PubMed Health⌕ Search

Biomedical subjects

P K Bender

Publications and source records attributed to P K Bender.

At least 19 recordsLinked to original sources
PubMed1996

Methionine-containing peptides can be used as methionine sources for protein accretion in cultured C2C12 and MAC-T cells.

Twenty-two methionine-containing di- to octapeptides were evaluated for their ability to be a source of methionine to support protein accretion in C2C12 myogenic and MAC-T bovine mammary epithelial cells. The cell cultures were incubated for 72 h at 37 degrees C in a humidified environment of 90% air: 10% CO2 for C2C12 cells or 95% air: 5% CO2 for MAC-T cells. The basal medium contained methionine-free Dulbecco's modified Eagle's medium and 6% desalted fetal bovine serum. Treatments included basal medium, the basal medium supplemented with one of the 22 methionine-containing peptides, or the basal medium supplemented with free L-methionine. Methionine-containing peptides with the exception of glycylmethionine and prolylmethionine in C2C12 cells were able to support protein accretion with responses ranging from 29.1 to 123.3% of the response of L-methionine. Dipeptides with methionine at the N-terminus promoted greater (P < 0.0001) protein accretion than dipeptides with methionine at the C-terminus. Stimulation of protein accretion by seven pairs of dipeptides with methionine at either the C- or the N-terminus was linearly (P < 0.0001) related to the hydrophobicity of the dipeptides. These results indicate that C2C12 myogenic and MAC-T mammary epithelial cells have the ability to utilize methionine-containing peptides as sources of methionine to support protein accretion.

Amino Acid Sequence↗
PubMed1996

Demonstration and characterization of a transport system capable of lysine and leucine absorption that is encoded for in porcine jejunal epithelium by expression of mRNA in Xenopus laevis oocytes.

Defolliculated Xenopus laevis oocytes were injected with size-fractionated poly(A)+ RNA (RNA) isolated from the jejunal epithelium of growing pigs (average BW 33.8 kg) to identify proteins capable of Na+ -independent amino acid transport. The ability of oocytes to absorb L-lysine (lysine) or L-leucine (leucine) from Na+- free media was quantified in oocytes after injection of RNA fractions or water. Specific RNA fractions were identified that induced saturable uptake of lysine (Kt = 52 microM) and leucine (Kt = 97 microM), whereas endogenous oocyte uptake was not saturable. Induced uptake of .05 mM lysine by oocytes was inhibited (P < .05) 68.1% by 5 mM leucine and 38.9% by .2 mM L-cystine (cystine). Induced uptake of .05 mM leucine was inhibited (P < .05) 83.1% by 5 mM lysine and 23.2% by .2 mM cystine. Although not significant (P > .05), 5 mM L-glutamate (glutamate) quantitatively stimulated the induced uptake of .05 mM lysine by 18.8% and the induced uptake of .05 mM leucine by 60%. To identify mRNA species responsible for this bo,+ transporter-like activity, oocytes were co-injected with the RNA fractions and degenerate DNA oligomers complementary (antisense) to the cloned human kidney bo,+ amino acid transporter, or (as a negative control) with a DNA oligomer complementary to the rabbit intestinal Na+/glucose cotransporter, or with water. Only those oocytes injected with two specific RNA fractions and the antisense DNA oligomer complementary to the bo,+ transporter displayed reduced (P < .05) uptake of lysine (45.7, 55.4%) and leucine (44.1, 65.9%). These results indicate that messenger RNA encoding for a protein capable of stimulating the competitive absorption of lysine and leucine is expressed by the jejunal epithelia of growing pigs.

Animals↗
PubMed1996

Demonstration and characterization of dipeptide transport system activity in sheep omasal epithelium by expression of mRNA in Xenopus laevis oocytes.

Research from this laboratory has recently demonstrated that the omasal epithelium of sheep is capable of absorbing dipeptides. In order to express proteins potentially responsible for the mediated absorption of small peptides, size-fractionated poly(A)+RNA (RNA) isolated from omasal epithelial tissue of sheep (average BW 67.5 kg) were injected into defolliculated Xenopus laevis oocytes. The ability of oocytes injected with RNA or water to absorb [14C]glycyl-L-sarcosine (Gly-Sar) from media (usually pH 5.5) was compared. After 4 d (P < .02) of culture, specific RNA fractions induced an increased (P < .02) rate of Gly-Sar absorption, as compared with water-injected oocytes. The dependency of Gly-Sar uptake on the presence of a pH gradient was evaluated at pH 5.0, 5.5, 6.0, 6.5, and 7.5. Inducible uptake increased (P < .001) in the presence of increasing proton concentrations, whereas endogenous uptake of Gly-Sar decreased (P < .001). At pH 5.5, induced Gly-Sar uptake was saturable (Kt = .4 mM), but endogenous uptake was not. The specificity of Gly-Sar absorption was studied by the co-incubation of .1 mM Gly-Sar with 5 mM levels of competing substrates (pH 5.5). Induced uptake was inhibited (P < .05) 44% by carnosine, 94% by methionylglycine, and 91% by glycylleucine, but not by glycine. Incubation of RNA with DNA oligomers that were complementary to the rabbit intestinal transporter completely inhibited (P < .05) induced Gly-Sar uptake. These results indicate that sheep omasal epithelial cells express messenger RNA that encode for proteins that are capable of H(+)-dependent dipeptide transport activity.

Animals↗
PubMed1995

The gamma subunit of phosphorylase kinase contains a pseudosubstrate sequence.

The catalytic subunit, gamma, of phosphorylase kinase is regulated by a complex set of interactions involving the calcium-binding protein calmodulin and two other subunits designated alpha and beta. These interactions regulate gamma activity that, at least for the calmodulin interactions, involves the regulatory domain in gamma spanning residues 302-366. Within this regulatory domain, we report the identification of a sequence (residues 326-334) that resembles the phosphorylation site in gamma substrates with the exception that a V residue (V332) occurs at the analogous position of the phosphorylated S/T residue. The inhibitory properties of the sequence were assayed with a 10-amino-acid peptide of the sequence. This peptide inhibits a truncated version of gamma, residues 1-300, which is missing the regulatory domain, more potently than it inhibits full-length gamma, and it is a better inhibitor of the full-length gamma at pH 8.2 than at pH 6.8. A similar peptide of the same sequence, except for a S substitution of the V residue, is a good substrate with a comparable Km and better Vmax than peptides of similar length that represent the phosphorylation site in the substrate of the enzyme, glycogen phosphorylase. A mutant gamma protein, with a S for V332 substitution ([V332S]gamma), was prepared using the baculovirus expression system. [V332S]gamma autophosphorylates by an intramolecular mechanism. This demonstrates that this sequence can occupy the catalytic site in the protein. Development of [V332S]gamma affords an experimental model in which the effects of the regulatory factors on autophosphorylation can be determined.

Amino Acid Sequence↗
PubMed1994

Three different calmodulin-encoding cDNAs isolated by a modified 5'-RACE using degenerate oligodeoxyribonucleotides.

In order to obtain the 5' ends of the three mouse calmodulin (CaM) cDNAs, we modified the standard 5' RACE (rapid amplification of cDNA ends) method to use degenerate synthetic oligodeoxyribonucleotides to prime cDNA synthesis of all three CaM mRNAs. In this modified method, the degenerate primers were annealed to mRNAs in an incubation step prior to the reverse transcription reaction. Separating the annealing step from the reverse transcription reaction allowed for greater stringency by using higher temperatures than could be tolerated if the reverse transcriptase were present. Annealing was also done with lower primer concentration and was driven by a longer incubation time. After the annealing step, cDNA synthesis was initiated by diluting the annealing mixture into a 42 degrees C buffer with reverse transcriptase. The synthesized cDNA was poly(dA)-tailed to allow PCR amplification of the first-strand cDNA with an anchor-dT17 primer and the degenerate primers. The CaM cDNAs were evident after this PCR. A second PCR, with nested gene-specific primers, was used to isolate the individual CaM cDNAs from the products of the first PCR. Three distinct CaM cDNAs were cloned and sequenced. By comparison of the 5' untranslated sequences between the mouse CaM DNAs and rat CaM cDNAs, the corresponding homologs were assigned. The results suggest that application of this modified RACE method could improve the success of isolating specific cDNAs in cases where use of a nested primer is not possible or when amino-acid sequence information is available and only degenerate primers can be designed for cloning cDNAs by the 5'-RACE method.

Animals↗
PubMed1994

Baculovirus-directed expression of the gamma-subunit of phosphorylase kinase: purification and calmodulin dependence.

A recombinant baculovirus containing a cDNA encoding the gamma-subunit of phosphorylase kinase from mouse skeletal muscle was constructed. Cultures of Sf-9 insect cells infected with the gamma-baculovirus produce an intact and soluble gamma-protein. A purification procedure is presented that yields a sample of gamma-protein which is devoid of interfering enzyme activity and which is not associated with calmodulin from the insect cells. The isolated gamma sample has a Km for phosphorylase b of 36 (+/- 6, S.E.M) microM at pH 8.2 and 140 (+/- 25) microM at pH 6.8. These values are similar to those reported for the activated phosphorylase kinase holoenzyme isolated from skeletal muscle tissue. However, the Vmax. of the baculovirus-expressed gamma is 65 and 80% of that of the activated holoenzyme at pH 6.8 and 8.2 respectively. These results indicate that one or more of the regulatory subunits alpha, beta, or calmodulin stimulate the activity of the catalytic subunit gamma in the activated holoenzyme. Addition of calmodulin to the baculovirus-expressed gamma stimulates its activity 1.5-2.0 fold at pH 6.8 in both the presence and absence of calcium. At pH 8.2, calmodulin has only minor stimulatory affects. The stimulation by calmodulin at pH 6.8 results from an increase in the Vmax of gamma with little effect on its Km. This result is unlike that for most calmodulin-stimulated kinases which bind calmodulin only in the presence of calcium and exhibit a decrease in their Km upon binding calmodulin. The change in Vmax. of gamma in the presence of calmodulin and in the absence of calcium presents a novel mechanism for the regulation of a calmodulin-stimulated kinase.

Animals↗
PubMed1991

Phosphorylase kinase activity in I/strain neonatal skeletal muscle with a deficiency in alpha/alpha' subunit mRNAs.

In the adult I/LnJ mouse skeletal muscle, phosphorylase kinase activity is 0.2% of that in normal. This deficiency results from a paucity of mRNA's for the phosphorylase kinase regulatory subunit- alpha and its isoform alpha'. However, in the I/LnJ neonatal skeletal muscle phosphorylase kinase activity is 20-25% of that in normal. During the first two months of development this activity decreases while in normal tissue it increases. The developmental differences in the magnitude of the I/LnJ deficiency indicate the possibility of stage specific mechanisms regulating the accumulation of alpha/alpha' mRNAs. To investigate this possibility, the abundance of alpha/alpha' mRNAs and of the catalytic subunit, gamma, mRNAs were compared by Northern Blot analysis. The results demonstrate that neonatal and adult I/LnJ skeletal muscle have a similar paucity of alpha/alpha' mRNAs whereas accumulation of gamma mRNAs is not significantly different from normal.

Aging↗
PubMed1989

I/Lyn mouse phosphorylase kinase deficiency: mutation disrupts expression of the alpha/alpha'-subunit mRNAs.

A cDNA encoding the alpha subunit of mouse skeletal muscle phosphorylase kinase was used to compare the expression of alpha mRNAs in normal and phosphorylase kinase-deficient tissues of the I/Lyn mouse. The results demonstrate that two different molecular weight species of poly(A)+ RNA in normal mouse heart and skeletal muscle hybridize to the alpha cDNA. These two mRNAs direct the synthesis of alpha protein and its isoform alpha' in a cell-free translation system. Thus, alpha and alpha' are encoded by two distinct mRNAs. The abundance of both of these mRNAs is reduced dramatically in the phosphorylase kinase-deficient skeletal muscle and heart tissues from the I/Lyn mouse strain. This result indicates that a mechanism common to both alpha and alpha' expression is disrupted by the I/Lyn mutation. The I/Lyn deficiency is inherited as an X chromosome trait. By Southern mapping of Chinese hamster-mouse cell hybrids the alpha gene was localized to the mouse X chromosome, supporting the possibility that the I/Lyn mutation is in the alpha gene. These results are discussed in terms of a cis or trans mutation influencing the expression of either a single alpha/alpha' gene or two genes encoding alpha and alpha'.

Amino Acid Sequence↗
PubMed1988

The abundance of calmodulin mRNAs is regulated in phosphorylase kinase-deficient skeletal muscle.

In the I/Lyn mouse strain a mutation on the X chromosome results in a deficiency of the major calmodulin-regulated enzyme in skeletal muscle, phosphorylase kinase. Calmodulin has been identified as the delta-subunit of phosphorylase kinase, and it is estimated that approximately 40% of the total calmodulin in rabbit skeletal muscle is associated with the phosphorylase kinase hexadecamer (alpha, beta, gamma, delta)4. The absence of phosphorylase kinase in I/Lyn skeletal muscle results in a reduction in the total amount of calmodulin. The mechanisms affecting this reduction were investigated by comparing the abundance and heterogeneities in calmodulin mRNAs between normal and phosphorylase kinase-deficient skeletal muscles. The results demonstrate that in normal tissue there are four species of calmodulin mRNA distinguished by their molecular weight. All four of these species are present in the deficient tissue, and none of them are preferentially reduced. However, there is a 54% reduction in all four mRNAs as well as in calmodulin in the deficient skeletal muscle relative to normal skeletal muscle. These results indicate that the expression of calmodulin mRNAs is coordinated with the expression of its major enzyme target in skeletal muscle.

Amino Acid Sequence↗
PubMed1988

Two calmodulin genes are expressed in Arbacia punctulata. An ancient gene duplication is indicated.

Calmodulin is highly conserved, and only in the sea urchin Arbacia punctulata have two distinct isotypes been reported. We have isolated and sequenced two cDNAs from a lambda gt 11 library constructed from RNA from ovary tissue of A. punctulata. One clone, designated alpha, encodes a calmodulin isotype previously designated A. It encodes an amino acid sequence that is identical with calmodulin of most vertebrates in positions 1 through 141; however, it does not encode the last seven amino acids. The other clone, designated beta, starts with an open reading frame and encodes the B form of calmodulin from position 11 through the C-terminal position 148. It has only four differences from vertebrate calmodulin, occurring at positions 78 (Asp, beta Glu), 99 (Tyr, beta Phe), 143 (Gln, beta Ala) and 147 (Ala, beta Ser). The nucleic acid sequences of the alpha and beta cDNAs differ at 46 nucleotide positions that are distributed throughout their coding sequences. We conclude that the corresponding mRNAs are not derived from post-transcriptional processing of a single gene, and we infer that they are transcribed from two non-allelic genes. The gene duplication is inferred to have occurred prior to the divergence of the vertebrates and the echinoderms. The expression of these calmodulin mRNAs in ovary tissue and eggs of a single animal differs as judged by hybridization of probes to RNA immobilized to filters.

Amino Acid Sequence↗
PubMed1987

Skeletal muscle phosphorylase kinase catalytic subunit mRNAs are expressed in heart tissue but not in liver.

A cDNA encoding the skeletal muscle phosphorylase kinase catalytic subunit gamma has been isolated and sequenced. It contains 57 nucleotides of 5' nontranslated sequence, the entire coding sequence, and 1004 nucleotides of 3' nontranslated sequence. Probes derived from this gamma-cDNA were used to investigate the expression of gamma-messages in liver, heart, and skeletal muscle tissues. The results demonstrate that the gamma-mRNAs expressed in heart tissue are homologous to the skeletal muscle gamma-mRNAs. However, in liver tissue, no homologous gamma-mRNAs were detected. The implications of these results for understanding gamma-isoform expression and the possibility of a liver-specific gamma-gene are discussed.

Amino Acid Sequence↗