectinesterase exhibited identical responses to pH, with activity only above pH 6. The formation of trichloroacetic acid-insoluble pectin in pea cell walls and their extracts is due to de-esterification of the pectin by pectinesterase and not to binding between pectin and a protein, as reported in the literature.     

A nucleoside triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. The mode of action of the enzyme.

The acid-soluble products of exhaustive digestion of native DNA with Bacillus laterosporus DNase consist of 6.5% of mononucleotides and 93.5% of oligonucleotides with an average chain length of 3.2. The results of viscometric studies and inactivation of transforming DNA indicate the existence of acid-insoluble intermediates and the selective degradation of the population of substrate molecules rather than a random nucleolytic action. Furthermore, sucrose density gradient analysis of partially digested DNA showed that the initial DNA added as a substrate disappeared progressively during the reaction, being replaced by much more slowly sedimenting acid-insoluble materials, which were eventually degraded into acid-soluble end products during the reaction; products intermediate in size between these two components were not detectable. Studies with DNA labeled at the 3'-terminus indicate that Bacillus laterosporus DNase does not attack DNA from 3'-hydroxyl ends to yeild acid-soluble or acid-insoluble materials in a random manner. The results presented in this paper indicate that the nature of the attack of B. laterosporus nuclease is similar to that previously proposed for Micrococcus luteus DNase. The possibility of the sequential release of acid-insoluble intermediate fragments as well as acid-soluble products from the terminal portion of DNA by the enzyme is discussed.     

Method for Restricting Incorporation of Radioactivity from 3H-Thymidine into Deoxyribonucleic Acid Only During Outgrowth of Spores of Bacillus cereus T

When heat-activated spores of Bacillus cereus T (thy(-)) were germinated and grown in medium containing (3)H-thymidine, a significant amount of radioactivity was incorporated into ribonucleic acid and deoxyribonucleic acid (DNA). A method was developed to restrict the incorporation of radioactivity from (3)H-thymidine into DNA only. This was accomplished by labeling the cells with (3)H-thymidine in the presence of 2 mg of 2-deoxyadenosine per ml, 250 mug each of uracil, cytosine, and guanosine per ml, and 500 mug of adenosine per ml. Under these conditions, 97% of the radioactivity incorporated into cold trichloroacetic acid-insoluble material was associated with DNA only. In the absence of these compounds, DNA contained only 72% of the total radioactivity incorporated into cold acid-insoluble material.     

Metabolism of high-density lipoproteins in cultured rat luteal cells

The uptake of cholesterol from high-density lipoproteins (HDL) labeled with 125I and [3H]cholesterol was examined in cultured rat luteal cells. Luteal cells were incubated with labeled HDL, following which the metabolic fate of the apolipoproteins and cholesterol moieties of the receptor-bound HDL were examined. About 50% of the originally bound HDL apolipoproteins were released into the medium in 24 h by a temperature-dependent process while only 5% of the HDL cholesterol was released unmetabolized. Inclusion of unlabeled HDL in the chase incubation resulted in increased release of apolipoprotein-derived radioactive products without significant change in the release of unmetabolized cholesterol. 60% of the apolipoprotein-derived radioactivity could be precipitated with trichloroacetic acid; the remaining trichloroacetic acid-soluble radioactive fraction was identified as [125I]iodotyrosine. Gel filtration chromatography of the chase-released material showed that the trichloroacetic acid-precipitable products, which contained no detectable amounts of cholesterol, eluted over a range of molecular sizes (9-80 kDa). No intact HDL was retroendocytosed. About 80% of trichloroacetic acid-precipitable products could be immunoadsorbed on anti-apolipoprotein A-I antibody immobilized on CNBr-activated Sepharose, suggesting the presence of fragments containing apolipoprotein A-I. This material was also capable of reassociating with native HDL. Lysosomal inhibitors were partially effective in inhibiting the amount of trichloroacetic acid-soluble products formed. The lysosomal degradation appeared to have no role in the uptake of HDL-derived cholesterol. These studies demonstrate preferential and total uptake of HDL cholesterol by luteal cells, with concomitant degradation of the lipoprotein.     

Phycobiliprotein synthesis in protoplasts of the unicellular cyanophyte, Anacystis nidulans.

Stable and metabolically active protoplasts were prepared from the unicellular cyanophyte, Anacystis nidulans, by enzymatic digestion of the cell wall with 0.1% lysozyme. The yield of protoplasts from intact algal cells was approx. 50%. Incorporation of L-[U-14C]leucine into cold trichloroacetic acid-insoluble material from protoplasts preparations was linear for 1.5 h and continued for an additional 2.5 h. Incorporation of radiolabeled leucine into hot trichloroacetic acid-insoluble material from protoplast preparations demonstrated protein synthesis in protoplasts in vitro. Phycocyanin is the principal phycobiliprotein and allophycocyanin is a minor phycobiliprotein in A. nidulans cells. The light-absorbing chromophore of both of these phycobiliproteins is the linear tetrapyrrole (bile pigment), phycocyanobilin. Radiolabeled phycocyanin and allophycocyanin were isolated from protoplast preparations which had been incubated with L-[U-14]leucine or delta-amino[4-14C] levulinic acid (a precursor of phycocyanobilin). The radio-labeled phycobiliproteins were purified by ammonium sulfate fractionation and ion-exchange chromatography on brushite columns. The specific radioactivity of phycocyanin and allophycocyanin in brushite column eluates (protoplasts incubated with radiolabeled leucine) was 106 000 and 82 000 dpm/mg, respectively. The specific radioactivity of phycocyanin and allophycocyanin in brushite column eluates (protoplasts incubated with radiolabeled delta-aminolevulinic acid) was 33 000 and 38 000 dpm/mg, respectively. Phycobiliproteins from protoplasts incubated with radiolabeled leucine were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 25% of the incorporated radioactivity in protoplast lysates and approx. 60% of the incorporated radioactivity in protoplast lysates and approx. 60% of the incorporated ratioactivity in phycocyanin and allophycocyanin (in brushite column eluates) comigrated with the subunits of these phycobiliproteins on sodium dodecyl sulfate-polyacrylamide gels. Chromic acid degradation of phycobiliproteins from protoplast preparations incubated with delta-amino[4-14C] levulinic acid yielded radiolabeled imides which were derived from the phycocyanobilin chromophore. Imides from radiolabeled phycobiliproteins isolated from protoplast preparations incubated with L-[U-14C]leucine did not contain radioactivity. These results show that both the apoprotein and tetrapyrrolic moieties of phycocyanin and allophycocyanin were synthesized in A. nidulans protoplasts in vitro.